SVS Reports 2010

Student: Yordan Sbirkov Supervisor: Dr Lindsay Nicholson Regulation of mRNA binding protein by CD200R ligation Background and Aims of the project Macrophages play a highly significant role in autoinflammatory disease, as they induce the largest part of the tissue damage involved in such conditions. Macrophages are regulated at many levels including via ligation of inhibitory receptors. A well-studied example of the latter is signalling through the TGFβ receptor. Ligation of the receptor results in increased expression of FXR1i (a mRNA binding protein) and culminates in post-transcriptional suppression of TNFα secretion (Tarnjit K. Khera et al - Fragile X-related protein FXR1 controls post-transcriptional suppression of lipopolysaccharide-induced tumour necrosis factor-α production by transforming growth factor-β1). Another receptor expressed on macrophages that exerts downregulatory functions is CD200 Receptor (CD200R). It has been demonstrated that CD200R ligation can decrease cytokine induced tissue damage (David Copland et al - Monoclonal Antibody-Mediated CD200 Receptor Signaling Suppresses Macrophage Activation and Tissue Damage in Experimental Autoimmune Uveoretinitis). The exact mechanisms underlying this reduction of cytokine secretion are unknown. Therefore, the aim of my project was to determine whether CD200R signalling, similarly to TGFβR signalling, could inhibit cytokine release by changing levels of mRNA binding protein expression. Description of work For the purposes of the project, monocytes were obtained from mouse bone marrow and cultured for 8 days in media containing M-CSF, to give a pure population of CD11b positive, F4/80 positive cells (macrophages). These cells were then stimulated with 5 different cytokines for up to 4 hours, in the presence of either CD200R agonist antibody (DX109) or isotype antibody. Supernatants were collected at intervals (30, 60, 120, 240 minutes) for ELISA and cells were lysed for mRNA extraction. To quantitate the changes in mRNA binding protein transcription levels, mRNA was converted into cDNA and then Q-PCR was used to measure the amount of cDNA. Results were analysed and data was presented as follows. Results Several experiments were carried out to interrogate the effects of cytokines (IL-6, IFNg and IL-4, IL-13 respectively) on TTP and FXR1 transcription. The data (Fig. 1) clearly shows 1) a specific pattern of TTP transcription. In response to IL-6, IFNγ and LPS, the levels of TTP mRNA rise several-fold compared to those after incubation with Th2 cytokines; 2) TTP levels increase very rapidly and remain relatively unchanged in cells treated with IL-6. The responses to LPS are somewhat slower; 3) at early time points after cytokine stimulation, CD200R ligation may be inhibiting the expression of the two mRNA binding proteins, but these results are not statistically significant. This effect is most clearly observed with FXR1 transcription levels at 30 min. Figure 1 A) TTP transcription levels

Q-PCR results from two experiments are shown. With increase of time, no or little change (i.e. icrease or decrease) is observed in TTP levels when cells have been treated with IL-4 or IL-13. Levels of TTP in cells treated with IL-6 rise rapidly and remain relatively unchanged throughout the experiment. IFNγ treated cells show a steady increase of TTP transcription over time, whereas we see peak of LPS induced TTP transcription after 1-2 hours of incubation.

i FXR1- Fragile X Related protein

B) FXR1 transcription levels

What the data suggests is that there is no or little change in FXR1 levels over time for all cytokines but perhaps IL-4 and IFNγ. LPS treated cells show upregulation of FXR1 transcription only after 4 hours of incubation. What is more interesting, however, is that when levels of FXR1 in DX109 and isotype antibody treated cells are compared, we see 30-50% inhibition of transcription in the IL-4, IL-6 and IFNγ treated cells in the first minutes of incubation. * The charts are comparing levels of TTP and FXR1 under different conditions to Plastic adherence levels where PA TTP is 1 and for example IL-6 TTP levels are ~2.5 times higher (or equal ~ 2.5)

The QPCR analysis from the different experiments produced errors that preclude definitive conclusions being drawn. The variation in the results may be due to some of the data being obtained at the time when I was still in the process of learning (first few weeks) rather than coming closer and keeping up to a good standard of laboratory dexterity. Another factor that may have contributed to the inconsistency of the data could be the fact that bone marrow from different mice (even if interbred) was used and it may have given rise to populations of cells at day 8 of incubation with slightly different responses. Supernatants were taken and ELISA for TNFα release after 4 hours of incubation was performed. The results show clearly that DX109 treated cells have several-fold decrease in TNFα release after LPS stimulation compared to isotype treated cells.

Fig 2 ELISA for TNFα

Future directions The experiments have to be repeated so that a more definite answer to the question whether CD200R ligation affects mRNA binding protein expression can be given. Moreover, if the new data confirms that FXR1 levels are repressed at 30 min, then the biological significance of this may be investigated. Outcomes of the studentship Thanks to the Biochemical Society I was given the opportunity to learn a vast range of commonly used lab techniques. I was introduced to cell culturing (but due to time constraints could not run tests with FXR1 KO cell lines), co-culturing of CD200 knock out and transgenic cells (time allowed only for a single experiment so data is not shown), proliferation assays using radioactive thymidine, cryostat section staining and scoring, TEFI and others. Moreover, by attending the weekly lab meetings, I managed to get an insight of what other members of the lab were working on, which helped me learn more about experimental design, analysis and data presentation. I feel much more confident that I can successfully tackle even rather challenging work at the bench (i.e. mRNA extraction, running good Q-PCRs, etc). What I believe is even better, however, is that the communication with the research staff and my supervisor gave me inspiration and encouragement to continue my degree with even more enthusiasm in October and start thinking of embarking on a PhD after that. Value of studentship to the lab Yordan participated fully in lab activities throughout this project. He was also able to observe several aspects of other projects. In the short time that he was in the laboratory, he made significant progress in mastering experimental techniques and the data he generated will inform additional investigations of CD200R signalling in macrophages. He was a pleasure to work with, was hardworking, attentive to instruction and careful in execution. His inquisitive nature provoked some interesting discussions and we would have been happy for him to stay

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throughout the summer holiday. I have found that enthusiastic students are always a source of inspiration and encouragement and Yordan was no exception.

Student: Alisa Crisp Supervisor: Martin Cann, University of Durham Biochemical Society Summer Studentship Report – The effect of inorganic carbon on T7 RNA Polymerase Aims: Adenylyl cyclases have been shown to be responsive to inorganic carbon. This studentship aimed to determine whether a structurally related enzyme, also with a palm domain, is similarly responsive. T7 RNA Polymerase (hereafter T7 RNAP) was chosen for study. The project aimed to express and purify the enzyme T7 RNAP for use in experimental assays and crystallography. Assays would then be performed to determine any change of activity in the presence and absence of inorganic carbon, and if time, crystallographic methods would be used to study the effect of inorganic carbon on the structure of the enzyme. Experimental Methods: BL21 cells which had been engineered to produce T7RNAP were grown in a liquid culture, induced to express a form of T7 RNAP with a histidine tag, and then harvested. After harvesting, the solution containing the soluble component of the bacterial cells was purified using a column containing Ni2+ NTA resin by metal ion affinity chromatography. The protein solution was further purified by both an anion exchange column and a size exclusion column (gel filtration) and visualised by SDS-PAGE. Template DNA was produced by performing PCR on the plasmid pQ3N1, and the DNA then purified by phenol:chloroform purification. Purified protein was used to run assays to test the response to inorganic carbon. Each assay contained 700ng purified DNA and 25µg protein as well as 30mM MgCl2 (as enzyme activity requires Mg2+), an RNase inhibitor (3 units RNAsin), 100mM Tris-HCl buffer at pH 7.5 and 32P-ATP in addition to 5mM each NTP. Assays were run for 30 mins at 37°C and activity stopped by pipetting onto filters which had previously been soaked in trichloroacetic acid (TCA) and dried. The filters were then washed with ice-cold TCA to remove any free NTPs, using a negative control which contained all assay components (but which had not been incubated) to judge when most of the excess nucleotides were removed. A scintillation counter was used, giving a result for the number of radioactive nucleotides incorporated into RNA by the enzyme during the assay. Salts (either sodium bicarbonate or sodium chloride) at the assay pH were then introduced to the assay methodology, allowing activity in the presence and absence of HCO3

- to be studied. Assays were performed with varying concentrations of Mg2+, and the pH of ‘cold’ assays (without 32P-ATP) was measured to determine whether pH changed significantly throughout the course of the experiment. Crystallography trays were set up with reservoirs containing ammonium phosphate and glycerol in various concentrations, ranging from 40-50% saturated ammonium phosphate and 27-30% glycerol. Four different sets of trays were set up, with the ammonium phosphate solutions at different pH levels: pH 7.5, 8.0, 8.5 and 9.0. Pure protein was concentrated into four solutions at these pHs each containing 40% saturated ammonium phosphate, 24% glycerol, 1mM DTT and 1mM EDTA.

Results: T7 RNAP was purified three times to a level acceptable for assays, and once to a level suitable for crystallography. Problems were experienced during purification as the protein had a tendency to form multimers known to disrupt crystal formation which had to be removed by gel filtration. Reliability of the assays during the first attempts proved very low, and a change to the protocol was introduced showing that the variation was due to inconsistencies within the washing and counting procedure, rather than differences between the assays themselves. Corrections were then made to the methodology leading to greater reliability of the results. Three experiments were run comparing the activity of T7RNAP in the presence of either NaHCO3 or NaCl. After method development and key controls, the results suggested that the activity of T7RNAP is reduced in the presence of HCO3

- ions compared to Cl-. The reduction of activity in the presence of HCO3- ions

may be due to a number of effects of these ions in solution rather than the direct effects of the ions. The pH of a solution may change, the Na+ ions may be the cause of the effect, and Mg2+ may be precipitated by a reaction with HCO3

- producing MgCO3. The reduction in concentration of the Mg2+ was calculated to be 0.17 mM used in an experiment using 40 mM NaHCO3. Experiments varying [Mg2+] in the presence of both salts suggested that such a small difference in [Mg2+] is not sufficient to produce the effects seen. The use of NaCl shows that any differences between the activities must be due to HCO3

- rather than Na+. Finally, NaCl assays at 4 pHs around the pH of reaction (pH 7.3, 7.5, 7.7, 8.0) were run as well as one NaHCO3 assay, and the start and end pHs measured. The pHs of the assays with NaCl changed by no more than 0.04 during the course of the assay, with the pH of the HCO3

- assay changing by only 0.05, suggesting that a change in pH cannot explain the difference in activity. Future Directions: This project has suggested that HCO3

- ions may cause a reduction in activity of T7RNAP. Further experiments are needed to prove this is the case. Crystallographic methods may show whether there is a direct interaction between the two molecules. Further work may also look at whether it is the bicarbonate ion or the related species, CO2, which is causing the effects, through disequilibrium studies. Finally, if these effects are shown to be true, it will be necessary to study other proteins with palm domains, for example RNA and DNA polymerases, to see if they are also affected by inorganic carbon and to investigate further the signalling pathways involved if polymerases are found to be responsive. Value of Studentship: This studentship has been a great experience for me, increasing my confidence in the lab as well as introducing me to many new biochemical techniques and I have learnt much about scientific research. The first hand experience I have had of a working lab has helped me with decisions regarding my future career, and although I have learnt a lot about the problems researchers often face, I have very much enjoyed working in the lab. This studentship has demonstrated that further research into palm domains and their regulation by inorganic carbon is necessary, and has shown that this is a fruitful area of investigation for the lab which they would otherwise not have known to study.

Student: Amber Shafi Supervisor: Michael Gordge Institution for placement: University of Westminster

Redox alteration of protein disulphide isomerase at the platelet surface

Introduction:

Protein disulphide isomerase (PDI) is an enzyme in the endoplasmic reticulum (ER) of eukaryotic tissues where it catalyzes the formation, breakage or isomerisation of disulfide bonds between cysteine residues within proteins. In recent years it has been found that PDI also exists in other locations other that the ER such as the surface of cells. Cell surface protein disulphide isomerase (csPDI) influences platelet function by the regulation of platelet aggregation responses as well as the delivery of nitric oxide signalling.

Aims:

To measure the activity of csPDI in platelets and MEG-01 megakaryocytes To investigate the effects of oxidative/nitrosative stress on platelet surface

Description of the Work

Preparation and generation of the fluorescent Glutathione disulphide (GSSG) was incubated with eosin isothiocyanate in phosphate buffer. The solution was passed down the G-25 Sephadex column (which had been washed with PDI assay buffer) where approximately 1ml aliquots. The fluorescence was measured with and without DDT where there was an increase in fold suggesting the completion of the reaction and the formation of the probe Di-E-GSSG. The eluted sample did not show an increase in fluorescence, therefore this showed that all the Di-E-GSSG had successfully been converted to EGSH. Thus the aliquots were combined together and quantified using the molar absorption coefficient of 88,000 M-1 cm-1.

MEG-01 megakaryocytes cell culture: The MEG-01 megakaryocyte cell line was obtained from ATCC and grown in RPMI-1640 medium supplemented with 10% v/v foetal calf serum (FCS), aqueous penicillin, streptomycin, and 2mM L-glutamine and incubated at 37°C with a humidiWed atmosphere of 5% CO2. The MEG-01 megakaryocytes were handled in a sterile environment (i.e fume cupboard, gloves and ethanol) involving the changing of medium, sub-culturing of cells and preparing the cells in order to measure csPDI. The cell count was adjusted to 8.0 x 106 I-1 by suspending in HBS. Preparation of washed platelets: Blood was taken from different volunteers (from a qualified person. About 25 ml of blood was collected and centrifuged at 1100 rpm (170g) for 10 minutes three times where the washed platelets were separated from platelet rich plasma each time. The pH was adjusted to 6.5 to inactivate fibrinogen receptors by adding 0.5M citric acid. Also, prostaglandin 1 and apyrase were added in known amounts to protect the platelets from aggregation. After further centrifugation the platelet pellet was obtained; HBS was then added. The solution was then put onto the Sepharose 2B column. The purified platelets were collected into an eppendorf tube (the platelets were a milky colour). The platelet count was adjusted to 200 x 106 I-1 by suspending in HBS; CaCl2

and MgCl2 were added thereafter. Static incubation: Fluorescence of csPDI activity of platelets and MEG-01 megakaryocytes with and without the presence of oxidative/nitrosative stress was measured in a 96 well plate reader. NaHS and SIN-1 were to be used as a source of redox stress; they were diluted in HEBES buffer in the fume cupboard (as NaHS has an unpleasant strong smell). For the redox reaction the platelets were also diluted in the buffer where there was a 1:2 dilution. The plate was incubated for 1 hour and

Student: Amber Shafi Supervisor: Michael Gordge Institution for placement: University of Westminster 30 minutes where the activity was monitored closely for the first 30 minutes. It seemed the csPDI activity was active up to 30 minutes where thereafter the activity would eventually level off. Results

The MEG-01 megakaryocytes cultured in the lab grew and multiplied healthily with no signs of contamination

By using the results from the fluorescence of the 96 well plate reader graphs were plotted for PDI activity under different concentrations as well as the PDI response with SIN-1 and NaHS. There seemed to be no on PDI activity in the presence of SIN-1 within the ranges of 0 - 500 µM. However there did seem to be an increase in PDI activity in the presence of NaHS over the range of 0 – 10 mM. The reason for this could be that NaHS works to directly reduce the probe or it regenerates csPDI active site thiols.

Departures from original proposal Unfortunately we did not have enough time to do platelet adhesion or investigate the role of antioxidants in PDI activity. Future directions of work and value of studentship

The title of ‘Redox alteration of protein disulphide isomerase at the platelet surface’ can further be researched in future by:

• Investigating whether platelet adhesion is altered in parallel with csPDI activity of platelets • Using an animal model to investigate the effect on thrombosis of altering csPDI activity. • Investigating the effects of other oxidative/nitrosative stress chemicals which may possibly

alter csPDI activity • Further investigating or replicating the experiment of NaHS on csPDI activity in order to try

and justify the increase in dose response of PDI. • Investigating the effects of antioxidants on platelet csPDI exposed to redox stress.

I am very grateful to have had the opportunity to gain work experience in the summer. My lab skills have immensely improved by practicing different lab techniques and coming in contact with equipment I have not used during my degree. I am now much more confident in carrying out dilutions and measuring out solids and liquids more accurately. I have enjoyed working in Dr. Gordge’s lab with other researchers and I hope that my contribution has been useful in highlighting possible future areas of research for PDI activity. I would like to thank the Biochemical Society, Dr. Gordge’s and post doctoral scientist Dr. F Xiao for providing me with the opportunity to gain such a invaluable work experience. No doubt will I be now applying for an MSc and PhD in scientific research after my degree References Raturi, A. and Mutus, B., (2007). Characterization of redox state and reductase activity of protein disulfide isomerase under different redox environments using a sensitive fluorescent assay. Free Radical Biology & Medicine. 43 (1), 62-70

Student: Amber Shafi Supervisor: Michael Gordge Institution for placement: University of Westminster Shah, C.M., Bell, S.E., Locke, I.C., Chowdrey, H.S., Gordge, M.P., (2007). Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery. Nitric Oxide : Biology and Chemistry / Official Journal of the Nitric Oxide Society. 16 (1), 135-142

Internalisation and recycling of two-pore domain potassium channel K2P3.1

Studentship Report Anna Williams Page 1

Student: Anna Williams

Supervisor: Ita O'Kelly

Institution for placement: University of Southampton

Aims of Project

The main aim of my project was to investigate the endocytic pathway(s) of the two-pore domain potassium channel, K2P3.1 (TASK1). The channel has a widespread tissue distribution and has been identified in neuronal, cardiac, pulmonary, genitourinary and gastrointestinal tissues (Duprat et al., 2007). When expressed on the plasma membrane, the constitutively active channel allows potassium efflux, which changes the electrochemical potential of the excitable cell to a more negative state and so regulates both basal and stimulated cell function. As, therefore, the protein is a regulator of the cells function, it is of paramount importance to understand the regulation of the channel itself, in particular the control of surface expression. Surface expression is maintained by balancing the forward transport of membrane proteins with their endocytosis. The team I joined have previously identified mechanisms of forward transport for the protein (O’Kelly et al., 2002) and so my aim of identifying mechanisms of internalisation would provide a more extensive knowledge regarding the regulation of surface expression of this protein. There are numerous forms of protein internalisation, including clathrin mediated or caveolae dependent endocytosis. By disrupting the function of proteins which function specifically in these different pathways, the mechanism of internalisation can be deduced. Furthermore, it is known that the potassium channel K2P9.1 is expressed in several types of human carcinomas (Pei et al., 2003) and so an additional aim to the project was to determine if this channel is expressed in cancerous oesophageal cells.

Description of Work Carried Out

Identification of K2P9.1 in OE19 by immunocytochemistry:

The first focus of the project was to identify K2P9.1 (TASK3) in an oesophageal cancer cell line, OE19. Both HEK (human embryonic kidney) and OE19 cell lines were cultured and then harvested to provide protein for analysis by Western Blot. Cells were lysed with a phosphate buffer saline solution (with the addition of NP40 detergent, EDTA and protease inhibitors), proteins separated by SDS electrophoresis and transferred to a nitrocellulose membrane. After a blocking step the membrane was incubated with the primary antibody overnight, washed off with Tris Buffer Saline 0.1% TWEEN, then incubated with secondary antibody before viewing the results using the chemidoc XRS. The experiment was repeated numerous-times to obtain optimal conditions, such as the use of different antibodies, different concentrations of loaded protein, different ECL solutions and different buffer solutions.

DNA transfection and Confocal Microscopy: To determine the mechanism utilised by K2P3.1 for endocytosis specific inhibitors of the major endocytic pathways were utilised followed by immunoflourescense and flow cytometry to detect any alteration in channel distribution. A viral transduction system (Cell Lights, Invitrogen) was used to fluorescently specify organelles (early endosomes, lysosomes, golgi, mitochondria) in HEK293 cells. These cells were then transfected with channel DNA (tagged constructs which introduced GFP and HA tags were utilised). 24 hours post-transfection cells were mounted to cover slips and viewed under the confocal microscope and channel co-localisation with lysosomes, mitochondria, golgi and endosomes was assessed in the presence and absence of specific treatments.

Dynasore, a specific inhibitor of dynamin shows an increase in green fluorescence indicating that endocytosis of the channel is via a dynamin dependent pathway. 8-Br cAMP antagonises cAMP dependent protein kinases and Gö6976 inhibits PKC. Treatment by both inhibitors results in an increase in green fluorescence, indicating that channel internalisation requires protein kinases; known regulators of endocytosis (Kumari et al., 2010). The experiment was also prepared with Brefeldin A (BFA), which disrupts forward transport by inhibiting vesicle movement from the endoplasmic reticulum (ER) to the golgi and inducing retrograde pathways back from the golgi to the ER. BFA visually disrupts the golgi when viewed under the confocal microscope.

Rate of Protein Internalisation: To determine the rate of protein internalisation from the cell surface, cells were transfected with DNA of channel constructs with a HA tag to be incorporated into an extracellular loop of the protein and a GFP tag on the N terminus of the channel (GFP.TASK1.HA). Brefeldin A (BFA) was included in the cell culture media to prevent forward transport. At timed points the cells were transferred from the incubator to ice and prepared them for quantification via



flow cytometry. This involved transferring the cells from petri dishes to falcon tubes, via cell scraping, then numerous wash steps with FACS wass (PBS 1% FCS). An antibody against the external HA tag was used to identify the amount of protein on the cell surface. The GFP tag identified the total amount of protein expressed. The results showed that BFA decreased the amount of protein on the surface within a five hour time period (not shown). The experiment was repeated, without a trypsining step, to remove the possibility that trypsinisation was damaging the channel and therefore causing endocytosis. The FACS experiment was repeated with inhibitors for specific players of different endocytic pathways (dynasore, BFA, and methyl B cyclodextrin) and a PKC inhibitor (Go6976) to quantify the amount and type of endocytosis that occurs. (Methyl B cyclodextrin depletes cells of cholesterol and so prevents caveolae dependent endocytosis).

Assessment of Results and Outcome of the Studentship:

K2P9.1 is present in the OE19 cell line; however it would be advantageous to perform another western blot with the optimum conditions to obtain clearer results.

The FACS experiment indicates the turnover rate of K2P3.1 is longer than 5 hours, however as results are visible it is clear the protein would, in the absence of endocytic inhibitors, be internalised within this time period.

Both the confocal microscopy and FACS experiments shows that K2P3.1 is internalised, which, by definition, is endocytosis. The next step was to determine the specific endocytic pathway utilised by the addition of inhibitors for different endocytic pathways to the cell media. As dynasore inhibits dynamin, it disrupts the dynamin dependent clathrin coated vesicle endocytosis. Methyl B cyclodextrin depletes cells of cholesterol and so prevents caveolae dependent endocytosis. The increase of surface expression of the protein in the presence of dynasore indicates endocytosis via a dynamin dependent pathway; two such pathways are clathrin coated endocytosis and caveolae dependent endocytosis.

Future Directions

This project can be continued in greater detail to identify the specific method in which the channel is endocytosed. Dominant – negative mutations of cell proteins which function in specific endocytic pathways can be utilised along with FACS and microscopy for both quantitative and qualitative results. By uncovering the method in which the protein is internalised it may be possible to interfere with the pathway and therefore regulate the function of cells which express this gene.

As K2P3.1 has been identified in the OE19 cell line, the next step could be to quantify the level of expression in comparison to a control, and compare it to cell viability and proliferation to give an indication as to whether the channel affects these factors. Inhibitors of endocytosis, such as dynasore or more specific dominant negative dynamin mutants could be added to the culture media to increase surface expression of the channel. Again, cell viability and proliferation could be assessed to determine if the channel has regulation of these important factors.

Departures from Original Proposal

In an effort to experience as many basic cell biology skills as possible, I undertook to examine the expression of K2P9.1 in an oesophageal cell line. This served to give me immediate cell culture experience and skills in protein purification, quantification and detection (through Western blotting) that I would not otherwise have experienced. This did however mean that there was less time to advance the proposed project and while I established that K2P3.1 was retrieved from the cell surface and showed that this was via a dynamin dependent pathway, inhibitors of other endocytic pathways were not tested.

Value of Studentship

Through this studentship I have learned many new laboratory techniques and gained confidence to carry out experiments independently. I believe my time in the lab has developed both my planning and analytical skills. By participating in meetings and seminars I have advanced my knowledge in the subject area and improved my presentation and communication skills. This experience has been invaluable to me as it has given me a great insight into research as a career and it has taught me about all aspects of life as a researcher. My team have been tremendously supportive and helpful and have made this experience very enjoyable and informative for me.

Student: Anneliese Flatt Supervisor: Dr Victor Gault, Biomedical Sciences Research Institute, University of Ulster Probing the biological actions of truncated forms of gastric inhibitory polypeptide (GIP) Background: Gastric inhibitory polypeptide (GIP) is an important gut hormone which has been shown to stimulate insulin secretion from the pancreatic beta-cells through stimulation of the second messenger cyclic AMP. However, circulating native GIP is very rapidly broken down by the enzyme dipeptidylpeptidase-IV (DPP-IV) which leads to the production of shortened GIP peptides, for example, GIP(3-42) (Figure 1). After early studies it was thought that these shortened GIP peptides were inactive, however, research from the Gault Laboratory has demonstrated that they may act as antagonists. No studies have investigated the actions of shortened or truncated GIP peptides beyond Ile at position 7. Therefore, the main objective of this study was to assess the biological actions of 2 truncated GIP peptides, GIP(8-42) and GIP(9-42) on the stimulation of cellular cyclic AMP production and insulin secretion.

Figure 1: Amino acid structure of human GIP(1-42) indicating primary DPP-IV cleavage site NH2-YA EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-COOH Aims: The aims of this study were to: • Purify and characterise native GIP, GIP(8-42) and GIP(9-42) by reversed-phase HPLC and MALDI-Tof MS; • Examine the actions of GIP(8-42) and GIP(9-42) on stimulation of cellular cyclic AMP production; • Determine ability of GIP(8-42) and GIP(9-42) to stimulate insulin secretion in pancreatic BRIN-BD11 cells.

Description of work: Native GIP, GIP(8-42) and GIP(9-42) were synthesised using Fmoc chemistry. Peptides were purified using repeated runs of HPLC (C-18 column, 4.6 x 250 mm; 0.1% TFA in 70% acetonitrile/H2O) and peaks collected and identified using MALDI-Tof MS (1µl sample / α-cyano-4-hydroxycinnamic acid). For cyclic AMP, BRIN-BD11 cells (100,000 cells/well) were seeded into 96-well plates and incubated with GIP peptides (10-

12 to 10-6 M) in the absence or presence of stimulatory native GIP (10-8 M). After 20-min incubation, medium was removed and cells lysed prior to measurement of cyclic AMP using a commercial HTS Immunoassay Kit. To assess insulin-releasing activity, BRIN-BD11 cells (150,000 cells/well) were seeded into 24-well plates and incubated with GIP peptides (10-12 to 10-6 M) in the absence or presence of stimulatory native GIP (10-8 M). Following acute incubation (20 min, 37°C, in 5.6 mM glucose), buffer was removed and insulin measured using radioimmunoassay. Results are expressed as means±SEM (n=8) and data compared using ANOVA followed by Student-Newman-Keuls post-hoc test. A P value less than 0.05 was considered to be statistically significant. Results: Table 1 shows that native GIP had a retention time of ~18.6 min which is consistent with published data. Retention times of GIP(8-42) and GIP(9-42) were ~18.9 and 19.2 min, respectively. All purified peptides exhibited well defined HPLC peaks and purity was estimated at >95%. The observed mass for each peptide using MALDI-Tof MS corresponded closely to predicted theoretical mass, thereby confirming identity (Table 1).

Table 1: Characterisation of GIP peptides by HPLC and MALDI-TOF MS MALDI-TOF MS Peptide HPLC Retention time

(min) Observed mass (Da) Theoretical mass (Da) Native GIP 18.6 4982.9 4982.9 GIP(8-42) 18.9 4072.1 4074.5 GIP(9-42) 19.2 3970.7 3969.4

Coupling of GIP receptors to adenylyl cyclase was examined by production of cyclic AMP after exposure of BRIN-BD11 cells to GIP peptides. As expected, native GIP dose-dependently stimulated cyclic AMP production (Figure 2A). Similarly, GIP(9-42) stimulated cyclic AMP production although this was much weaker than that of native GIP. In contrast, GIP(8-42) very weakly stimulated cyclic AMP production with cyclic AMP formation only reaching approximately 10% of maximal stimulation evoked by the native peptide (Figure 2A). When incubated in the presence of a stimulatory concentration of native GIP, GIP(8-42) significantly (P<0.001) inhibited cyclic AMP production, with maximal inhibition of 67±0.5% (Figure 2B). In contrast, GIP(9-42) did not affect the stimulatory actions of the native peptide. As shown in Figure 3A, native GIP evoked significant stimulation (P<0.01 to P<0.001) of insulin secretion in BRIN-BD11 cells compared to 5.6 mM glucose control. GIP(8-42) significantly reduced insulin secretion (P<0.001) compared to both 5.6 mM glucose and an equivalent concentration of native GIP. In contrast, GIP(9-42) stimulated insulin-release across the entire concentration range (P<0.05 to P<0.001)

DPP-IV

compared to 5.6 mM glucose. To test for potential antagonist activity, GIP(8-42) and GIP(9-42) were incubated in the presence of a stimulatory concentration of native GIP. As shown in Figure 3B, GIP(9-42) did not affect the insulin-releasing action of native GIP. However, GIP(8-42) significantly inhibited (P<0.001) GIP-stimulated insulin secretion. These combined cyclic AMP and insulin secretion data clearly show that GIP(8-42) behaves akin to a GIP antagonist, whereas GIP(9-42) appears to exert weak agonist properties.

Figure 2: Effects of GIP(8-42) and GIP(9-42) on cyclic AMP production

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Figure 3: Effects of GIP(8-42) and GIP(9-42) on insulin secretion

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GIP(8-42) and GIP(9-42) were incubated in the absence or presence of native GIP (10-8 M) in BRIN-BD11 cells. ***P<0.001 compared with 5.6 mM glucose, ∆∆∆P<0.001 compared to equivalent concentrations of GIP and ΨΨΨP<0.001 compared to native GIP (10-8 M). Future investigation: Further clarification of the precise role of circulating and physiological concentrations of truncated GIP peptides is necessary. Both acute and chronic experiments using truncated GIP peptides in animal models of diabetes/obesity will be essential to further our understanding in this area. Importantly, development of specific assays to measure circulating concentrations in both normal and diabetes states will be useful. Deviation from original proposal: Due to time constraints and the poor quality of extracted mRNA, it was not possible for to examine the long-term effects of GIP peptides on expression of key glucoregulatory genes. Value of studentship to me: I am very grateful to the Biochemical Society for giving me the opportunity to carry out this research project from which I have benefited greatly. This studentship has been a very interesting and rewarding experience which has enhanced my biochemical knowledge and allowed me to develop skills in a range of laboratory techniques, including, HPLC, mass spectrometry, cell culture, measurement of cyclic AMP, general laboratory skills, data presentation, and statistical analysis as well as highlighting the importance of thorough planning, effective time management and aspects of health and safety. The confidence and experience I have gained from this studentship will be invaluable to my intercalated degree project in biochemistry next year and has further reinforced my interest in pursuing an active research career. Value of studentship to the lab: This Biochemical Society Studentship has generated important preliminary data which will be fundamental for possible future research grant applications. Although it was not possible to

perform gene expression studies, the data clearly demonstrate the importance of truncated GIP peptides and the need to further understanding of their precise physiological role.

Student: Benjamin Durham

Supervisor: Dr Ian Wood

Institution for placement: University of Leeds

Biochemical Society Summer Vacation Studentship Report, Summer 2010

Project title: The Role of REST in Brain Cancer

Description of project and background

Medulloblastoma is derived from undifferentiated proliferating neural stem cells (NSC) of the cerebellum. In some medulloblastoma cell lines (e.g. DAOY), REST a transcriptional repressor of neuronal differentiation genes is over-expressed in comparison to normal differentiated neural cells. This over-expression of REST in combination with other factors is thought to play a role in tumorigenesis. Cohen et al. (2005) developed REST-VP16, which is a recombinant transcription factor that binds to the same target genes as REST but activates transcription rather than repressing it. It is not clear if the results generated using REST-VP16 are an artefact resulting from the artificial protein or are truly a result of inhibition of REST function.

This summer project will determine whether knock down of REST produces the same effects as those when REST-VP16 is expressed to help us understand the role of REST in medulloblastomas.

Aims

1) Use shRNA transfection to produce a stable cell line of DAOY medulloblastoma cells which have REST knockdown.

2) Test for REST knockdown at the protein and mRNA levels using Western blots and RT-PCR respectively.

3) Use RT-PCR to determine expression levels of β-tubulin a neuronal differentiation marker in both REST knockdown and non-REST knockdown stable DAOY cell lines.

4) Perform a crystal violet proliferation assay to determine effect of REST knockdown on cell proliferation.

Description of the work carried out and results

DAOY cells were transfected with psuper.puro a control plasmid or pshREST.puro a plasmid producing siRNA for REST. Over several weeks these cell lines were selected using puromycin for stable genome integration of the plasmid. A Western blot for REST and β-actin (control) was carried out (figure 2).

RT-PCR of C showed a 34.8% knockdown of REST and a 147% increase of β-tubulin (neuronal differentiation marker) at the mRNA level relative to U6 as a control. An increase of β-tubulin expression in DAOY cells with REST knockdown indicates a loss of REST’s repressor activity on this gene and possible neuronal differentiation. Further RT-PCR of markers and observations will be required to study this possible differentiation.

Figure 1: A schematic illustrating the involvement of REST in the onset of medulloblastoma and how REST-VP16 blocks tumorigenesis.

Fuller, GN, Su, X, Price, RE, Cohen, ZR, Lang, FF, Sawaya, R, Majumder, S (2005). Many human medulloblastoma tumors overexpress repressor element-1 silencing transcription (REST)/neuron-restrictive silencer factor, which can be functionally countered by REST-VP16. Molecular Cancer Therapeutics, 4(3), 343-349.

REST

A B C Figure 2: Western blot of a whole cell protein extract from A (pshREST.puro transfection 1), B (p.super.puro transfection) and C (pshREST.puro transfection 2). This was analysed and a 50.5% and 76.1% knockdown of REST was produced in A and C respectively. REST=Top Band

A proliferation assay using crystal violet was carried out; 3000 and 6000 DAOY, psuper.puro and pshREST.puro cells were initially seeded into wells of a 96 well plate and the OD (570 nm) was measured after 24 and 48 hours of incubation and growth in 10% FCS, the average fold change between OD measured at 24 and 48 hours is displayed in figure 3;

A similar fold change of OD (thus growth) for all cell lines can be seen, in this run REST knockdown did not effect proliferation or cell death, repeat experiments will be needed, the assay can be extended above 48 hours and the assay can be repeated in growth limiting medium (1% FCS) to assess REST knockdown and cell death more closely.

Departures from original proposal

There were no departures from the original proposal but it took time, some experimental modification and several attempts of shRNA transfection to obtain a stable DAOY cell line with REST knockdown.

Further directions in which the project can be taken

Repeats of the above experiments are needed for statistical analysis. New stable transfections can be performed and selection carried out using puromycin and colony rings, this will allow selection of cells with the greatest REST knockdown and remove undesired cells from the experiment. Apoptosis assays (promega) and alternative proliferation assays can be carried out.

Value of the studentship to the student

This summership has been absolutely amazing and I have really enjoyed the challenge. It has definitely opened my eyes to the world of research and how scientists have to be extremely patience and persevere. Developing a research mindset, using critical techniques (siRNA transfection, Western blotting and RT-PCR) and discovering how a lab works in general will defiantly help me in a future career in research which I am wanting to do more than ever now.

Value of studentship to the lab

Ben worked very hard during his time in the lab and although he didn’t have much experience of molecular techniques before starting in the lab he picked them up very quickly. Overall it was a thoroughly enjoyable experience to have someone in the lab so full of enthusiasm and motivation and very keen to learn new things. Ben produced

β-actin

Figure 3: Average fold change in OD ±SEM between 24 and 48 hours incubation for 3000 and 6000 DAOY, psuper.puro and pshREST.puro cells initially seeded (n=3)

some publication quality data (see above for some examples) and was able to show that the shRNA we had designed to REST was effective in knocking REST expression down. He also showed there was a functional consequence in REST knockdown because he showed the mRNA levels of a REST target gene were increased in response to REST knockdown. Finally Ben produced some stable cell lines using our shRNA expression plasmid. Although it seems as though the stable cells lines produced do not express high levels of the shRNA this has provided a very solid foundation and convincing functional data that we can now build upon.

Student: Betty Gration Laboratory: Sarah Newbury BSMS

Biochemical Society Summer Vacation Studentship Report,

JulyAugust 2010

Comparison of various techniques used to isolate microRNAs in the blood for use as a diagnostic markers for multiple myeloma and MGUS

Aim The overall project aim is to establish peripheral blood miRNA levels as a novel diagnostic/prognostic test. For my 6 weeks in Dr Newbury’s laboratory, my own personal aim has been to determine the best technique for isolation of miRNAs from the blood.

Work Carried out Background information Multiple myeloma is a cancer of the plasma cells (cells responsible for

production of immunoglobulins) whereby excessive numbers of plasma cells accumulate in the bone marrow as a tumour. The average age for this cancer is 70 but it accounts for 10% of all haematological cancers. Symptoms include bone pain, skeletal destruction, anemia, hypercalcemia and eventual death through renal failure and infection.

The first pathogenetic step in myeloma development is the occurrence of a limited number of plasma cells in the bone marrow, clinically known as monoclonal gammopathy of undetermined significance (MGUS). Of all MGUS patients, only 1% per year progress to have myeloma but there is not yet a prognosis test to determine if and when this will occur. In addition the diagnosis tests for myeloma are poor with the primary test being a bone marrow biopsy which is painful, invasive and cannot be done routinely.

Overview of my experimental work I have centrifuged whole blood to leave plasma from which total RNA was extracted using 3 different techniques. The yield and purity of the total RNA was then evaluated using a nanodrop spectrophotometer. Using probes and primers specific for miRNAs, I carried out reverse transcription of the miRNAs into cDNAs which were then subjected to real-time PCR. Real-time PCR involves a fluorescent Taqman probe allowing quantitative assessment of the miRNA levels extracted by the 3 different techniques.

In addition to evaluating which is the best technique for detection of miRNAs in the plasma, this method was performed on multiple different blood samples to see how the following variables affect the miRNA level detected by the 3 techniques:

• Storage To see if the way in which the plasma is stored affects the level of miRNAs detected; samples from the same patient were divided into two with one half being immediately frozen at -80°C and another being refrigerated for several days at 4°C before freezing.

• Centrifugation Speed To compare a low speed centrifugationusing falcon tubes with a higher speed using eppendorf tubes.

• Patient sample Comparison between normal healthy control people and hospitalised patients with no paraprotein in their blood (i.e. patients that have been referred to a haematologist to test for myeloma or MGUS due to symptoms but appear not to have this specific cancer).

Assessment of results i. Which method was best

• Nanodrop spectophotometry showed that the Trizol technique of total RNA extraction produced the most impure sample with high phenol contamination

• RT PCR showed that Exiqon and mirVANA PARIS kit methods extract a larger amount of miRNAs from the plasma than the Trizol method

• Exiqon uses the least amount of sample therefore will be best for future work in this project as sample is limited

ii. Storage makes no difference to the miRNA level, although the total RNA yield appeared to differ, the level of miRNAs detected did not change.

iii. Increasing centrifugation speed increases total RNA extraction. iv. Normal healthy control patients have no detectable miRNAs in their plasma whereas hospitalised

patients do have a detectable level.

Future Directions In the knowledge that Exiqon is the best method for use in this project and after confirming that healthy control patients have no detectable miRNA levels at all, this project can progress to isolating a specific group of miRNAs that are biomarkers of the disease and appear only in myeloma/sick patient samples. This project is most likely to be followed up by another student starting in September and hopefully next year will receive industrial funding to proceed.

Departures from original Proposal No significant changes but after writing the grant application it was noted by Dr Newbury that due to the extremely small amounts of miRNA in plasma, a very robust reliable isolation method is required. Therefore rather than comparing miRNA levels in myeloma and MGUS patients with 1 technique my project focussed more on comparing 3 techniques under a multitude of conditions.

Value of the project to Bettyand SFN lab This summer has been rewarding in many ways. The project has been educational in that I have learnt more about multiple myeloma as a disease and the existence of microRNAs in the peripheral blood. I have also been able to experience life working in research which will be highly beneficial for me when deciding my career choices next year after my Masters. Finally, I have been taught very well how to carry out techniques such as real time PCR and general laboratory tips such as keeping up with my lab book which will set me in good stead for my Masters in Oxford in September.The experiments I have carried out are crucial for the project as it will be important to use a technique that is sensitive enough to detect microRNAs at very low concentrations and

TRIZOL METHOD spectrophotometry result

EXIQON METHOD spectrophotometry result

to be able to register a very small change in microRNA levels that may indeed indicate an important change in medical condition. Identifying the Exiqon method as best for the SFN lab has thus been very valuable.

Student: Brad Schweers

Supervisor: John Christodoulou

Institution for placement: University College of London

Towards in-cell NMR spectroscopy of alpha-synuclein RNC Brad Schweers; in association with JC Group

Background. Nuclear magnetic resonance (NMR) spectroscopy unlike other biophysical methods has a key advantage in its ability to investigate biological macromolecules in near physiological conditions. It utilizes the magnetic properties isotopic nuclei and its ability to resonate electromagnetic radiation creating a signal or chemical shift. Coupled nuclei such as 1H and -15N or 1H and 13C can produce multi-dimensional correlation or heteronuclear single quantum coherence (HSQC) creating higher resolution images of macromolecules by limiting overlapping chemical shifts. By selectively labeling the desired macromolecule its spectrum can be identified against the relative zero-background of in vitro study or in the crowded cellular environment. In-cell NMR thus provides high resolution snapshots of residue specific conformational dynamics of the macromolecule brought about by biological interactions within the cellular environment in a time dependent manner (Selenko and Wagner 2007).

The JC Group has focused on studying alpha-synuclein (αsyn) via in vitro NMR spectroscopy. Alpha-synuclein is an intrinsically disordered protein of 140 residues commonly associated with Lewy bodies in Parkinson’s disease. The JC Group has extended its research in to developing techniques to stall nascent chains on the corresponding “ NMR silent” ribosome (RNC) in vivo(Cabrita et al. 2009) observing any co-translational folding or interaction the emerging protein might have with the ribosome at different points of translation by NMR spectroscopy. RNC samples are synthesized by introducing the SecM sequence onto the C-terminus of the nascent chain in question, stalling the emerging protein on the P site of the ribosome (Evans et al. 2005). This rationale of creating RNC sample is not useful for in-cell NMR as it is not possible to determine the extent of intact RNC samples during the experiment. In this investigation we attempt to create a new and effective method of producing in-cell RNC samples for NMR

spectroscopy using αsyn in E. coli. Using pulse gradient NMR spectroscopy we demonstrated that αsyn is localized in the cytoplasm of the E. coli cell. However due to time constraints we were not able to determine whether our method is effective in producing in-cell RNC samples.

Results & Discussion. It has been shown that NMR spectroscopy can be used to measure the diffusion of molecules by applying a series of gradient pulses along the one axis of the NMR tube as the spin experienced by the nuclei is dependent on the spatial orientation of the nuclei (Yoshizaki et al. 1982). The phase created on the first encoding pulse cancel out on the second decoding phase however if the molecule nuclei has moved the net phase it obtains is dependent on the displacement leading to loss of signal intensity (Yoshizaki et al. 1982). Taking this into account we tested if αsyn is present in the E. coli cytoplasm by creating three in-cell samples using existing protocol (Serber et al 2006) with BL21 E. coli cells overexpressing 15N-labeled αsyn were produced. One sample was treated with lysozyme, another was flash frozen following thawing, and the last sample was unmodified. These samples were then analyzed separately using pulsed gradient NMR spectroscopy. Αsyn was overexpressed in DH5α E. coli cells grown in 15N- labeled M9 media and purified using ion exchange chromatography (3.9 mg/mL) and analyzed by pulse gradient NMR spectroscopy. As the free αsyn diffuses throughout the NMR sample tube its signal lost. The signal intensity loss experienced by the in-cell sample exposed to lysozyme and the flash-frozen sample is less than that of the purified αsyn sample. Lysozyme causes cells to burst by osmotic imbalance releasing the contents of the cell, and αsyn, into the NMR sample tube. The cells and the NMR sample solution rapidly reach osmotic equilibrium resulting and no further αsyn is released from the cell, as suggested by the constant reduced signal intensity. The results of

the flash-fozen in-cell sample indicate that roughly 50% of the cells burst releasing αsyn to diffuse through the sample tube. The unmodified in-cell sample shows no loss of signal intensity of αsyn demonstrating its presence in the cytoplasm.

The SecM sequence contains a single tryptophan which produces a unique HSQC due to the indole ring. The JC Group obtained αsyn-RNC plasmids containing tryptophans engineered in the place of key phenylalanine (4, 125, 136) residues in the hopes that each residue would produce a discrete chemical shift to that of the tryptophan in the SecM. The αsyn FxW-RNC and αsyn-RNC plasmids were transformed into super competent E. coli and cultures grown. However other members of the JC Group discovered a mutation of valine 182 to a methionine in the original αsyn-RNC used to create the αsyn FxW-RNC plasmids. The original plasmids were fixed using SDM-PCR and transformed.

Once colonies were obtained the purified plasmids were sent to sequencing. An additional set of colonies of were grown with a form of the releasable nascent chain from the ribosome by mutating the final proline of the SecM to an alanine, to observe possible changes to the chemical shifts of the different tryptophan residues; in addition to the M182V fix. Unfortunately achieving growth of both the stalled and releasable form of the αsyn FxW-RNC colonies took longer than anticipated. Once obtained the sequencing indicated that neither mutations were successful. The JC Group is now continuing these experiments in my absents.

Acknowledgements. I would like to thank John Christodoulou, Chris Waudby, Lisa Cabrita, Luke Goodsell, Maria Karyadi, Helene Launay, and Xiaolin Wang of the JC Group for their enormous patience, advice, and assistance in the laboratory. Furthermore I want to thank the Biochemical Society and Alexa Hime for the financial support of the project.

References

1. Cabrita LD et al (2009) Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy. Proc.Natl.Acad.Sci.U.S.A 106 (52):22239-22244.

2. Evans MS et al (2005) Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro. Nat.Methods 2 (10):757-762.

3. Selenko P and Wagner G (2007) Looking into live cells with in-cell NMR spectroscopy. J.Struct.Biol. 158 (2):244-253.

4. Yoshizaki K et al (1982) Application of pulsed-gradient 31P NMR on frog muscle to measure the diffusion rates of phosphorus compounds in cells. Biophys.J. 38 (2):209-211.

0 0.1 0.2 0.3 0.4 0.5

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Fig. 1 – Graph of the intensity of the signal of the pulse gradient NMR experiment against the strength of the gradient pulse of the in-cell samplesBlue marks -unmodified in-cell sample, Geen marks – lysozyme exposed in-csample, Pink marks – flash-frozen in-cell sample, Red marks – purified αsyn sample

Figure 2 - A. (top) Preparation for the in-cell αsyn NMR sample. B. (bottom) Sonication of the αsyn plasmid containing cell pellet, resuspended in lysis buffer, for the purification of αsyn.

Student: Ms Chandni Patel Supervisor: Prof Steve Busby

Bench supervisor: Dr David Lee

Institution for placement: University of Sussex

Biochemical Society Summer Studentships 2010

Locating targets on bacterial chromosomes

Student: Ms Chandni Patel Supervisor: Prof Steve Busby Bench supervisor: Dr David Lee

Background

The lab has been using the binding of fluorescently tagged transcription factors to locate specific targets on the chromosome of Escherichia coli. It had been planned that I would construct a new tagged transcription factor. However, at the last moment, it was decided that I would switch my project to the construction of targets for a tagged transcription factor, the cyclic AMP receptor protein (CRP), that had been recently constructed by one of the PhD students in the lab. CRP is a DNA binding transcription factor that recognises and binds to specific DNA target sequences (CC site in the diagram) and activates genes in response to glucose starvation. A promoter is located upstream of genes. It is made of specific DNA sequences and directs transcription machinery to the correct location.

At many target promoters, CRP directly interacts with the alpha subunits of RNA polymerase (RNAP), via an amino acid residue on the surface of CRP (Histidine 159: star on diagram). This enables RNAP to begin transcription of these genes.

The Busby lab found that CRP can function when bound to DNA far upstream of promoter, moreover, 2CRP’s can work together to increase promoter activity. In some cases the second CRP was inhibitory (ie -122.5: 122.5BP upstream of the transcription site), as opposed to further enhancing promoter activity Such promoters are ideal for use as targets for fluorescently tagged CRP.

Experimental plan

CC site

RNAP

RNAP

Key = CRP = α subunit =Histidine 159

CC site

My aim was to construct new promoters containing multiple DNA sites for CRP that could be used in location studies and also that would allow us to understand the cause of the inhibitory effects due to upstream-bound CRP.

It is known that wild-type CRP protein cannot bind to an altered DNA binding site (a QQ site). However a mutant version of the CRP, which has a glutamate to valine substitution at position 181, can bind. Our strategy was to mutate the DNA site for CRP at position 122.5 to the QQ version, and use CRP protein derivatives that have the Val181 substitute to establish why CRP is inhibitory at the position.

In order to make the QQ site an oligonucleotide was designed with a mismatch in the sequence of the second CRP site. The oligonucleotide was cloned using PCR. The cloned PCR fragment was digested with EcoRI and HindIII. The PCR fragment was then inserted into the plasmid pRW50 which has β-galactosidase activity. The PCR fragment is located immediately downstream of the LacZ gene. Transcription from the promoter will result in production of β-galactosidase activity which can be used

to measure the β-galactosidase; hence promoter activity could be measured.

Controls were used to eliminate alternate explanations. A wild-type CRP was used as a positive control. The EV181 binds to the QQ site whereas the HL159 and TA158 abolish interaction with alpha subunit of RNAP therefore transcription stopped.

Conclusion

My results show that as the distance of the CRP site reaches (-122.5) the activity decreases. With CRP EV181 ta158 and CRP EV181 HL159 the activity is at the lowest therefore shows that α subunit is not interacting with the CRP and so transcription is not taking place. The next step would be to transfer my new promoter constructs to the E. coli chromosome and to study the location of fluorescently tagged CRP.

Experience of lab

My experience in the lab has been very beneficial and rewarding. I now appreciate and understand the range of techniques used to obtain accurate results. I look forward to using the skills I have developed and improved in my third year project as well as any future work. Working in a lab environment has made me realise that I would like to direct my career path towards research. I would like to thank Prof Steven Busby and for giving me the opportunity to work in the Busby lab and Dr David Lee for the help and guidance throughout the project. I would also like to thank the Biochemical Society funding my studentship and allowing me to gain invaluable experience.

References

Figure 1: A graph to show the rate of activity when the α subunit of the RNAP binds to the second CRP site at different positions

Hollands, K, Busby, S & Lloyd, G (2007) New targets for the cyclic AMP receptor protein in the E. coli K-12 genome. FEMS Letters 274 89-94

Tebbutt, J, Rhodius, V, Webster, C & Busby, S (2002) Architectural requirements for optimal activation by tandem CRP molecules at a Class I CRP-dependent promoter. FEMS Lett. 210 55-60

Student: Chelly van Vuuren Supervisor: Professor Kevin F. Sullivan Biochemistry Society Studentship Report 2010 Investigation of Centromere Replication by Fluorescence Photodynamic Approaches Background The centromere is a permanent locus on the chromosome where sister chromatids are joined and the kinetochore is transiently assembled during mitosis. Centromere mediated segregation of sister chromosomes during mitosis and meiosis ensures that the correct number of chromosomes will be allocated to each daughter cell. A variety of studies have shown that the identity of the centromere is dictated epigenetically. The histone H3 variant CENP-A, which contains a histone fold domain, is a marker of centromeric DNA. It has been proposed that CENP-A or other chromatin proteins could play a role in epigenetic centromere inheritance. A number of proteins are closely associated with CENP-A chromatin. CENP-T, and W contain histone fold domains and are therefore of particular interest. A histone fold is composed of three helices, the largest being flanked by two smaller helices. This feature is present in histones and is thought to be involved in protein interactions and binding. siRNA mediated knock down of CENP-T, and W has been shown to be detrimental to the formation of the centromere. The cells were frozen in mitosis by mitotic checkpoints. It has recently been illustrated that CENP-W forms a complex with CENP-T. Further exploration of the properties of these proteins might provide information regarding centromere assembly. In studying these proteins and their potential role in centromere assembly, their dynamic properties become important. In order to better understand the nature of the residency of Centromere proteins (T, and W) at the centromere, a photodynamic approach is suitable. Aims The aim of the project was to characterize the dynamic profile of CENP-T and CENP-W with the direct application of the surveillance of a particular generation of protein. Fluorescence recovery after photobleaching (FRAP) experiments have been instrumental in establishing a dynamic profile for each of these centromeric proteins. The initial goal was to conduct a complementary fluorescence loss after photoactivation (FLAP) experiment utilizing photoactivatable GFP (PAGFP) tagged CENP T, and W. The “Bucket List”, time permitting, was to conduct some FRET experiments coupled with a conditional labeling scenario to investigate the nature of CENP-A nucleosomes from a cell cycle specific perspective. Departures from the original proposal Unfortunately, as often occurs in science, time did not permit. The FRET experiments were not carried out because of time constraints. Experimental Procedure Hela cells expressing PCNA-Cherry were transfected with CENP-T or W-PAGFP plasmids using an AMAXA Nucleofector with program I-013. 24 hours later, FLAP experiments were carried out using the confocal microscope (LSM 710 Meta; Carl Zeiss, Inc.).

Cell judged to be in late S using PCNA staining were marked. Once the patterning in the cells had dissipated (indicating the cells passage into G2), PAGFP at ~ 5 centromeres was activated using the 405nm laser. A Z stack image was taken with the 488nm laser to record the GFP intensity at centromeres. Once cells reached mitosis, another image was taken with the same parameters. If the cell did not enter mitosis, a picture was taken a minimum of 3 hours post activation. The data analysis was accomplished using Metamorph software to find the average intensity of centromeres postactivation and then again in the last image. The remaining fluorescence in the second time point was then expressed as a percentage of that in the first. In order to further investigate this behavior a FLAP FRAP hybrid experiment was designed and the pilot experiment was carried out. FRAP experiments are highly useful to examine the rate at which a fluorescent protein moves into a region. This can provide information about the timing of loading/exchange of proteins to the centromere. However, its detailed interpretation is difficult because loading is a combined function of the “on rate” and the “off rate”, which cannot be directly observed. The elegance of the FLAP FRAP experiment is that the on and off rate can be measured simultaneously in the same cell. And, even better, at the same centromere. As the dynamic behaviour of these proteins is dictated by cell cycle timing, cell cycle markers were necessary. Immunofluorescence using PCNA and H3P atibodies was conducted. A Matek cell culture dish was used. It contains a grid etched into the cover slip, which allowed us to pin point individual cells after immunofluorescence. Results Both CENP‐T and W molecules were shown to dissipate from centromeres during G2. This was a surprising finding, but one that boosted later double FRAP results that indicated an exchange rather than loading process for CENP T in G2 and CENP W in S. There remains a discrepancy between CENP‐W FRAP data, which shows recovery in S phase only, and FLAP data which shows a decrease in fluorescence during G2. However, this may be the missing link. Previous experiments clearly indicate an absence of heritability of these proteins from one cell cycle to the next. Could this be the dissipation of a portion of the pool of parental protein prior to mitosis? It would be valuable to test this hypothesis. The FLAP FRAP procedure is robust and can be developed and used as a tool for establishing protein dynamic profiles. Value of Project to the Student The experience of working in a foreign country is invaluable to an emerging Biochemist. Science knows no borders, and a healthy science career will often take one far from home. The opportunity to work with the Jena microscope was an enriching experience. Learning to master their data analysis systems will stand me in good stead for research projects I undertake in the future. I gained experience in experimental design, as well as learning the importance of pilot experiments. I learned that Biotechnology is multifaceted and exportable. The enthusiasm of my mentors in the intricacies of our research niche was contagious. The experience has solidified my resolve to pursue a career in Science. I intend to undertake a doctorate as my next step towards scientific enlightenment. Value of Project to the lab The project has helped consolidate an important collaboration between the Sullivan and Diekmann labs and introduced new methodology, fluorescence photoactivation, to both. The work demonstrating an off-rate in G2 for CENPs –T and –W is now included in a manuscript about to be submitted for

publication. The combined FRAP/FLAP experiment is an important new approach that will be incorporated into ongoing assembly analysis of other members of the centromere protein family. A

B

Figure 1: CENP-T and W are not stably associated with centromeres during G2.

(A) Comparison of average fluorescence directly after activation and 4 hours later. (B) Activation of CENP-W PAGFP at centromeres during G2 illustrates an acute loss of

fluorescence at foci when imaged 4 hours later.

FRAP FLAP Pilot Experiment A B

C

D Figure 2:The FRAP FLAP pilot experiment proves viable.

(A) Photobleaching and activation are successfully managed in concert.

(B) The live cell chamber was rigged to accommodate the Matek dish with a grid etched cover slip.

(C) Immunofluorescence of cell of interest for cell cycle staging.

(D) Data analysis of average intensties at the three centromeres of interest indicate an increase in cherry by T120

A

Figure 3: (A) A night out with the lab. (B) A symbolic representation of my supervisor in Jena (Volker Doring), who has become fairly retiring since I requested a photo of himself. I am sure he will approve of the placeholder. (C) My P.I. in Jena, Professor Stephan Diekmann, Head of the Department Molecular Biology at the Liebniz Institute for Age Research. (D) Professor Kevin F. Sullivan and Chelly van Vuuren

Student: Clare Rogerson Supervisor: Dr Katherine Bowers. Dept. Structural and Molecular Biology, UCL. Biochemical Society Summer Studentship 2010 Regulation of the yeast intracellular sodium/ proton exchanger Nhx1p in protein trafficking Introduction Cellular function in eukaryotic cells requires the correct trafficking of newly synthesized proteins for their function, modification or degradation. This involves sorting many proteins via the secretory and endocytic pathways, through a series of vesicles and vesicular maturation. It has been proposed that the pathway of proteins through this system is controlled in part by the lumenal pH of these vesicles, which is thought to alter receptor ligand association and/or coat protein binding. NHE’s are involved in the control of the lumenal environment of organelles. NHE’s are a family of sodium/ proton exchangers that catalyse the electroneutral exchange of protons for sodium ions across a membrane, down their concentration gradients. The only NHE in yeast is called Nhx1p. Nhx1p has been shown to be essential in correct protein trafficking to the vacuole (deletion mutants contain a large aberrant endosomal compartment where proteins accumulate and are not trafficked to the vacuole) and important in salt tolerance (mutants are increasingly salt sensitive). Nhx1p, localised to the late endosome in yeast, has 12 transmembrane domains and an as yet uncharacterised C terminal tail, which has been previously described as lumenal (i.e. inside the endosome rather than cytosolic). Another research group have proposed that the RabGAP protein Gyp6 binds to the C terminus of NHX1 and regulates its role in protein trafficking; however Gyp6 is a cytosolic protein. The aims of my project were to study further the role of the C terminus in protein trafficking and salt tolerance, and to identify specific regions or residues essential for these functions, making use of mutants already available in the lab containing truncations of the protein’s C terminus or point mutations in highly conserved residues at the end of the C terminal tail. Methods

• Plasmids containing the mutated nhx1 genes were transformed into the yeast S. cerevisiae. The plasmids studied contained point mutations: K606A, P607A, V608A, F609A, and deletion of the first half, second half and entire length of the C terminus. Other constructs of interest, such as a plasmid expressing HAL5, (known to rescue nhx1Δ salt sensitivity), were also transformed. Wild type yeast strains and mutants containing empty plasmids were also analysed as controls.

• These yeast strains were then studied for correct protein sorting and salt tolerance using two assays: o Protein trafficking was assayed in yeast strains containing a fusion of the vacuolar protein

Carboxypeptidase Y (CPY) to the reporter protein invertase. In wild-type yeast CPY is correctly targeted to the vacuole but in nhx1Δ mutants it is secreted. The invertase produced by the cell during a defined time is used to hydrolyse sucrose in the assay mixture to glucose. Added glucose oxidase causes the oxidation of glucose producing hydrogen peroxide as a by product. Added horseradish peroxidase in the assay uses hydrogen peroxidase to oxidise the chromogen o-dianisidine. The product of this is a red precipitate which can be quantified by measuring the absorbance at 590nm.

o The assay to determine salt tolerance of the yeast strains involved growth in liquid culture in a medium containing high salt. A preliminary assay suggested that medium with 800mM NaCl gave a significant difference in yeast growth between wild-type and nhx1Δ strains and this concentration was used for all subsequent experiments. Cultures were grown in a plate reader at 30°C, shaking occasionally, for at least 50 hours and the absorbance at 600nm recorded every 2 hours.

Results and discussion

Results of the invertase assay to study protein trafficking function are shown in Figure 1. The amounts of precipitate measured at 540nm in total and secreted fractions of each of the strains were then compared to give the % secretion of CPY. Wild type cells (BHY10) secreted about 8% of the total CPY, whereas nhx1Δ (third column) secreted around 20%. As can be seen clearly from the bar chart all mutant forms of nhx1 studied rescued the protein trafficking function. These values are significantly different from the nhx1Δ mutants or nhx1Δ with pRS416 (empty plasmid). We conclude that the protein is correctly localised and functional in all the mutants, even those with the whole C terminus deleted (all C del). The HAL5 construct did not rescue the secretion of CPY so, although known to rescue Nhx1p function in salt tolerance, it cannot rescue protein sorting phenotypes indicating that the two roles of Nhx1p may be independently controlled by the protein. The second assay was to investigate the ability of the mutant forms of Nhx1p to rescue salt sensitivity of nhx1Δ strains. These results are shown in the three graphs, Figures 2, 3 and 4. These graphs show the growth of each of the strains over a 60 hour period in a plate reader. In Figure 2 it is clear that the empty plasmid (pRS416) does not rescue salt sensitivity, unlike wild type and HA tagged forms of Nhx1p (NHX1 and NHX1HA). HAL5 is shown to partially rescue sensitivity as expected. As shown in Figure 3, strains expressing the nhx1 point mutants K606A, P607A, V608A and F609A all grew at a very similar rate to the strain with the wild type protein and much faster than strains with empty plasmid. Hence it is proposed that these point mutations do not affect function of Nhx1p in salt tolerance. As shown in Figure 4, yeast strains expressing the Nhx1p mutant lacking the first half of the protein (1st C del) seems to grow at an almost identical level to wild type, whilst that lacking the second half (2nd C del) seems to grow at a slower rate and to a lower end point; the rate and endpoint of the mutant lacking the entire C terminus (all C del) seems to be even lower than this again. This suggests that the second half of the C terminus may be important in salt tolerance (and thus in the control of Nhx1p ion exchange) although further repetitions of this experiment are required to back up this conclusion as the graphs shown are an average of only two assays. Overall, the assays discussed showed that four highly conserved residues towards the end of the C terminal tail are not essential for Nhx1p function in salt tolerance or protein trafficking and that the entire C terminus is not required for protein trafficking but may be involved in salt tolerance, especially the second half. Further Work Following the results of this project it is clear that the topology of the protein needs to be clearly defined, as whether the C terminus is lumenal or cytosolic would greatly affect the regulation of the protein. All other defined members of the NHE family contain targeting sequence in their C terminal regions, hence the ability of Nhx1p to be correctly localised and fully functional without a C terminus would be an intriguing area for further investigation. Value of the Studentship This studentship has enabled me to spend my summer on a very interesting placement and greatly improved my practical skills. I have gained a lot of experience from my involvement with this project. I now feel that I understand the processes involved in lab work, from planning and application for grants to data collection and analysis, and have gained much more confidence in and around the lab. Everyone in the lab contributed to making this a great experience and showed me what day to day work in the lab is like. This has helped me to see what doing a PhD would mean and what in practical terms it would involve, and has inspired me to apply once I graduate. Value of the studentship to the lab The summer vacation studentship awarded to Clare this summer enabled her to generate some very useful results during her 8 weeks in London. This was good for us, as we can use the data she generated for our further studies and hope to include it in a future publication. I also believe that Clare learnt a lot this summer: after learning and practising the relevant assays and techniques, she was able to work very independently and showed a great ability to manage her time effectively. This opportunity has also given her a valuable taste of research that will be a huge advantage for the completion of her undergraduate studies and beyond.

Biochemical Society Summer Studentship 2010 Regulation of the yeast intracellular sodium/ proton exchanger Nhx1p in protein trafficking Student: Clare Rogerson Supervisor: Dr Katherine Bowers. Dept. Structural and Molecular Biology, UCL. Figure 1

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Biochemical Society Studentship Report, Summer 2010

Towards smFRET Studies of Tetratricopeptide repeat proteins (TPR)

Student: Danny Zust Supervisor: Dr. Ewan Main i) BACKGROUND

In the last ten years there has been substantial interest into an extremely abundant class of proteins called repeat proteins. These ubiquitous folds are present in nearly all forms of life and act as protein interaction scaffolds and adapter molecules in numerous cellular processes. Such proteins are composed of multiple repeated motifs whose final 3D structure is formed from the stacking of repeats into tandem arrays that form elongated non-globular domains (Figure 1a).

The modular construction of repeat proteins causes them to have radically different structural properties in comparison with “normal” proteins (Figure 1b). For example, the super-helical coils produced from the stacking of certain repeated units have been shown to have elastic / spring-like qualities. The fundamentally different structural and mechanical properties of repeat proteins, as outlined above, present a unique opportunity to explore the exciting spring like characteristics of this class of protein.

ii) AIMS

This project aimed to attach light sensitive dyes to a model TPR protein and then, by measure the energy transfer efficiency between the 2 chromophores (donor and acceptor), begin to observe and characterize its folding and flexing (thus probing its spring-like qualities).

iii) PROTOCOL / METHODS

Attachment of dye molecules & purification of labelled protein: Two Alexa Fluor dyes (maleimide derivatives of AF555 and AF647) were attached to the protein and using affinity chromatography, the doubly-labelled protein was separated from unlabelled protein.

‘Bulk’ experiments: Dilutions on the doubly labeled sample were carried out to determine the detection limit of FRET interactions.

Single Molecule Experiments: The final experiments were conducted on the single molecule apparatus to monitor and confirm any FRET interaction.

iv) RESULTS

The dye solutions were prepared by adding 100 µl of DMSO to 1 mg of each of the 2 dyes. They were added to the protein solution simultaneously, to ensure that a high yield of doubly labeled protein was obtained (23%). The reaction was carried out in an inert atmosphere and left to stir for 2 hours at room temperature. To stop the reaction, an excess of glutathione and DTT was added to the reaction mixture. This was to make sure there was no reactive species (like unreacted dye) in the separation and purification step, which was carried out using a MonoQ 5/5 column, an anion exchange column for use with moderate pressure chromatography systems. A linear salt gradient (1M NaCl in 100 mM phosphate pH7), 0-100% sodium chloride was used.

UV280 spectroscopy and fluorimetry (fig.1) were used to prove the presence of doubly labeled proteins. The samples containing these proteins were diluted to see the detection limit, and it was found that FRET was detectable down to a concentration of 8 nM (fig.2).

Samples for single molecule studies were also prepared, and although some FRET interaction could be detected, the sample preparation would have to be improved; the proximity of the glass surface on which the sample was immobilized induced the unfolding of the protein due to electrostatic attractions between the charged protein surface and the glass surface. This results in an increased distance between the donor and acceptor dyes at which sometimes makes energy transfer not possible.

.v) DEPARTURES FROM ORIGINAL PROPOSAL

Initially, it was planned to attach one dye first, separate the mono-labelled protein from the doubly-labeled and non-labeled proteins and only then attach the second dye to this purified sample. However, due to unforeseen problems with the mass spec, the fractions containing uniquely mono-labeled proteins could not be identified for definite. This is why a second attempt was made, by adding the 2 dyes simultaneously, which gave a decent yield of the protein doubly –labeled by the AF555 and AF647 dyes, as proven by UV spectroscopy and fluorimetry. (23% yield)

vi) FUTURE RESEARCH

The proteins have been successfully labeled with the 2 alexa dyes. The single, double and correctly doubly labeled samples were separated and the FRET interaction and the sensitivity could be determined. This process of bio-conjugation of dyes to the proteins needs to be optimized, so that higher concentrations and high degrees of labeling of doubly-labeled CTPRs are obtained. To characterize the relationship between dynamics and the size of proteins, more CTPR proteins would need to be synthesized and analyzed as well. Also, to get the most similar environment to the natural environment of proteins, single molecule studies must be conducted on freely diffusing solutions. This can be achieved by coupling FRET with fluorescence correlation spectroscopy (FCS)

vii) VALUE OF THE STUDENTHSIP TO THE STUDENT

This studentship was a good opportunity to give me some insight into the real-life experience of working in a lab. I have learnt many new, invaluable skills that will be of use for my further studies, particularly to my final year laboratory project and prospective PhD studies. Apart from all the new skills learned, I have also learnt and realized that real research is very different from following undergraduate laboratory practicals, and that no matter how good an initial protocol is, critical thinking is very important to surmounting difficulties with new and innovative ideas. This was one of the most challenging and satisfying parts of the project for me.

viii) VALUE OF THE STUDENTSHIP TO THE LAB

It was a great pleasure to host Danny in our lab this summer. He had a great enthusiasm for science and overcoming the innumerable small problems that occur on a day to day basis in the lab. His results were of great significance to us as he managed to design and rectify a labeling protocol that will enable us to now study the dynamics of TPR proteins on the single molecule level. Thus, this studentship has enabled the laboratory to obtain a preliminary protocol that should produce labeled protein for further experimentation.

Fig2. Graph showing how the intensity of the FRET signal changes on dilution ; it is detectable down to a conc. of 5 nM (green curve)

Fig1. Fluorescence em. Spectrum of doubly-labeled protein showing absorptions at 555nm and 647 nm, proving FRET interaction between the 2 dyes attached.

Student: Elizabeth Carroll

Supervisor: Derek Brazil

Institution for placement: Queen’s University Belfast

Introduction

Diabetes is hyperglycaemia disease of defective glucose metabolism, and is characterised by high blood glucose or hyperglycaemia. The injurious effects of hyperglycaemia cause serious secondary complications classified as macrovascular complication, affecting the heart and lower limbs and microvascular complications affecting the kidney (diabetic nephropathy) lower limb, (neuropathy)and the eye (diabetic retinopathy (Fowler, 2008)).

Diabetic retinopathy is one of the most common micro vascular complications of diabetes and is the leading cause of blindness in working age population’s worldwide (Fowler, 2008). Exhaustive research has been carried out in this field and as of yet there is no cure for diabetic retinopathy. There is a call for new research to develop novel therapies. Endothelial progenitor cells (EPCs) which were first described in 1997 (Pearson, 2009), are a subgroup of circulating cells that are recruited to areas of restricted blood flow in the eye, heart and lower limbs. These cells could potentially represent a “rapid response” repair mechanism to damaged endothelium in areas of restricted blood flow. They are as a result being investigated for their therapeutic potential in the treatment of vascular damage in the eye (Hookham et al., 2010). The recruitment of EPC’s to areas of restricted blood flow is stimulated by hypoxia (Ceradini and Gurtner, 2005). The retina of a dietetic experiences hypoxia and this stimulates the recruitment of EPC’s to the area of ischemia. EPC’s from diabetic patients are present in lower numbers than healthy individuals and are not capable of facilitating blood vessel repair. In order for a potential novel therapy involving ex vivo repair of these cells for autologous transplant into patients suffering from diabetes the molecular basis of this deficit in diabetic EPC’s must be elucidated. The focus of this project is on protein kinase B/AKT as a key mediator of cellular responses to growth factors, such as insulin, and under conditions of hypoxia. The important role mediated by PKB/Akt in hypoxic cells has been previously established (Barry et al., 2007).

Aim of the project

To characterise the effect of hypoxia on endothelial progenitor cells

Experimental methods

Outgrowth endothelial progenitor cells were used in this project. These were grown by Dr. Hookham and under her supervision the basic principles of sterile technique and cell culture were learned. OEC’s can be isolated from both adult and umbilical cord blood.

(a)OEC’s were cultured on collagen coated plastic wear and exposed to varying times of 1% O2 to mimic low oxygen concentrations in the diabetic retina in a hypoxic chamber. Protein lysates of these cells were prepared. Radio Immunoprecipation Assay Buffer (RIPA) (50mM Tris HCL, 1% NP40, 0.25% sodium deoxcolate, 150mM NaCl, 1mM EDTA) was used to lyse the cells. Proteins present in the cell lysates were then analysed using Western Blotting.

(b)Using the technique of Western Blotting well established markers of hypoxia such as hypoxia-inducible factor-1 (HIFIα) induction was measured; activation of protein kinase B (PKB/AKT) was measured using phospho-Thr308 and phospho-Ser473. Changes in Akt substrate phosphorylation of GSK3β and MDM2 were also examined. OEC’s were grown in the same manner as the previous

experiment and exposed to varying times of 1% O2 in an hypoxic chamber. HeLa cells were grown in identical conditions and functioned as a positive control for the experiments. HMEC cells were grown in parallel in hypoxic conditions (1% O2) and normoxic conditions (21% O2).Western blot analysis was carried out using both the Odyssey LiCOR system and chemiluminescent detection in X-ray film using Immobilon Western HRP (horseradish peroxidase) chemiluminesence substrate.

(c) A stimulation experiment was then carried out. 8 OEC replicates were grown up, 4 of which were then exposed to 1% O2 and the remaining maintained in normoxia (21% O2). 2 of each of the OEC replicates were stimulated with pervanadate in hypoxia and normoxia for 30min. Pervanadate was used as to mimic strong tyrosine kinase receptor activation . The samples were then lysed with RIPA buffer to release intracellular contents. Using western blot analysis the effect of pervanadate stimulation was examined in the same manner as in the previous experiment.

Results

H

Figure1. OECs grown for Oh, 8h, 24h and 48h at 1% O2 and HeLa cells grown in normoxia (21% O2). Expression of HIF1α is up at 8hr hypoxia and is maintained up to 48hr. Phospho-Ser473 was used to measure activation of PKB/Akt during hypoxia. β-actin functioned as the control in this experiment.

Figure 2. OECs grown for Oh, 8h, 24h and 48h at 1% O2 and HeLa cells grown in normoxia (21% O2), HMEC’s grown in normoxic and hypoxic conditions. There is a marked decrease in the phosphorylation of GSK3 β, a substrate of PKB/Akt. Changes in the phosphorylation of Mdm2 and 70S6K substrates of PKB/Akt are also observed. There was a marked decrease in the phosphorylation of both these Akt targets in HMEC cells. The levels of total GSK3 β appear to remain constant throughout the various stages of hypoxia in both the OEC and HMEC cells.

Figure3. OEC’s exposed to normoxic (21% O2) and hypoxic (1%O2) conditions and then stimulated with pervanadate. In both normoxia and hypoxia there is a marked increase in Akt phosphorylation (pThr308 and pSer473) and in phosphorylation of eNOS (nitric oxide synthase) a target of PKB/Akt.

Discussion

The results of Western blot analysis on OECs exposed to hypoxia illustrated a strong increase in the well established marker of hypoxia HIF1α. This increase confirmed a robust hypoxia response in OECs. There was an increase in the phosphorylation of PKB/Akt on the Ser-473 residue. Phosphorylation of this residue and Thr-308 leads to the activation of this protein.

In OECs and HMECs there was a decrease in the phosphorylation of GSK3β a substrate of PKB/Akt. Changes were observed during hypoxia in both OECs and HMECs in the phosphorylation of 70S6K and Mdm2 both of which are targets of PKB/Akt. This indicated a decrease in activity of PKB during hypoxia. The levels of total GSK3β appeared to remain relatively constant throughout the varying stages of hypoxia in OECs.

In the stimulation experiment the increase in the activation of PKB/Akt and its target eNOS in response to growth factors such pervanadate (which mimics a number of different insulin effects) demonstrates that in both normoxic and hypoxic OEC’s there is a strong response to growth factors. The response in “normal” cells, those exposed to 21% O2 is slightly stronger than hypoxic OECs.

Future directions

The next step is to determine the importance of the patterns of hypoxia-induced gene expression in OEC cells. These genes have the potential then to form the basis of comparison between control and diabetic OEC’s, to identify variations in diabetic cells that may explain their inability to repair vascular damage. The data obtained from these experiments will aid in the development of an ex vivo therapy for the repair of diabetic OEC’s and autologous transplant into patients suffering from diabetic retinopathy.

Outcomes of the studentship

The 8 weeks spent working with Dr. Brazil and Dr. Hookham has been an invaluable experience for me. I have had the opportunity to witness and carry out techniques and experiments that I would never had the opportunity to do otherwise. I have gained experience in experimental procedures and techniques that are of great importance in the coming year as I complete my final year project in university. I was fortunate to work in a very friendly and helpful environment. This was an experience that I will stay with me throughout my studies an undergraduate and in the years to come as I hope to carry on my studies.

References

Barry, R.B., Allan, B.B., Cummins, E.P., Kattla, J.J., Giblin, A., Scally, N., Taylor, C.T and Brazil, D.P.2007. Enhanced sensitivity if protein kinase B/Akt to insulin in hypoxia is independent of HIF1α and promotes cell viability. European Journal of Cell Biology.86.pp393-403.

Ceradini, D.J., and Gurtner, G.C.2005. Homing to Hypoxia: HIF-1α as a mediator of Progenitor Cell Recruitment to Injured Tissue.TCM.15 (2).pp57-63

Fowler, M.J. Microvascular and Macrovascular Complications of Diabetes. 2008. Clinical Diabetes.26 (2).pp77-82.

Hookham, M.B., O Brien, A.C., Carroll, E.C., O Neill, C.L., Medina, R.J., O Doherty, M., Stitt, A.W., and Brazil., D.P. Endothelial Progenitor Cell Responses to hypoxia: Time course dependent changes in AKT signalling and gene expression.

Kato, J., Tsuruda, T., Kita, T., Kitamura, K., and Eto, T. 2005. Adrenomedullin protective factor for blood vessels. Arterioscler Thromb Vasc Biol.25 (12).pp2480-7.

Pearson, J.D. Endothelial progenitor cells-an evolving story.2009.Macrovascular research.79.pp162-168.

Student: Emma Tredgett Supervisor: Felicity Watts Institution for placement: University of Sussex Aim The aim of this project was to determine whether minichromosomes based on chromosome I form isochromosomes, as is observed in the Watts lab with a minichromosome based on chromosome III. Specifically, this required the construction and analysis of an appropriate ChI minichromosome (ChIUG). During the course of this work, it became apparent that the ChIUG minichromosome was lost at a much higher frequency than the minichromosome based on chromosome III, making analysis of isochromosome formation difficult with this construct. Therefore the project was widened to include the construction of another minichromosome based on chromosome III which could be used to screen an S. pombe gene deletion library for mutants displaying increased frequency of isochromosome formation. Results 1. Construction of ChIUG

PCR primers were designed to integrate the G418R marker into the rad50 gene on the right arm of the minichromosome pSp(cenI)-7L, which already has ura4 on the left arm. PCR was then undertaken to amplify the G418R cassette from pFA6-KanMX. The PCR product was used to transform a wt strain containing pSp(cenI)-7L, and G418-resistant transformants selected. Two methods were used to test for integration of the G418R cassette: co-instability with the ura4 marker and Southern blotting. Since the rad50 gene is present not only on the pSp(cenI)-7L minichromosome, but also on the normal chromosome III, it was necessary to identify transformants where the G418R marker had integrated into the minichromosome and not into chromosome III. Eight G418R transformants were streaked onto non-selective medium (YEA) and allowed to form single colonies. These were then replica plated onto YEA, ura-, and G418 plates. Five of the eight G418R transformants showed co-instability of ura4 and G418 consistent with integration of G418R on the minichromosome rather than chromosome III. During the replica plating, it became apparent that the pSp(cenI)-7L minichromosome and the G418 derivative are very unstable as they are lost at high frequency from cells grown under non-selective conditions. The presence of G418R on the minichromosome was confirmed by analysing the minichromosomes by pulse field gel electrophoresis and Southern blotting. As in the previous experiment the pSp(cenI)-7L minichromosomes appeared very unstable. 2. Construction of Ch16UH

Since it appeared that pSp(cenI)-7L-derived minichromosomes might be difficult to work with, a parallel project was initiated. Previous work in the Watts lab had demonstrated that a minichromosome based on chromosome III spontaneously forms isochromosomes, and that this frequency is increased in pli1-d (defective in a SUMO E3 ligase) and rad22-d (deleted for the homologue of the homologous recombination gene RAD52). One of the aims of the Watts lab is to identify additional genes required for the prevention of isochromosome

formation. To do this it was proposed to screen an S. pombe gene deletion library. Since the library was constructed by deleting genes with G418, it would not be possible to use the current Ch16UG minichromosome, since it contains G418R. I therefore designed PCR primers to use to replace G418R on the minichromosome with HphR to create Ch16UH. PCR was carried out as before and the product was used to transform wt cells containing Ch16UG. Six hygromycin-resistant transformants were selected and checked for loss of G418R and co-instability of ura4 and HphR as in (1). All six transformants showed loss of G418R and co-instability of the two markers. Before further use of the minichromosome, it was necessary to ensure that it behaved as the original Ch16UG (i.e. that the frequency of isochromosome formation was the same for the two minichromosomes). This was confirmed by carrying out fluctuation tests on wt cells containing either Ch16UG or Ch16UH. 3. Introduction of Ch16UH into pli1-d cells Having established that the two minichromosomes behave similarly in wt cells, Ch16UH was introduced into pli1-d cells, to check whether it also resembled C16UG in the pli1-d background. Further fluctuation tests and pulse field gel analysis of the resulting minichromosomes indicated that again Ch16UH resembles Ch16UG. 4. Introduction of Ch16UH into library of deletion mutants Before introducing Ch16UH into the deletion library, it was necessary to (a) learn how to manipulate the library using the Singer RoTor HDA station and (b) get the Ch16UH minichromosome into the correct genetic background. I was taught how to use the robot by a PhD student from Tony Carr’s lab (which is adjacent to ours). In order to cross the Ch16UH minichromosome into the library the strain needed to contain mat1_m-cyhS, rpl42::cyhR. wt cells containing Ch16UH were therefore crossed with AMC400 (ade-, leu-, ura-, mat1_m-cyhS, rpl42::cyhR (sP56Q), smt0), and tetrads dissected. The resulting colonies were next replica plated and hgh+, ura+, CyhS, h- colonies identified. One of these was selected for further study and was crossed with the strains in the deletion library. Unfortunately, due to the lack of time, it was not possible to continue with these experiments. However, a PhD student in the lab has recently shown that Ch16UH has been successfully introduced into most of the deletion strains. Summary In summary, two modified minichromosomes (ChIUG and Ch16UH) were created. ChIUG was analysed by pulse field gel electrophoresis and found to be very unstable. Ch16UH was checked to ensure that in both wt and pli1-d cells it behaved as Ch16UG. It was introduced into an appropriate genetic background and crossed with the deletion library. I feel lucky to have had this experience and would like to thank the Biochemical Society and Felicity Watts and her lab for making it possible. I would recommend doing a summer internship like this to other undergraduates; it has improved my confidence in the lab, I now feel more able to do my final year project and it has helped me decide that I would like to go on to do research after I’ve finished my degree. Techniques I have learned

Primer design, PCR, agarose gel electrophoresis, S. pombe transformation, pulse field gel electrophoresis, Southern blotting, fluctuation tests, tetrad dissection and use of the Singer RoTor HAD robot.

Student: Ghulam Karim Khan, University of Florida Supervisor: Professor Peter Knight, Institute of Molecular and Cellular Biology, University of Leeds Biochemical Society Summer Studentship Report 2010 Impact of Cardiomyopathy Mutations on the Assembly of Myosin Tails Student: Ghulam Karim Khan, University of Florida Background: Genetic mutations in the cardiac myosin heavy chain gene result in Hypertrophic Cardiomyopathy, a familial heart disease which is the most common cause of death of people younger than thirty. About one sixth of mutations result in changes in the light meromyosin (LMM; the filament forming region of myosin). Prof Knight collaborates with Prof Michelle Peckham to study six LMM mutations and their impact on the thick filament. It is thought that R1382W destabilizes interactions between myosin molecules by interfering with the formation of the hydrophobic seam of the coiled coil. Mutations E1555K and E1768K probably result in ionic interactions between the chains of the coiled-coil tail. R1500W, E1356K, and N1327K probably affect the formation of thick filaments and the interaction between different molecules, as the mutations are located on the outside of the coiled-coil. Aims: The aim of this project was to observe and understand the impact the aforementioned mutations have on the formation of filaments and polymerization. This was done through experiments in the lab and electron microscopy. The experiments included using glutathione S-transferase (GST) tagged LMMs to create synthetic thick filaments in various pH and ionic strengths and assaying polymerization by both centrifugation and light scattering spectrophotometry. Samples were also negative stained and metal shadowed for viewing in the electron microscope. Description of work: Samples of various GST-LMMs produced by Dr Melanie Colegrave in Prof Michelle Peckham’s lab were dialysed against a high salt concentration buffer to ensure they were monomeric. The samples were then diluted with into a range of lower salt concentrations. Following this, the samples were fixed with glutaraldehyde in order to create dimers or larger species if the molecules were polymerised. Samples were then run on gradient SDS gels and the resulting band intensities were measured in order to determine the amount of polymerization at the various salt concentrations. This was done using several different samples and dialysis buffers. Assaying polymerization of the GST-LMM using spectrophotometry was conducted by recording the absorbance levels in the light scattering region of polymerized GST-LMM, to which a concentrated salt solution was incrementally added. Assaying GST-LMM polymerization through centrifugation was done by diluting the GST-LMM in different salt conditions, centrifuging it, and then resuspending the pellets. Samples of the diluted LMM, supernatant, and resuspended pellet were run on SDS gels in order to determine the solubility of LMM in different salt concentrations.

Wild type GST-LMM and N1327K GST-LMM were examined by electron microscopy. Samples were negative stained by applying a drop of the sample to a UV treated, carbon-filmed copper grid and then flicking uranyl acetate stain across the grid. Metal shadowing was also performed, by spraying sheets of mica with the sample, drawing a vacuum around the mica in an Edwards Coating Unit, depositing platinum onto the sheets of mica, and then coating the mica with carbon. These carbon-platinum replicas were floated off the mica and placed onto copper grids. Both negative stained and metal shadowed grids were examined in the electron microscope. Results: The gels of the dialysed GST-LMM’s hinted that LMM solubility increases with salt concentration, but not much dimer was formed by the fixing. This led to a series of experiments concerning the fixative agent and the quencher. The light scattering spectrophotometry proved inconclusive, as the decrease in absorbance due to increasing salt levels (increasing solubility) was not statistically significant. Assaying polymerization of the GST-LMM through centrifugation was successful, in that a trend in solubility was discovered for all of the LMM’s: solubility was low (~0%) in 100mM NaCl, around 50% in 250mM NaCl, and high (~100%) in 300mM NaCl. Negative stain showed that the GST-LMM formed filaments, albeit narrower than expected and often in large networks. Different conditions were used for the negative stain in order to determine whether the networks were a result of some external condition. After viewing fresh, unfrozen, undialysed wild type GLMM and observing filaments and no networks, I concluded that dialysis or freezing likely caused the networks to form. Metal shadowing was largely unsuccessful, as spraying the sample in low salt (100mM NaCl) onto the mica caused the large filaments to be broken and not enough metal was deposited to see small filaments in higher salt (250mM NaCl). While no obvious difference between the wild type and the mutants was discovered, the impact of salt concentration on filament formation was explored thoroughly, resulting in the determination of the aforementioned solubility trend. Further directions for research: More research can be done in exploring the solubility trend for GST-LMM by using more precise methods for determining polymerization, and filaments formed by the wild type and the mutants can be further examined and compared. Departures from the original proposal: Apart from the addition of several additional experiments, such as determining the fixative properties of glutaraldehyde, the original proposal was followed. However, some of the techniques discussed in the proposal, such as immunofluorescent confocal imaging of cells, were not fully explored due to time constraints. Value of the research to the student: After completing this studentship, I now feel confident working in a microbiology/molecular biology laboratory. In addition to learning and practicing skills and techniques that I can now apply in my course work and research at the University of Florida, I have had the opportunity to use equipment, such as the transmission electron microscope, which I may never have had. My discovery that GST-LMM forms filaments like intact myosin, rather than

the bulky polymers formed by LMM, is valuable to the Leeds group as it shows the GST-LMM is likely to be a good model system for studying cardiomyopathy in future.

Fig 1: Showing the level of activation and inhibition using the Tn mutants. Series 1-2 b Tn6b, series 3-4Tn7b, series 5-6 Tn8b, series 7-8 Tn10b.

Student: Hannah Elam

Supervisor: Mohammed El Mezgueldi

Institution for placement: University of Leicester

Background: Muscles are specialised cells built up of different proteins organised in a specific way. The functional unit is the sacromere which is built up of interchanging thick and thin filaments. Thick filaments consisting of myosin while thin filaments are made of actin, tropomyosin (Tm) and troponin (Tn). Muscles contract when the thin filaments slide between the thick filaments, filament sliding driven by myosin heads binding to actin. The binding of myosin heads to actin is coupled to the intrinsic ATPase activity of the myosin head. The binding to actin is regulated by the components Tm and Tn which change conformation when Tn binds Ca2+. The activity of a muscle can be investigated through the use of ATPase assays mimiciking the intrinsic ATPase activiy in the myosin head.

Muscle myopahtise are a range of diseases which are cause by mutations in the muscle components making muscle activity impaired. Common mutations causing said diseases being found in regulatory components such as Tm and Tn.

Aim: The aim of this project was to investigate in what way different Tm and Tn mutations cause alterations in muscle activity and how this may relate to different skeletal muscle myopathise.

Departures from aim: The functional effects of Tm mutations were not possible to investigate as there did not seem to be binding of Tm to actin. For this reason the investigation concerning Tm became about understanding why it did not bind and at what conditions it would rather than explaining any alterations in muscle activity it may cause.

Description of work: In this project rabbit skeletal muscle actin and myosin head was used. Actin and myosin head were used in combination with Tm mutation (TPM1 and TPM3) and Tn mutations (Tn 6b, Tn 7b, Tn8b and Tn 10b). Tm mutations expressed in SF9T baculovirus purified using chromatography. Tn mutations expressed in E.coli. Tissue purifed Tn was also used.

The proteins were used in ATPase assays. 7 samples were set up for each Tm and Tn mutation. Each sample containing myosin head, actin, Tm and Tn. In samples using Tm mutant the tissue purified Tn was used. Tissue purified Tm was used in samples containing a Tn mutant. To start the reaction 5 μL was added to each sample making final volume 100 μL. Final cocentration for Tm was then 4 μM in the first set of assays. The same assays were run again with a final concentration of 8 μM Tm. Reaction was run for 5 min after which it was terminated by adding 500 μL TCA. After a calorimetric method OD was read at 700nm for each sample.

Co-sedimentation was performed using samples containing Tm mutants, tissue purified Tn and actin. Concentrations for Tm were 4 μM and 8 μM.

Results: The ATPase assays were run to be able to see if Tm and Tn mutations caused altered muscle ativity. Figure 1 shows the assays run for Tn mutants

Fig 2; Showing levels of activation and inhibition using Tm mutants. Series 1-2 TPM3 low concentration, series 3-4 TPM1 and series 5 TPM3 high concentration

Fig 3: Showing the results of co-sedimentation. P standing for pellet and S for supernatant. A is low TPM3 concentration, b being the double concentration. M is marker.

6b, 7b, 8b and 10b. The graph is showing the affect of increasing Tn concentration in each assay. All mutations show similar pattern of activiy, only slight vaiance is present. Overall the mutations cause a lower activation that would be found in the same assays with non mutant Tn, suggesting the mutations cause a more inhibited state of the muscle.

Figure 2 is showing the assays using the Tm mutants TPM1 and TPM3. As can be noted no great activity was presented. The same pattern in presented with the doubled concentration shown in fig 2 series 3-5. Overall this suggest that there is no binding of the Tm mutants to the thin filament components in the assay samples. Figure 3 shows co-sedimentation results for TPM1 and TPM3 at both lower concentration and

double concentration. As can be noted there are thicker bands present in the mutant samples compared to the Tm tissue samples. From this is can be further concluded that the Tm mutants are not binding to the thin filaments in the assay samples.

The reason for Tm mutants not binding to actin are most likely explained by failure of expression. The levels of expression had been very high, acetylation and other post translational modifications may have been compromised due to this high level of expression. In the event of post translational modifications being compromised the Tm mutants will be left lacking properties necessary for proper binding to actin. This can however, not be taken for granted until further evidence has been obtained.

Future directions of the experiment: As has been discussed in the results the reason for the most likely reason for the Tm mutants inability to bind to actin can be explained by failure of expression. To be able to see this as fact the Tm mutants have to be investigated further. Lack of acetylation seems to be the most likely cause. To investigate this mass spectrometry can be performed. When it has been established which properties the mutants lack re-expression may be performed with slight alterations for a more successful outcome.

Value of the studentship to the student: These 7 weeks in the lab have provided me with great insight to have “science” is run. Something that previously seemed daunting and rather intimidating is now something which I find highly enjoyable and could see myself having a future in. When studying at undergraduate level lab experience is limited and does not really resemble real day to day lab work as it is restricted to a few hours a week. Being able to design experiments and set them up over weeks have made me understand the logic behind coming up with publishable studies, something which previously baffled me. Without this summer studentship I feel I would have struggled with my upcoming third year project, a project I now look forward to doing. I also now feel a Phd is a natural root for me to take.

Value of the studentship for the lab: The studentship has been valuable to the lab as the work I performed showed that the tropomyosin mutants were not expressed properly. This saving future studies from being side tracked.

1

Student: Heather Bruce

Supervisor: Daniel Ungar

Institution for placement: University of York

Biochemical Society Student report 2010

Vesicle targeting specificity in the Golgi – a job for more than one tethering factor

Student: Heather Bruce Supervisor: Dr Victoria Miller

Background

In eukaryotic cells the Golgi apparatus is essential for sorting glycolipids, proteoglycans and glycoproteins for intracellular trafficking and secretion. It is also responsible for modifying these proteins to increase their stability and for their normal function. Within each cisterna of the Golgi there are different modification enzymes and their localisation is maintained by retrograde vesicular transport (figure 1).

The vesicles meant for retrograde transport are targeted to specific cisternae by the interactions between the COG complex, specific Rab GTPases and tethering factors belonging to the golgin family. The COG complex is an essential tethering factor important in maintaining the structure and function of the Golgi apparatus (Ungar et al. 2006). Specific Rab GTPases and golgins are required for targeting vesicles to different cisternae. During the project, the interaction between the golgin TMF and the COG complex was investigated.

Objectives

The prime objective is to characterize the interaction between COG and the golgin TMF in cells, and to generate reagents to study the functional relevance of this interaction in vivo.

Aims

1. Biochemical characterisation of COG-TMF interactions 2. Generation of COG mutants unable to bind TMF 3. Localisation of TMF in cells with mis-localized COG subunits

1. Biochemical characterisation of COG-TMF interactions

An immunoprecipitation technique was carried out following previous work that showed an interaction between TMF and the COG subunits Cog2 and Cog6 using a yeast two hybrid assay. Human Embryonic Kidney (HEK) cells stabling expressing HA-tagged Cog1 were transiently transfected with GFP tagged TMF, and Cog1-HA immunoprecipitated using an anti-HA antibody to precipitate the COG complex. Samples were probed with anti-Cog3 to demonstrate that the full COG complex was immunoprecipitated and also with anti-TMF (figure 2).

3

TMF co-immunoprecipitated with Cog1-HA(* in figure 2) but was not present in the control experiment, therefore we conclude that COG interacts with TMF. This method was also extended to transfect Cog1-HA cells with a GTP locked form of Rab6 in addition to being transfected with TMF-GFP, as Rab6 and TMF are known to interact (Fridmann-Sirkis et al. 2004). We wanted to see whether this interaction had an effect on the interaction between COG and TMF. There appears to be a negative effect on the interaction between TMF and COG when Rab6 is bound to TMF.

2. Yeast two hybrid assay to look at the interaction of Cog2 with different

golgin domains To generate Cog2 mutants unable to bind TMF using the reverse yeast two-hybrid method we first decided to map the binding site for TMF and also with other golgins.The approach taken was to make 11 plasmids, each containing a different protein domain from one of the three golgins; TMF, GM130 and p115. The golgin domains were amplified by PCR, underwent a restriction digest and were ligated into a yeast two-hybrid vector. Successful insertion was confirmed by colony PCR or restriction digest. The plasmids were then sequenced and finally transformed into yeast. I constructed five plasmids that I transformed into yeast with various bait proteins (empty vector or Cog2). The interaction of golgin fragments vs Cog2 by yeast two-hybrid is shown in figure 3. Colonies were grown at 30°C on media lacking tryptophan and leucine (WL), WL and uracil (WLU) and WLU and adenine (WLUA). On the -WL plates colonies have grown indicating that both types of plasmid were successfully transformed into yeast. However on the -WLU and -WLUA plates, no colonies are present which suggests that the binding site may lie outside these regions.

3. Localisation of TMF in cells with mis-localised COG subunits

The optimum conditions for transfecting CHO and ldlB cells with GFP tagged TMF was determined however I was unable to image these cells in the time available.

Future directions

Given more time I would have liked to have made all 11 plasmids for the yeast two hybrid assay and also have transfected the CHO and ldlB cells with GFP tagged versions of TMF to see where TMF localises in these cells.

The IP will be repeated with different Rab GTPases and the cell staining and cloning will be continued.

Value of studentship to the student

I feel more independent and confident in the lab after this positive experience and have learned many new techniques such as immunoprecipitation, western blotting, cloning and yeast two hybrid assays. I have also gained a greater appreciation of the importance of scientific research, maintaining a complete and up-to-date lab book and planning experiments. I have really enjoyed this time spent in a research environment and as a result am looking forward to my final year research project and intend to do a PhD after my degree. I would like to thank the Biochemical Society for funding the project, Dr Daniel Ungar for his advice and the opportunity to work in the Ungar lab and also Dr Victoria Miller for her patience and constant support throughout the project.

References Ungar D., T. Oka, M. Krieger and F.M. Hughson. (2006) Retrograde transport on the COG railway. Trends Cell Biol.,

16, 113-120 Y. Fridmann-Sirkis, S. Siniossoglou and H. Pelham. (2004) TMF is a golgin that bings Rab6 and influences Golgi

morphology. BMC Cell Biology, 5:18

Biochemical Society Summer Vacation Studentship Project Report 2010 Name: Holly White

Date: 7th June – 30th July 2010 Supervisor: Dr Maria O’Connell, Senior Lecturer in Pharmaceutical Cell Biology Location: School of Pharmacy, University of East Anglia

Regulation of the transcription factor Nrf2 by protein kinase C alpha Background Nrf2 is a transcription factor which regulates oxidative stress and inflammation. In response to oxidative stimuli, Nrf2 activates a plethora of antioxidants, detoxifying enzymes and other proteins which protect the cell. These proteins are switched on during infection and inflammation and this dampens down the production of pro-inflammatory cytokines. The O’Connell group have previously shown that the Gram negative bacterial cell wall component lipopolysaccharide (LPS) induces the cellular anti-oxidant haem oxygenase-1 (HO-1) and phase II detoxification gene NAD(P)H:quinine oxidoreductase 1 (NQO1) in human monocytes and THP-1 monocytic cells through Nrf2. LPS also induces de novo synthesis of Nrf2. Furthermore, the group have shown that overexpression of HO-1 or Nrf2 suppresses pro-inflammatory cytokine expression in these cells1. In resting cells, Nrf2 is constantly targeted for degradation by its cytoplasmic inhibitor Keap1. However, in response to redox modulators Nrf2 is phosphorylated on its ser40 residue, preventing association with Keap1 and this allows it to translocate into the nucleus. There, Nrf2 heterodimerises with small Maf bZip proteins and binds to specific DNA sequences termed antioxidant response elements (ARE) in the regulatory regions of cytoprotective genes. Consequently, cytoprotective genes including HO-1 and NQO1 can be transcribed2. Protein kinase C (PKC) encompasses a family of serine/threonine kinases. The kinase isoforms are activated in various ways and differ in their location in tissues. Recently, it has been shown that Nrf2 dissociation from Keap1 is regulated by PKCδ in response to the anti-oxidant t-BHQ3. Research in the O’Connell lab has also demonstrated that LPS induces Nrf2 and HO-1 expression through PKC in human monocytes and THP-1 cells4. Inhibition of the PKCδ isoform using rottlerin regulated Nrf2 activation by the anti-oxidants curcumin and epigallocatechin in these cells5,6. However, it did not regulate LPS induced HO-1 expression or Nrf2 binding to the ARE4. The isoform of PKC that is responsible for LPS-induced Nrf2 activation is therefore currently unknown and so this provides a key area to explore. Further work from the group found that the classical PKC inhibitor Go6976 (an inhibitor of

PKC α and β1) suppressed LPS-induced HO-1 expression but that LY333531 (which inhibits PKCβ isoforms) had no effect, suggesting that PKCα was the isoform involved. PKCα knockdown using siRNA confirmed that this isoform regulates LPS-induced HO-1 and NQO1 expression. We therefore hypothesise that Nrf2 activation by LPS is regulated by PKCα. Aims of the Project The main aim of this study was to determine if PKCα plays a role in LPS-induced Nrf2 activation in the human monocytic cell line THP-1. The placement aimed to analyse different possible signalling pathways that involve PKCα and Nrf2 phosphorylation (Fig 1). We first examined whether LPS activates PKCα. We considered whether PKCα regulates LPS-induced de novo synthesis of Nrf2. Finally, we assessed whether PKCα acts in a similar way to PKCδ by inducing phosphorylation of Nrf2.

Fig 1 Possible interactions of PKCα on LPS-induced Nrf2 signalling pathways Results LPS induces PKCα phosphorylation in THP-1 cells THP-1 cells were serum starved and seeded in 24 well plates. Cells were treated with LPS (10 µg/ml) for 15 min and whole cell extracts were made. The proteins were separated by SDS-PAGE using the Invitrogen Novex Minigel system. Immunoblot analysis was carried out using anti-human phosphorylated PKCα

(Ser657) antibodies and anti-human non-phosphorylated PKCα

antibodies (Santa Cruz) and blots were imaged using a GBox iChemi Chemilluminescence imager (Syngene).

Fig 2 LPS induces PKCα (Ser657) phosphorylation in THP-1 cells. LPS induced PKCα phosphorylation within 15 min. PKCα levels did not change. Role of PKCα in LPS-induced Nrf2 mRNA expression To determine if PKCα plays a role in LPS-induced de novo synthesis of Nrf2, THP-1 cells were seeded in 24 well plates and treated for 30 min with Bisindolyl-maleimide I (Bis I, a highly selective inhibitor of several PKC isoforms, including PKCα). Following this, cells were stimulated with LPS for 4 hours. Total RNA was extracted, reverse transcribed to cDNA and quantitative realtime PCR analysis carried out on a Rotorgene Q

thermocycler (Qiagen) using SYBR Green technology. Nrf2 mRNA levels were normalised to GAPDH mRNA expression.

Fig 3 PKC isoforms (including PKCα) do not regulate LPS induced Nrf2 mRNA expression. There was no significant dose-

dependent inhibition of LPS-induced Nrf2 mRNA expression, suggesting that

PKCα (and other PKC isoforms) are not involved in LPS-induced Nrf2 de novo synthesis. Regulation of LPS-induced Nrf2 phosphorylation by PKCa THP-1 cells were serum starved and seeded in 24 well plates. Cells were stimulated with LPS for various times and whole cell extracts were made. Proteins were separated by SDS-PAGE and western blot analysis carried out using anti-human phosphorylated Nrf2 (Ser40) antibodies (Abcam) or anti-human GAPDH antibodies (Sigma). LPS induced Nrf2 phosphorylation within 15 min. However, it was more strongly induced by 1 hr and remained phosphorylated at 4 hr (data not shown). Cells were pre-incubated with varying concentrations of Bis I or Go6976 (an inhibitor of PKCα and β1) for 30 min prior to stimulation with LPS for 4 hours and Nrf2 phosphorylation measured by western blot analysis.

Fig 4 PKC inhibitors suppress LPS-induced Nrf2 (Ser40) phosphorylation. (a) Bis I reduced LPS-induced Nrf2 phosphorylation at 5-10 µΜ (b) Go 6976 (1-10µM) suppressed Nrf2 phosphorylation following LPS stimulation. Discussion PKC is emerging as an important regulator of the Nrf2 pathway. Previous work in the lab suggested that PKCα may play a role in LPS-induced Nrf2 activation. Here, we demonstrate for the first time that PKC is not involved in LPS-induced Nrf2 de novo synthesis. We found that LPS induces PKCα phosphorylation. Furthermore, we also report for the first time that LPS induces Nrf2 phosphorylation and that inhibitors of PKCα suppress this, suggesting that PKCα regulates LPS-induced Nrf2 signalling through phosphorylation on Ser40, similarly to PKCδ in response to anti-oxidants. Further studies are required for confirmation.

Future directions in which the project can be taken • Since the results from this study support a role for PKCα,

further studies will compare the kinetics of PKCα with Nrf2 binding to the ARE and the co-localisation of PKCα, Nrf2 and Keap1 using confocal microscopy. It was our intention to progress these experiments but due to time constraints we were only able to optimise the PKCα antibody for immunofluorescence

• It would be advantageous for experiments to be also conducted in primary monocytes. Primary monocytes taken from the blood will enable a more realistic representation of an in vivo environment.

• Due to time constraints, we were unable to transfect cells with PKCα siRNA to confirm the role of PKCα in LPS-induced transcription of Nrf2.This will be carried out in the near future.

My experience Throughout the course of the placement I have learned how to conduct biological techniques such as quantitative realtime PCR, western blotting and Immunofluorescence. I have been able to practice efficient cell culture and have experienced the day to day activities involved in maintaining the lab. Despite the frustration when experiments were not successful or when results were not as expected, the excitement of discovering new trends and patterns was extremely rewarding. I am now able to understand the importance of accurate note taking, time management and keeping up to date in your field of knowledge by reading journals and attending conferences. At the start of the placement I was nervous and unsure of my ability in the lab; however, I gained an enormous amount of confidence through the project and feel far more prepared for my research project next year. Presenting my results in the weekly group meetings was daunting at first, but I began to feel great afterwards for participating in them and I especially enjoyed hearing about the rest of the lab’s research. The positive and energetic atmosphere in the lab made my placement thoroughly enjoyable and so I would like to thank the entire team for their friendship and support. Overall, the studentship has enhanced my determination to pursue a research PhD and I look forward to the opportunities that complementary membership to the biochemical society will provide. Value of the studentship to the lab This studentship has progressed our understanding of the regulation of the Nrf2 pathway in inflammation. The experimental results point towards PKCα playing a role in LPS-induced Nrf2 activation, and the work carried out by Holly will form the basis for the first experiments of a new PhD student in the laboratory to confirm this. This research has contributed towards an abstract to be presented at the BSI annual congress. Holly was a great member of the team and also optimised new antibodies for immunofluorescence, helped in the routine maintenance of the lab and contributed regularly to discussions during our weekly group meetings. References 1 .Rushworth SA et al.. J Immunol 2008, 181, 6730-7. 2. Kaspar JW et al. Free Radic Biol Med. 2009, 47,1304-9 3. Niture SK et al. J Cell Sci. 2009, 122, 4452-64. 4. Rushworth SA et al. J Immunol 2005, 175, 4408-4415. 5. Ogborne RM, et al.Biochem Biophys Res Commun 2008, 373, 584-588. 6. Rushworth SA, et al Biochem Biophys Res Commun 2006, 341, 1007-1016.

Report by Mr James Harrison supervised by Dr Luke Alderwick at the School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. Biochemical Society Summer Vacation Studentship 2010 Lab report Investigation of polysaccharide translocation systems in Mycobacterium tuberculosis: are they ideal drug targets? Background Mycobacterium tuberculosis is the causative organism of tuberculosis (TB), an infectious disease which causes more illness and death than any other infectious organism worldwide. It is estimated that TB currently infects a third of the world’s population and results in around 1.7million deaths annually. With the rise of multi-drug and extensively-drug resistant forms of TB, it has never been more important for us to identify new drugs in order to combat this deadly pathogen. The unique cell wall of M. tuberculosis acts as a primary defensive barrier to the host immune response and its unusual chemical properties account for its impermeability and resistance to common antibiotic therapies. The M. tuberculosis cell wall is composed of peptidoglycan which is covalently

attached to arabinogalactan (AG). AG is made up of ~ 30 alternating β(1 5) and β(1 6) linked galactofuranose (Galf) residues forming the galactan domain which is decorated with three arabinan domains at the 8th 10th and 12th Galf residues. Mainly

composed of α(1 5) linked arabinofuranose (Araf) residues, mycobacterial arabinan is an elaborate polysaccharide with distal and

proximal α(1 3) branching before terminal β(1 2) Araf caps. AG acts as a molecular scaffold for the attachment of long chain mycolic acids, which forms a second hydrophobic “pseudo” membrane and intercalates with other lipid-linked antigens which are important for M. tuberculosis pathogenesis. Although many of the genes involved in AG biosynthesis have recently been characterised, such as the arabiniofuranosyltransferases AftA, AftB and AftC, our understanding of how large lipid-linked polysaccharide intermediates are translocation across the cytoplasmic membrane remains fragmented. Since the synthesis of AG is already a target for the front line TB drug ethambutol (EMB), cell wall biosynthetic processes, such as the translocation of AG intermediates, represent potential targets for the development of new drug therapies. In order to determine the validity of these targets as being “druggable”, we must first investigate the essentiality of genes involved cell wall assembly. In this regard, generating gene knock out mutants in laboratory model organisms such as Corynebacterium glutamicum and Mycobacterium smegmatis, followed by subsequent phenotypic analysis, provides a system to study putative flippase candidates involved in the translocation of AG intermediates. Aims The main aim of this project is to determine the essentiality and function of the putative flippase system responsible for galactan translocation across the cytoplasmic membrane. In M. tuberculosis two open reading frames (Rv3781 and Rv3783) represent mycobacterial homologues of the Pseudomonas aeruginosa O-antigen flippase system wzt and wzm, respectively. Orthologues are also present in C. glutamicum (NCgl0197 and Ncgl0198) and M. smegmatis (MSMEG6366 and MSMEG6369). We will investigate the essentiality of these genes by:

1. Generation of C. glutamicum and M. smegmatis null mutants. 2. Generation of merodiploid strains of C. glutamicum and M. smegmatis null mutants expressing a second complementing

copy of the gene on an inducible vector. 3. Cell wall and polysaccharide phenotypic analysis of conditionally expressed strains of M. smegmatis MSMEG6366 and

MSMEG6369 mutants. 4. Generation of plasmids for the over-expression and purification of recombinant MSMEG6366 and MSMEG6369 protein in

Escherichia coli.

Work carried out For the genomic disruption of NCgl0197 and NCgl0198, we constructed two plasmids pK18mob-NCgl0197-int and pK18mob-NCgl0198-int, respectively. The resulting DNA fragments were treated with AvaI/KpnI and ligated into the non-replicative vector pK18mob, which was similarly treated with AvaI/KpnI. The resulting plasmid was transformed into C. glutamicum by electroporation and selected for resistance to kanamycin on brain heart infusion sorbitol agar. After several independent rounds of electroporation we eventually obtained two colonies for pK18mob-NCgl0197-int and one colony for pK18mob-NCgl0198-int. However, subsequent genotypic analysis of each of these clones revealed that each recombination occurred in a poorly defined region of the genome suggesting that both of these gene products are required for growth and integrity of C. glutamicum. We used specialised

transduction for the genomic deletion of MSMEG6366 and MSMEG6369 in M. smegmatis. Upstream and downstream flanking regions of MSMEG6366 and MSMEG6369 were amplified by PCR and treated with Van91I. Each upstream and downstream region of each respective gene was ligated into the temperature sensitive phasmid (p00045), which is used to deliver an allelic exchange

marker designed to replace targeted genes with a hygromycin resistance cassette at 30 °C. After several attempts to obtain

transductants of M. smegmatis grown at 37 °C, we were unable to obtain any hygromycin resistant clones in M. smegmatis for both MSMEG6366 and MSMEG6369. Therefore, we carried out a conditionally expression-specialised transduction essentiality test (CESTET) to determine if we could modulate the expression of both MSMEG6366 and MSMEG6369 in M. smegmatis using an acetamidase inducible “rescue” plasmid. We amplified the ORFs encoding MSMEG6366 and MSMEG6369 which were then treated with EcoRV and HindIII. The mycobacterial expression plasmid pSD26 encodes the inducible acetamidase promoter which was removed by double-restriction digest using XbaI and EcoRV and subsequently dual-ligated into the mycobacterial expression vector pMV306 pre-treated with XbaI and HindIII. The process of generating these “rescue” plasmids for both MSMEG6366 and MSMEG6369 was fraught with difficulties and seriously delayed aims 2 and 3 of the project. We eventually obtained constructs for both pMV306-acet-MSMEG6366 and pMV306-acet-MSMEG6369 which will be transformed into M. smegmatis prior to the transduction of the temperature senbsitve phasmid (harbouring appropriate flanking regions of either MSMEG6366 and

MSMEG6369, respectively) and selection on acetamide at 37 °C. In addition, the XbaI and EcoRV doubly-restricted PCR amplicons for MSMEG6366 and MSMEG6369 were also ligated into both pMALp2x and pMALc2x expression vectors. Both pMALp2x and pMALc2x allow for the in-frame cloning of MSMEG6366 and MSMEG6369 both with N-terminal maltose binding protein (MBP). pMALp2x carries an additional N-terminal signal peptide for the secretion of the recombinant protein into the periplasm of the E. coli expression host. In this regard, E. coli C41 (DE3) cells were transformed with either pMALp2x-MSMEG6366, pMALp2x-MSMEG6369, pMALc2x-MSMEG6366 and pMALc2x-MSMEG6369. These

clones were then cultured in 5 mL of LB-broth and induced with 0.5 mM IPTG for either 4 hrs at 37 °C or 10 hrs at 16 °C. Cells were harvested by centrifugation resuspended in TRIS-buffered lysosyme containing protease inhibitors and lysed using the freeze thaw

procedure. The clarified lysate (CL) fractions containing soluble proteins were obtained after 4 °C centrifugation of the whole cell lysate (L) at 27,000 x g for 20 mins. The over-expression of N-terminally-tagged MSMEG6366 and MSMEG6369 was analysed by 10 % SDS-PAGE followed by coomassie blue staining. The predicted molecular weights of MBP-tagged MSMEG6366 and MSMEG6369 were calculated to be 73 kDa and 74 kDa respectively. The molecular mass of MBP is 43 kDa. As depicted in Figure 1, no expression was observed for either MBP-tagged MSMEG6366 (Fig. 1A) or MSMEG6369 (Fig. 1B) using the pMALp2x expression vector. However, in a control experiment using just pMALp2x, we were unable to observe expression of MBP, suggesting that the expression host was unsuitable for over-expression of periplasmic secreted proteins or the subsequent extraction procedure was also unsuitable. Apart from a clear expression of MBP using pMALc2x, we were also unable to see any expression of either MBP-tagged MSMEG6366 (Fig. 1C) or MSMEG6369 (Fig. 1D) using the pMALc2x expression system. This data suggests that other E. coli expression systems are required for the over-expression of recombinant MSMEG6366 and MSMEG6369.

Future directions The immediate future of this project would be to carry out a completed CESTET analysis of MSMEG6366 and MSMEG6369 using the acetamide-inducible “rescue” plasmids. This would allow us to deplete the expression of both of these genes for us to carry out a phenotypic analysis of the reduced expression strains thus enabling us to assign a biochemical function for each of these genes. In addition, a range of E. coli expression hosts will be expored for the over-expression of recombinant MSMEG6366 and MSMEG6369 protein. Once the essentiality and biochemical characterisation of these flippase components have been fully investigated further work will be carried out to determine the suitability for the development of drugs that target this translocating system. Value of studentship I feel that the studentship has been of great benefit to me, as it has allowed me to sample the highs and lows of

Figure 1. 10 % SDS-PAGE analysis of cell lysates (L) and clarified lysates (CL) of E. coliC41 (DE3) cells transformed with pMALp2x-MSMEG6366 (A), pMALp2x-MSMEG6369 (B), pMALc2x-MSMEG6366 (C) and pMALc2x-MSMEG6369 (D).

scientific research, and has given me a great insight into how research is conducted. I feel that I am now much better prepared for any laboratory work I may be presented with in the future because of the skills I have learnt during this project. I feel that the laboratory benefited from the studentship, as I have persevered with the molecular biology side of the project. The work carried out in this project provides a platform for future research associated with M. tuberculosis cell wall biosynthesis and drug development.

Student: James Wood

Supervisor: Peter Roach

Institution for placement: University of Southampton

Site specific alkylation of DNA using a propargyl S-adenosylmethionine analogue

Background

S-adenosylmethionine (SAM) is the major methyl group donor for biological methylation. These reactions are catalysed by methyltransferase enzymes and are used in biological systems to place methyl groups selectively onto DNA, proteins and other biological molecules. The DNA methyltransferases are highly selective, recognising specific sequence motifs in the genetic code. DNA adenine – N6 methyltransferase (dam) was the enzyme selected to catalyse DNA alkylation during this project. Dam transfers the methyl group from the cofactor (SAM), onto the N6 site of an adenine base in a DNA chain. Dam recognises the DNA sequence motif GATC in double stranded DNA.

This project aimed to use an analogue of SAM, replacing the methyl group with an alkyl chain containing an alkyne (Fig. 1). This alkyne could subsequently be used for a click chemistry reaction to attach a fluorophore to the DNA strand. The fluorophore would allow for a quantitative measurement of fluorescence increase during the transfer step, enabling the investigation for enzyme kinetics.

Aims

• Express a sufficient quantity of Dam to catalyse the transfer of the alkyl chain. • Synthesise and purify the propargyl-SAM analogue. • Assay the activity of Dam in the propargyl group transfer. • Use click chemistry to attach fluorophore.

Results

A plasmid containing the dam gene and a kanamycin resistance gene was constructed by restricting one plasmid containing the dam gene and inserting the gene into the pET24d vector in order to achieve better expression of the gene. The purified gene and backbone were ligated and transformed into competent TOP10 E. coli cells to replicate the DNA containing the dam gene. The DNA was isolated from the TOP10 cells using a mini-prep kit and restricted to ensure the plasmid contained the correct gene The plasmid was then transformed into E. coli strain BL21(DE3) for overexpression of the target gene.

Under the control of the T7 promoter, IPTG induction was investigated but yielded very low levels of expression. Auto-induction (as described by Studier [1]) was then attempted. A small scale expression

N

NN

NO

S+H2N

O OH

NH2

Nu:

N

NN

N

NH

R

+N

NN

NO

HO OHSH2N

O OH

NH2

Propargyl SAM analogue

S-adenosylhomocysteine (SAH)

Dam

HO OH

Fig 1. Proposed mechanism of transfer of the propargyl group onto the N6-position of an adenine base in a DNA chain. The N atom with a lone pair at the 6 position of the adenine base is the nucleophile shown.

study yielded higher expression levels and the optimal temperature for growth was found to be 37 °C. A large scale expression experiment was trialled using the auto-induction method at 37 °C (see figure 2).

The propargyl-SAM analogue was produced by reacting S-adenosylhomocysteine with propargyl bromide and using formic acid as the solvent (figure 1). Silver triflate was added to remove the excess bromide from the reaction mixture. This reaction produced two products of the same mass, potentially these may be diastereomers. These compounds were purified by reverse phase HPLC (see figure 3).

Deviation from the original proposal and future directions

The Dam enzyme was expressed and the SAM-analogue have been synthesised. However, the compound was not stable unless under acidic conditions and kept at low temperature. Therefore, due to the time restraints on the project, the transfer and click reaction requires further optimisation but the foundations are present for future students to develop this methodology.

Value of studentship to myself and the lab

Completing this studentship, as well as being enjoyable, has been beneficial to me in many ways. It has helped me to improve skills such as analysis and organisation which are vital for a career in chemistry as well as learning a number of new ones. The biological aspect of the project has allowed me to broaden my skills and knowledge, therefore deviating from straight chemistry. The combination of biology and chemistry could be very useful in future projects that I may undertake. It has also allowed me to have a taste of life working as a postgraduate which will aid me in my decision as to if and where I would like to become a PhD student. The research group that I have been working in will benefit from the contribution I have made to this project which can be continued with when possible and will be of great use when it can be put into practice.

References

[1]. “Protein production by auto-induction in high-density shaking cultures”. F. W. Studier. Protein Expression and Purification 41 (2005) 207–234.

Fig. 2. SDS-PAGE gel of large scale auto-induction. Lanes 1-4 are 1% inoculations of 1.25 L of auto-inducing media. Auto-induction works on an overnight timescale and so little protein has been expressed after 3 hours, and much more after 19 hours. The desired protein has the approximate molecular weight of 30000 g mol-1. The highlighted band (32000) has a molecular weight of 32000 g mol-1.

Fig. 3. HPLC trace of reaction mixture. Peaks were shown to have the same mass by LCMS.

Biochemical Society Summer Vacation Studentship Report

Interactions between Deubiquitinases and Smads in TGFβ signaling pathway by Jia Yueh Wong (Supervisor: Dr. Sylvie Urbé, University of Liverpool)

Introduction The Transforming Growth Factor-β (TGFβ) signaling pathway is involved in the regulation of many cellular processes (Dennler et al., 2002). Smads are key regulatory proteins, which modulate TGFβ signaling and act as transcription factors. Involvement of reversible ubiquitination in regulation of TGFβ signaling is proven and several deubiquitinases (DUBs) have been shown to regulate and/or interact with components of this pathway (Ibarrola et al., 2004; Dupont et al., 2009). DUBs are a class of enzymes emerging as potential drug targets in treatment of various diseases such as cancer (Komander et al., 2009). The aim of my research project is to carry out a systematic analysis of potential interactions between DUBs and Smads (by Yeast 2 Hybrid and co-immunoprecipitation), and to identify potential functional relationships, between DUBs and Smads (by siRNA library screen). Screening for Interaction between DUB and Smads by Yeast 2 Hybrid (Y2H) To begin with, I first carried out a systematic Y2H screen to look for interactions between 53 DUBs, representing members from all 5 classes of DUBs (93 known human DUBs as yet) and 9 Smads (including 5 regulatory Smads – Smad1/2/3/5 and 9; 1 common Smad – Smad4; and 3 inhibitory Smads – Smad6-short/long and 7). Open reading Frames (ORFs) of 53 DUBs had been inserted into a Y2H bait vector and transformed into MATa yeast strains while all Smad ORFs were inserted into a prey vector and transformed into MATα yeast strains. These yeast strains were mated and eventually transferred to a triple selection plate using a HIS3 and ADE2 reporter system. Growth of yeast colonies on triple selection plate would indicate possible interaction.

siRNA DUB library Screen to Identify DUB(s) Regulating Smad2/3

Checking Interaction between Smad2/3 and DUBs by co-Immunoprecipitation (co-IP) The interaction between YOD1 and Smad3 as identified by Y2H warrants further validation since the system used is artificial and hence any interaction picked up might not be physiologically relevant. Also, as is the case for the DUB, USP9X, which regulates and interacts with Smad4 (Dupont et al, 2009), functional relationships between Smad2/3 and few of the top candidate DUBs identified in the siRNA screen, may likewise suggest possible physical interactions.

Therefore, I then progressed to further validate/determine interactions between the selected DUBs and Smad2/3 by co-immunoprecipitation. HEK293T cells were transiently transfected with myc-Smad2/3 and GFP vector or GFP-

Figure 1. Screen of Smad3 against all 53 DUBs. This figure shows a representative image of a plate with HIS3 reporter after 14 days. YOD1 interacts weakly with Smad3 (position D10, circled in red). Growth observed at positions B3 (USP13), B11 (USP25), E11 (AMSH-LP), E10 (AMSH) and F9 (AMSH) was due to autoactivation since growth was detected on control plates as well (not shown). Single colonies were not taken into consideration since growth was likely due to mutation. No novel interaction between DUBs with any other Smads was observed (Data not shown). YOD1 is also found to interact with Smad2 (Data not shown).

In parallel to the Y2H experiment, a siRNA DUB library screen was performed to identify DUBs involved in regulating stability of Smad2 and 3, since no DUB has yet been identified for these 2 Smads and the antibody* is available in the lab. A549 cells were transfected with 40nM pool of siRNA oligos (comprises of 4 single oligos), each targeting 1 of 92 DUBs. Cells were lysed in NP40-buffer 72 hours post-transfection and lysates were prepared for SDS-PAGE, followed by immunoblotting (IB) and densitometry for quantitation. [*Antibody used recognizes both Smad2 and Smad3]

A

B

Figure 2. DUBs regulating Smad2/3 stability. (A) Representative immunoblot (B) Change of Smad2/3 level (normalized to actin) relative to control. Knockdown of OTUD7B resulted in significant depletion of Smad2/3 level, in steady state conditions. Deconvolution experiment, which uses individual single oligos for knockdown, was subsequently performed and 3 out of 4 oligos reproducibly recapitulated the pool knockdown effect, confirming the observed Smad2/3 loss following knockdown of OTUD7B using pool oligos was not an off target effect (not shown).

tagged DUBs for 24 hours (results are only shown for OTUD7B and YOD1). Cells were starved overnight and treated with (+) or without (-) TGFβ for 1 hr prior to lysis. The IP was carried out using anti-GFP antibody and samples were subsequently processed for SDS-PAGE and IB. Experiment was done in duplicate.

Figure 3. Determining Interaction between Smad2/3 and DUBs by co-IP. Immunoblot for (A) co-IP and (B) total cell lysate. OTUD7B interacts with Smad3 regardless of TGFβ stimulation (red box), but not with Smad2. No interaction was observed between YOD1 and Smad2 and 3. Note that cells expressing GFP-

OTUD7B had higher level of myc-Smad2 or myc-Smad3 (blue box), but this was not observed with other GFP-YOD1 and GFP-vector. This is a promising observation, demonstrating the effect of overexpression of OTUD7B, which stabilizes Smad2/3 level, as opposed to the effect of its knockdown (Figure 2A and 2B), which leads to significant depletion of Smad2/3.

Conclusion The results obtained thus far suggest a functional relationship between OTUD7B and Smad3. Possibly, OTUD7B stabilizes Smad3 by deubiquitination and thereby rescuing it from degradation (Figure 3B), while its knockdown by siRNA resulted in significant depletion of Smad2/3 level (Figure 2A and 2B). The fact that OTUD7B interacts with Smad3 as revealed by co-IP (Figure 3A) adds further confidence on the plausible functional relationship between Smad3 and OTUD7B. The fact that this interaction was not picked up by Y2H may be due to several possible reasons, such as requirement for post-translational modification and participation of other protein components.

In the future, an in vitro pull down assay using purified OTUD7B and Smad2/3 can be performed to confirm interaction between OTUD7B and Smad3, and also to give an indication whether the interaction is direct or not. To firmly establish the functional relationship between the two proteins, a rescue experiment can be done using a siRNA-resistant construct with respect to OTUD7B knockdown, and see whether that stabilizes Smad3. Simple experiment can also be done to determine the effect of overexpression of OTUD7B and a catalytically inactive mutant on endogenous Smad3 level. If OTUD7B is a bona fide interacting partner and regulator of Smad2/3, its functional role in the context of TGFβ signaling modulation would be one key question to answer. Moreover, a recent publication has shown chain specificity for K11-ubiquitin chain exhibited by OTUD7B and this ubiquitin chain linkage has not yet been implicated in TGFβ signaling (Bremm et al, 2010). This would present itself as a very intriguing and exciting avenue to explore.

Value of Studentship to Student and Lab The 8 week research experience in Dr. Urbé's lab has been a fantastic opportunity for me to gain an in depth insight into the field of scientific research. I was given very good guidance in learning various laboratory techniques, ranging from proteomics (Y2H) to cell biology (siRNA) techniques. Moreover, I also had the chance to attend journal club and seminars in the department, which again has provided me with valuable flavor of fascinating nature of science. This has ascertained my decision to do a PhD research degree following completion of my undergraduate degree.

Currently, there is a PhD student in Dr. Urbé's lab whose research focus is to study regulation of TGFβ signaling by DUBs. The finding of my research project, if confirmed, will provide another level of understanding of the pathway, and possibly, open up a different branch for TGFβ signaling related research since it is a novel finding. References Bremm A., Freund S. M. V. & Komander D. (2010) Lys11-linked ubiquitin chains adopt compact conformations and are preferentially

hydrolyzed by the deubiquitinase Cezanne. Nature Structural & Molecular Biology. 17: 939-947. Dennler S., Goumans M.J. and Dijke P.t. (2002). Transforming growth factor ß signal transduction. Journal of Leukocyte Biology. 71: 731-740 Dupont S., Mamidi A., Cordenonsi M., Montagner M., Zacchigna L., Adorno M., Martello G., Stinchfield M. J., Morsut L., Inui M., Moro S.,

Modena N., Argenton F., Newfeld S. J., and Piccolo S. (2009). FAM/USP9x, a Deubiquitinating Enzyme Essential for TGFβ Signaling, Controls Smad4 Monoubiquitination. Cell. 136: 123-135.

Ibarrola N., Kratchmarova I., Nakajima D., Schiemann W.P., Moustakas A., Pandey A., and Mann M. (2004) Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor beta signaling. BMC Cell Biology. 5:2.

Komander D., Clague M. J. & Urbé S. (2009) Breaking the chains: structure and function of the deubiquitinases. Nature Review Molecular Cell Biology. 10: 550-563.

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Student: Joanne Hood, University of Manchester Supervisors: Dr Joseph Costello and Dr Graham Pavitt

Project Title: Genetic analysis of novel regulators of the translation initiation factor eIF2B bio

Background

Translation is a complex, highly regulated process, which enables the incorporation of new amino acids to a growing polypeptide chain. The main steps of protein synthesis, initiation, elongation, termination and recycling occur only in the presence of soluble initiation and elongation factors each with a specific role in the production of a mature polypeptide chain. A key regulated step in the initiation phase is the activation of eukaryotic initiation factor eIF2 by the guanine nucleotide exchange factor eIF2B1,2(Figure 1). When activated eIF2-GTP binds initiator methionyl-tRNA necessary for each translation initiation event. The GCN4 gene, which encodes a transcriptional activator of amino acid biosynthetic genes, is highly sensitive to eIF2B activity, enabling yeast cells to respond to amino acid depletion3,4. Upstream open reading frames in the GCN4 mRNA mediate this response. My project's main aim was to characterise new mutants to assess whether any impact on this nutritional starvation response using molecular biology, genetic and biochemical assays.

.

Aims of the project The main aim of my project was to assess whether genes of interest play a role in translational control of gene expression. Nine different gene knockout strains of S. cerevisiae each deleting a different known or putative mRNA binding factor were selected. The His3 mutations in each strain were first complemented to facilitate this analysis. The His3p competitive inhibitor 3-aminotriazole provided a simple tool to monitor GCN4 translational control on Petri dish assays. Candidates from this screen were to be further assessed with specific GCN4 reporter gene studies.

Description of work Use of cloning techniques to create His+ strains Gene deletion strains were previously created for all yeast genes and have been a vital resource for a wide variety of studies. However these deletion strains also bear mutations in several amino acid biosynthetic pathway genes which prevents systematic analysis of some pathways. As we wanted to assess the effects of gene deletions on amino acid biosynthesis and the control of master regulator GCN4 it was necessary to complement the his3 mutation with the wild-type gene (HIS3). The plasmid pUN90 vector contains a HIS3 cassette with flanking restriction enzymes sites, which can be used to excise the cassette and then integrate into the yeast genome by homologous recombination. This had previously been used in the Pavitt lab to create His+ strains, but suffers from a high background due to the presence of a yeast replication origin enabling growth of non-integrated transformants. By careful inspection of the plasmid sequence it was found it was possible to remove the yeast replication origin DNA and create a zero background deletion vector. Standard molecular biology techniques were used to complete this: plasmid miniprep (QIAprep® Miniprep), digestion with XmaI and NgoIV restriction enzymes, agarose gel electrophoresis and band excision and clean up (QIAquick gel extraction kit), DNA ligation and transformation of E. coli cells (XL-10 Gold Ultra competent cells). The identity of the resulting plasmid was confirmed with restriction enzyme digests (BglII, XhoI and BamHI). A pre-digested plasmid stock was created using restriction enzymes XhoI and BamHI which release the 1.4kb HIS3 cassette which can then be used to transform each test yeast knockout strain. Yeast transformations were performed using Li Acetate and successful integrants were be selected for on SCD-His medium. Polymerase chain reaction was used next to assess whether the HIS3 cassette was integrated at the correct position within the yeast genome. Phenol/chloroform extracted genomic DNA from selected transformants was used as the template DNA for PCR reactions. The first two attempts of PCR were unsuccessful however the problem was resolved by altering the thermocycler settings, lowering the annealing temperature from 60°C to 55°C. The procedures were repeated so that 9 new HIS3+ strains were constructed and confirmed. These were next used for translational control analysis.

Assessment of translational control phenotypes with 3-Aminotriazole (3AT) Plates

Growth on medium containing 3AT requires GCN4 activity and a wild-type HIS3 gene. The newly created strains were

grown in liquid culture to a set optical density, then serially diluted and spotted onto yeast growth plates ± 3AT (10mM,

25mM and 50mM). Unfortunately the 3AT plates did not yield conclusive results. Possibly due to the strains carrying residual

histidine from being grown in YPD medium before plating. Therefore the experiment requires repeating, but time ran out...

Figure 1. eIF2 is converted to active GTP bound form by GEF eIF2B. eIF2-GTP binds initiator methionyl-tRNA. and then the 40S ribosome to initiate protein synthesis.

Student: Joanne Hood, University of Manchester Supervisors: Dr Joseph Costello and Dr Graham Pavitt

Project Title: Genetic analysis of novel regulators of the translation initiation factor eIF2B


The proposed day-to-day supervisor (Dr Mohammad-Qureshi) was on maternity leave and Dr Costello substituted this role.

The integration of the His3 cassette into the gene mutants took longer than originally planned and therefore several assays

planned(eg reporter gene assays and SDS-PAGE gels) were not completed.

Value of project to student

My summer project was hugely beneficial to me. I thoroughly enjoyed my project and have decided to pursue a career in

research. Working alongside post doctoral and PhD students was invaluable in making this decision as I was able to see the

work they are doing and ask them questions about careers in science. One of the most surprising aspects of the project for

me was the realisation that research takes a lot longer than I had imagined with each step being checked and sometimes

repeated. I have learnt an incredible amount and developed many practical lab techniques that will prove very useful in my

final year project enabling me to work efficiently and confidently when I carry out my final year project in January.

Assessment of results

The first phase of the work was successful. A plasmid tool was generated to efficiently restore the HIS3 gene in yeast cells. This was shown to be effective by the production of 9 new HIS+ yeast strains. A PCR confirmation of correct integration and growth in the absence of histidine showed this. Unfortunately this phase took longer to complete than anticipated leaving only a short time to evaluate the new strains. These results were inconclusive as the negative control grew when it was meant to show no growth (Figure 2). A simple explanation suggested that this was due to excess histidine being present in the medium used to set up the experiment, causing the drug (3AT) to be ineffective. This analysis needs to be repeated.

Value of Student to Laboratory

It was a pleasure to host Joanne in the Lab this summer. She clearly has an enthusiasm for science and learned a lot

while here about how science is conducted in a laboratory setting and the day-to-day lives of scientists in the

laboratory. Dr Joe Costello also gained mentoring and supervision experience which will help him develop further as a

research leader of the future.

Future directions

Future work that could be conducted on this project includes

repeating the 3AT plates to identify genes of interest, then possibly

inserting reporter plasmids with specific mutations in the GCN4

upstream open reading frames fused to the lacZ gene into selected

strains. This would allow any changes in GCN4/eIF2B control to

assessed quantitatively and perhaps provide clues to the

mechanisms involved.

SD+10mM 3AT

WT

Scp160 Δ

5061Δ

SLF1Δ

MRN1Δ

VTS1Δ

YLL032CΔ

Negative

Control

Figure 2. Sample results of knockout strain growth

on 3-Aminotriazole plates showing growth of

negative control.

References

1. Sonenberg N, Hershey J, Mathews MB, Translational Control of Gene Expression, 2000, Cold Spring Harbour Laboratory Press,

New York, Pg 187-193. 2. Jackson RJ, Hellen CU, Pestova TV, The mechanism of eukaryotic translation initiation and principles of its

regulation, 2010, Molecular Cell Biology, Vol 10, Pg 113-127 3. Richardson JP, Mohammad SS, Pavitt GD, Mutations causing

childhood ataxia with central nervous system hypomyelination reduce eukaryotic initiation factor 2B complex formation and

activity, 2004, Molecular and Cellular Biology, Pg 2352-2363. 4. Hinnebusch AG and Natarajan K, Gcn4p a master regulator of gene

expression is controlled at multiple levels by diverse signals of starvation and stress, 2002, Eukaryotic Cell, Vol 1, Pg22-32.

Student: Joshua Dunne

Supervisor: Dr. Nicholas Watmough

Institution for placement: University of East Anglia

Determinates of superoxide formation by electron transfer flavoprotein:ubiquinone oxidoreductase (ETF-QO)

Background:

ETF-QO is a 64KDa protein of the inner mitochondrial membrane which contains an iron sulphur cluster (4Fe4S) and FAD(Flavin Adenine Dinucleotide) cofactors. The role of ETF-QO, along with electron transfer flavoprotein(ETF) is to transfer electrons from the primary acyl-CoA dehydrogenases of fatty acid beta oxidation and amino acid catabolism to the ubiquinone pool of the main respiratory chain. In addition ETF-QO is one of seven proteins which are associated with superoxide formation in the mitochondria. However, the amount of superoxide produced by ETF-QO is not fully understood nor is the site of superoxide formation in the enzyme known. It was proposed to isolate membranes from E.coli that contain recombinant ETF:QO from Rhodobacter sphaeroides (a close homologue of the mitochondrial enzyme) and use it along with purified ETF and a primary dehydrogenase to develop an in vitro assay of superoxide formation. Once established the assay would be used to if changing the reduction potential of either the FAD or 4Fe4S cofactors influenced superoxide formation. Secondly, various oxygen concentrations would be used in the assay to see how this effects the formation of superoxide.

Aims

1. Establish a reliable assay for the formation of superoxide by ETF-QO during turnover.

2. Evaluate the relative contributions of the FAD and 4Fe4S cofactors to superoxide formation using engineered forms of the protein that affect the potential of the cluster.

3. Determine the effect of oxygen concentration on superoxide formation.

Details of work carried out:

An expression vector containing the gene encoding wild type R.sphaeroides ETF-QO and two variants P390L and P390T were used to transform two strains of E.coli C43(for protein expression) and HB101(to propagate plasmid) and transformants selected on antibiotic resistant plates. The first round of transformations of, HB101 produced colonies from P390L and P390T but no wild type ETF-QO colonies.Crucially the negative control in the C43 transformations yielded colonies, meaning the colonies produced on those plates were not useable for my experiments. This was due to the fact transformed bacteria with the required plasmid and those with ‘naturally’ occurring resistance could not be told apart.This caused two problems, firstly HB101 was unable to express ETF-QO(due to HB101 being incompatible with the pET type expression vector, in which ETF-QO gene is cloned) and secondly I had no cells which contained the wild type plasmid to extract plasmids from. Over the few weeks multiple failed attempts to transform C43 with ETF-QO wild type plasmid and the two mutant plasmids were conducted, until successful transformants were obtained and tested for their validity. A large grow up of the C43 cells, transformed with the ETF-QO wild type plasmid, was performed.

The cells were tested for ETF-QO by running a cell lysate against a pure ETF-QO sample on a SDS-PAGE gel. Once it was confirmed the cells were lysed and the membrane containing the ETF-QO was isolated, ready to be used for superoxide assays. In the mean time the plasmid pJR46-1, which contains the genes for human ETF(Electron transferring flavoprotein) was used to transform the E.Coli strain TB1 and used for large scale batch culture. The growth (6L of cells) failed to produce ETF. The procedure was repeated and ETF expression was detected using SDS-PAGE in the same manner as ETF-QO. The cells were lysed and ETF along with many other proteins were isolated from the cell. ETF purification was initiated by running the cell lysate through a DEAE column, however it wasn’t complete due to the 8 weeks expiring.It was agreed that it would be most useful for me to gain experience of developing the superoxide assays using components provided by Dr Watmough.

An assay was constructed and tested in stages to ensure its legitimacy. The first stage was to test if glutaryl CoA was being oxidised by glutaryl CoA dehydrogenase and transferring electrons to an acceptor molecule(PMS). This process was monitored by the acceptor molecule passing electrons to a dye(2,6 dichlorophenolindophenol) causing a colour change monitored by UV-vis spectroscopy. This stage proved that electrons were being transferred to the dye. At this stage the acceptor molecular was removed and replaced with ETF. The rate of dye colour change was again measured. The rate was lower than the previous experiment but it was assumed at this time that it was due to the ETF being less efficient at transferring electrons than the acceptor molecule. The dye was then removed and replaced with ETF-QO and UQ1 (ubiquinone-1), a decreased absorbance of the ubiquinone as it was reduced would indicate a transfer of electrons through the system. However the rate of absorbance change was minimal. It was then decided to go back and further test the components of the system to see if my assumption about the transfer of electrons was correct. It was discovered that the glutaryl-CoA dehydrogenase passed electrons directly to the dye, but at a slow/lower rate than the acceptor molecule. This caused me to initially assume that ETF was accepting electrons and passing them to the dye, but actually the ETF had degraded and the observed rate was due to transference from glutaryl CoA dehydrogenase to the dye. Hence no observable rate was observed when adding ETF-QO and Q1 because the electrons could not be transferred or transferred at a far slower rate than ETF. The final assay would of consisted of the following components; glutaryl CoA, glutaryl CoA dehydrogenase, ETF, ETF-QO and UQ1. The electrons would be passed sequentially through this assay. If this assay had been established superoxide dismutase and Amplex Red would have been added. This would allow the reaction between superoxide with superoxide dismutase forming hydrogen peroxide, this hydrogen peroxide could then directly react with Amplex red causing the colour to change from blue to pink. This reaction could be directly monitored in a UV-vis spectrophotometer and thus the rate of superoxide formation could be established.

Results and Outcome

A successful transformation of the bacteria was confirmed by the plasmid being isolated and then digested by restriction enzymes. An example of a successful digest run on an agarose gel with ethidium bromide is show below. The plasmid digested was that isolated from a C43 cell with the ETF-QO P390L mutant. The plasmid insert is shown on the diagram.

The first large growth of E.coli TB1 with the pJR46-1 (ETF) plasmid was tested for protein production. However the SDS-PAGE gel shown below could not confirm if ETF was present or present in any significant quantity, it was thus decided it would be quicker to repeat the grow up than to start purification only to find that there was either no protein produced or insufficient quantity of the product.

The second large growth of TB1 with the pJR46-1 plasmid was tested in the same way as the first the resulting gel is below.

Marker

Control

Marker

Pure ETF

Cell lysates of varying concentrations

Marker

Pure ETF

Cell lysate First run through DEAE column

NdeI HindIII

Double

Insert of ETF-QO P390L

Unexpected fragment, possibly due to the presence of a HindIII restriction site within the insert. Only visible in the P390L mutant.

Future work

Future work on this project would begin with the purification of ETF. Once this had been done the assay could be used to ensure electrons are transferred from one side of the system to the other as is currently accepted. When that is confirmed, the rate of superoxide formation can be measured by setting up the assay as described with the addition of superoxide dismutase to reduce the O2

- to H2O2, this reaction can then be directly monitored by the change in colour of Amplex Red which reacts directly with H2O2. Another minor change was to the original assay proposal, it was stated in the proposal that we were to use octanoyl CoA and its dehydrogenase but instead we used glutaryl CoA and its dehydrogenase as either work for our assay. This is because the acyl-CoA and its dehydrogenase were to be used to transfer electrons to ETF and downstream thereafter, thus as long as the acyl-CoA could be dehydrogenated by an acyl-CoA dehydrogenase and pass electrons to ETF then it is irrelevant which acyl-CoA they came from.


Unfortunately due to time constraints a complete superoxide formation assay was not established. This was due to the fact ETF wasn’t purified in time and the sample of ETF provided by Dr. Watmough had unfortunately degraded.

Value of studentship to myself

I believe this experience will prove invaluable to organising my third year project as it allows me firsthand knowledge of techniques that I can use for my project. Also the studentship has better informed me on what it is like to work in the research lab and has prompted me further into pursuing a career in research. I appreciate better the amount of hard work that goes into producing publications. The experience has given me a summer I will never forget.

Value of the studentship to the lab

I believe the value the studentship had on the lab was that they could use someone of my samples for further research, primarily with that of a new PhD student starting in October. Also the most successful methods I found for doing particular tasks can be passed onto a new PhD so that they are able to get a head start when reproducing methods to ensure the desired outcome is more probable.

The Biochemical Society Summer Vacation Studentship 2010 Report

Student: Karishma Asiani (Supervisor: Dr. Rajnikant Patel)

Project title: Examination of the intracellular localization of Pds5a in the mammalian cell cycle using live cell imaging

Description of Work Carried Out/ Results:

In order to understand the localization of the fluorescent Pds5a protein, HeLa cells were transfected with either the wild type or mutant Pds5a, and then fixed 24 hours after transfection. Following fixation and staining the HeLa cells were observed under a fluorescence microscope. The DNA was stained using Hoechst 33342 (blue), and the microtubules were stained with an α-tubulin antibody (green) In addition, cells were also stained with an antibody against native Pds5a, in order to determine its intracellular localization. Initially, the results were not very positive. This is because the transfection efficiency and/or expression of the Pds5a proteins (wild type and mutant) were low. I attempted to optimize the transfection efficiency by using different concentrations of plasmid, different transfection reagents and various transfection times. However, both the expression level of the Pds5a proteins and the transfection efficiency remained relatively low. Nevertheless, I did manage to obtain some good data with valuable results. Upon analysis, the results for both the native Pds5a and wild type M-Cherry Pds5a showed that Pds5a was present primarily in the cell nucleus during the cell cycle (Figure 1A and 1B). The fact that these results match tells us that the expressed full-length construct is localizing in the same manner as native Pds5a, hence the N-terminal fluorescent tag was not interfering with protein localization. The data obtained for the mutant M-Cherry Pds5a illustrates how this variant of the protein delocalizes (Figure 1C). This result indicated that the C-terminus, containing the DNA hooks, was important for correct protein localization.

Summary of Project Description/ Background:

Precocious dissociation of sisters 5 Homolog A (Pds5a) is thought to be a sister chromatid cohesion protein, that regulates the maintenance of cohesin - the protein complex which is responsible for binding sister chromatids during the S phase, G2 phase and into M phase of the cell cycle. Cohesin is composed of four core components, which are: Smc1, Smc3 (members of the Structural maintenance of chromosomes family), Scc1 (a member of the kleisin protein family) and Scc3 (SA1 and SA2 in vertebrates). These have been suggested to form a ring-like structure to hold the sister chromatids together until the Scc1 subunit is cleaved by the protease separase during mitotic metaphase, allowing sister chromatid separation and the onset of anaphase. Correct chromosome segregation during mitosis is vital for maintaining genome integrity in eukaryotic cells. Errors in the process can result in aneuploidy and ultimately lead to the development of cancer or birth defects. The study of the localization of Pds5a should enable us to understand the function of Pds5a in greater detail.

Project Aims and Objectives:

The aim of this project was to generate two fluorescent-Pds5a (M-Cherry, a GFP variant) mammalian expression constructs (one wild type - full length and the other a mutant - truncated Pds5a, lacking the C-terminus) for transfection in HeLa cells. The cells expressing the chimaeric protein would be monitored using live cell imaging, allowing the determination of the intracellular localization of Pds5aand its effect (if any) on the cell cycle.

The Biochemical Society Summer Vacation Studentship 2010 Report

Departures from Original Proposal:

No significant departures were made from the original plan. However, over-expression of the Pds5a protein (Myc-tagged Pds5a) was carried out to observe any affects on its localization, during the cell cycle. Nevertheless, due to the limited time available for this experiment, insufficient data was obtained in order for the results to be discussed.

Figure 1.Immunofluorescence Image Showing Localization of Native Pds5a (A), Wild Type M-Cherry Pds5a (B) and Mutant M-Cherry Pds5a (C)

Cells were also co-stained with anα-tubulin antibody (green) and Hoechst 33342 (blue).

Scale Bar: 10µm

Value of Studentship to the Student:

This Studentship has presented a real taste of life in a working Research Laboratory. It has been an extremely stimulating placement whilst also proving to be very rewarding and beneficial, by providing numerous skills such as: experimental design, optimising experiments, troubleshooting, data analysis, presentation skills and good laboratory practice in general. This firsthand experience has also allowed the discovery of the necessities of patience and time management required in a lab-based environment. The Studentship has been a fantastic opportunity on the whole and a confidence boost for my desire to carry out a PhD and eventually obtain a Biomedical Research based occupation.

Value of Studentship to the Laboratory:

The localization of Pds5a has permitted us to understand its intracellular localization and its role in regulating sister chromatid cohesion. Further analysis of these biochemical events should increase our understanding of Pds5a and hence, aid in understanding the mechanism of chromosome segregation in human cells.

Future Directions for the Project:

Live-cell imaging can be carried out to follow the localization of Pds5a throughout the different stages of the cell cycle, following further optimization of transfection efficiency. Over-expression of the Pds5a protein (Myc-tagged Pds5a) will also be carried out to observe its localization during the cell cycle.

Figure 1. An antibody against human drebrin cross-reacts with fly drebrin. Immunoblot of rat neonatal brain proteins (R) and fly larva proteins (F) probed with an antibody against human drebrin. Left-hand tracks are x1.5 amount of protein in right-hand tracks.

Biochemical Society Summer Studentship 2010

Student: Katie Mooney

Supervisors: Professor Phillip Gordon-Weeks and Professor Guy Tear

Department: MRC Centre for Developmental Neurobiology, King’s College London

Understanding the function of the vertebrate F-actin-binding protein drebrin by studying the Drosophila homolog

Introduction

Drebrin is an actin-binding protein that has important roles in neuronal development and synaptic plasticity in the adult nervous system but its molecular mode of action is unknown. Drebrin couples filopodial actin filaments to dynamic microtubules in growth cones by binding directly to the +TIP protein EB3 (Geraldo et al., 2008). Dominant-negative and knockdown experiments have shown that the interaction between drebrin and EB3, and hence dynamic microtubule-actin filament coupling, underlies neuritogenesis and axon growth (Geraldo et al., 2008). Recently, drebrin homologs have been identified in C. elegans (Wang et al., 2008) and Drosophila (unpublished observations). The homology extends across the molecule but is particularly high in the N-terminal ADFH domain and the proline repeat domain. This opens up the possibility of using the tractable genetics of these model organisms, particularly the fly, to understand drebrin biochemically, which will form the basis of my project.

Aims

The aim of this project is to determine the expression and distribution of the fly drebrin homolog. In addition, I will be experimenting with fly strains that harbour P element insertions or chromosome deletions which might interfere with the transcription of drebrin and hence be potential loss of function alleles.

Description of Work

I will be screening drebrin antibodies whose epitopes are conserved between vertebrates and fly to identify species cross-reacting antibodies. For example, rabbit polyclonal anti-drebrin (Abcam ab11068) was raised against amino acids 22-42 of human drebrin and this region has 7 identical and 10 homologous amino acids in common with fly drebrin. The antibodies will be screened by immunoblotting and immunofluorescence. I will also be performing in situ hybridisation experiments with fly embryos, using drebrin homolog riboprobes, to give a visual perspective of where and when drebrin is expressed in developing Drosophila embryos. In addition, I will have the opportunity to learn PCR techniques in order to make RNA probes that will be used for in situ hybridisation. Results

Proteins extracted from fly larva and rat neonatal brain, as a control, were separated by SDS PAGE and immunoblotted with a rabbit polyclonal antibody against human drebrin. As expected, a single band of approx. 130 kDa, corresponding to drebrin, was seen in rat brain (Fig. 1). Drebrin runs anomalously high in gels since its predicted molecular weight is 95 kDa. Fly drebrin has a lower predicted molecular weight than rat drebrin and so it was interesting that the rabbit antibody also recognised a single band in fly larva of approx. 100 kDa (Fig. 1).

In situ hybridisation of fly embryos was preformed using an RNA probe (GC10083) made by the addition of a

T7 RNA polymerase consensus site to a PCR product. In wild type embryos there was clear expression of drebrin RNA in the central nervous system at various stages of Drosophila development, in both brain the lobes and the ventral nerve cord (Fig. 2). Towards the later stages of development, expression steadily decreased, indicating that drebrin might be down-regulated towards the end of embryonic development as it is in vertebrates.

In embryos with a deficiency for the drebrin gene there was no labelling after in situ hybridisation but labelling of embryos with a P element insertion in the promotor region of the drebrin was similar to wild type.

Wild type embryos labelled with the anti-drebrin antibody showed a distinct fluorescence in the brain and ventral nerve cord of the CNS, in confirmation of the in situ hybridisation results (Fig. 3). The labelling of the deficiency and the P element insertion were not successful enough to make any strong conclusions.

Future Objectives

Future experiments will include the determination and understanding of the function of the P-element insertion by phenotypic analysis of fly lines. Also repeating the in situ experiments and immunofluorescence with the deficiency embryos from a fly line balanced over a marked balancer to identify the homozygote embryos and confirm fly drebrin expression.

Value of Studentship

My summer studentship was a fantastic experience. To have the opportunity to work with some brilliant people in a world recognised research centre was great. The summer programme has allowed me to develop an extensive range of laboratory skills that will prove invaluable for the future. To have the opportunity to perform some fascinating experiments – particularly the in situ hybridisations was thoroughly enjoyable. Not only was this a great experience to broaden my skills within the laboratory, it also allowed for development of my problem solving skills as well as improving my ability to solve complicated calculations in reference to making the appropriate solutions. Furthermore, having the opportunity to take part in laboratory meetings with various members of the department to discuss my experiments and results gave a great boost in my confidence. I am very grateful for the opportunity and it has further pushed my aspiration for a career within scientific research. References

Geraldo, S., Khanzada, U. K., Parsons, M., Chilton, J. K. & Gordon-Weeks, P. R. (2008). Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis. Nat. Cell Biol., 10, 1181-1189.

Figure 2. Drebrin is expressed in the fly CNS. In situ hybridisation of stage 13 fly embryos with a drebrin ribroprobe. Cells, probably neurons, in the ventral nerve cord (arrowheads) and in the brain lobes (arrow) are labelled. a) ventral view, b) lateral view. Anterior is to the left.

Figure 3. Drebrin is expressed in the fly CNS. Fluorescence images of stage 14 fly embryos immunolabelled with rabbit antibody to human drebrin. Cells, probably neurons, in the ventral nerve cord (arrowhead) and in the brain lobes (arrow) are labelled, as are cells in the gut (asterisk). a) ventral view, b) lateral view. Anterior is to the left.

Wang, W., et al. (2008). ITSN-1 controls vesicle recycling at the neuromuscular junction and functions in parallel with DAB-1. Traffic 9, 742-754.

Student: Kittiphat Chanthong Supervisor: Dr Jeremy Brown Institution for placement: Newcastle University Biochemical Society summer studentship report 2010 Purification of pre-SRP complexes using RNA-aptamers

Background The signal recognition particle (SRP) is a ribonucleoprotein that recognises the hydrophobic sequence on the N-terminal of membrane and pre-secretory proteins during their translation and targets them to the endoplasmic reticulum. The complex itself is mainly assembled in the nucleolus within the nucleus. Studying the biogenesis of SRP in yeast by work in the laboratory has determined that it is subjected to quality control. Several conditions have been identified under which biogenesis can be blocked, including overexpression of La protein, a protein that binds to RNA polymerase III products, or depletion of complexes involved in processing of nuclear RNA such as TRAMP and the exosome1. These conditions lead to nuclear accumulation of the pre-SRP complexes.

The purification of specific RNAs can be achieved by modifying them such that they include aptamers, short sequences of RNA that binds tightly to specific molecules. One such sequence, the ‘S1-aptamer’ binds tightly to streptavidin, and can also be dissociated from it by competitive binding using biotin2. Using this method, the approach to be taken in this project was to purify the pre-SRP complexes and associated proteins accumulated in the nucleus when biogenesis was disrupted, to provide an insight to the biogenesis and quality control processes of SRP and its RNA.

Aims

1. Purify pre-SRP complexes from samples obtained from Saccharomyces cerevisiae using variants of scR1 that includes the S1-aptamer that binds to streptavidin.

2. Study the purified pre-SRP complexes and any associated proteins using mass spectrometry.

Departure from the original protocol At the start of the project, a yeast strain lacking the endogenous copy of the SRP RNA gene (SCR1) but containing instead copy on a plasmid with the URA3 auxotrophic marker, was separately transformed with three different plasmids. These plasmids contain SCR1 with the S1 aptamer inserted at different positions corresponding to loops of the secondary structure of the RNA. The URA3-marked plasmid in the strain was removed by plasmid shuffle, using a counter-selection (5-fluoro-orotic acid) leaving the tagged version as the only copy of SCR1. Attempts to purify the tagged RNA from extracts of these cells were unsuccessful. The aptamer present in the initial constructs was slightly abbreviated over the full-length S1 aptamer. Though published information suggested that it should be fully functional, the protocol was reviewed to include following objectives:

1. Purification of pre-SRP complexes using MS2-GFP tag instead of RNA-Aptamer 2. Imaging of yeast strains that expresses MS2-GFP tagged SRP complexes under conditions that blocks

biogenesis of SRP complexes

Work carried out Cell Imaging: The MS2 phage coat protein binds to a specific short hair-pin motif, and has been used extensively for RNA localisation and purification. A yeast strain was available in the laboratory containing a modified SCR1 that included MS2 binding sites and which was known to bind an MS2-GFP fusion protein, which was co-expressed in the strain. This strain was transformed with a multicopy plasmid containing LHP1, the yeast La gene, that thereby allows over-expression of Lhp1p or a control empty vector. These strains were then analysed by fluorescence microscopy. The DNA of the cells was stained with DAPI, providing a marker for the nucleus. Cells over-expressing La revealed a strong nuclear accumulation of GFP, indicating that not only was the MS2-GFP fusion able to bind the tagged scR1, but it was able to enter the

a)

b)

Figure 1 Overexpression of Lhp1p leads to nuclear accumulation of scR1. Cells of a yeast strain containing both a modified scR1 with MS2 binding sites and GFP-MS2 transformed with either an empty vector (a) or a multi-copy plasmid containing LHP1 (b) were grown, fixed and analysed by fluorescence microscopy. DAPI reveals the localisation of DNA, GFP fluorescence the SRP RNA.

DAPI GFP Merge

nucleus and bind to the RNA there. This accumulation was not seen in cells that are transformed with control plasmid (fig.1), and neither did MS2-GFP accumulate in the nucleus of cells lacking MS2-tagged scR1 (not shown).

SRP Purification: An attempt was made to purify SRP using the MS2-GFP fusion by immunoprecipitation using anti-GFP antibodies. This initial experiment was carried out using cells expressing MS2-GFP and the MS2-tagged SRP RNA, alongside controls lacking either one of these components. Immunoprecipitated material was resolved on an SDS-PAGE gel, blotted to a nitrocellulose membrane and probed with anti-GFP antibodies as well as anti-SRP antibodies. The MS2-GFP fusion protein was detected from the western blot obtained from the experiment. However, SRP proteins were not detected on the blot.

Analysis of results and Outcomes

Successful imaging of GFP-MS2 associated with pre-SRP in the nucleus indicates that the MS2-tag strategy is appropriate for purifying these complexes. Anti-GFP antibodies should provide a good way to purify them, as these antibodies are highly specific.

Future Directions

Now that the images of live cells over-expressing Lhp1p are obtained and showed nuclear accumulation of scR1, the conditions can be altered to see the effect of over-expression or depletion proteins that are involved in quality control processes of nuclear RNA, such as depletion of Rrp44p. As for purification of pre-SRP complexes, construction of scR1 variant with full length RNA-aptamer should be carried on and test whether this is successful as RNA-aptamers would provide us with a simple tool to study proteins involved in biogenesis and quality control of ribonucleoproteins.

For the purification of SRP, an alternative would be to use a different MS2 fusion protein, such as a TAP-tagged MS23. The Tandem affinity purification tag is another tried and tested route for purification of complexes from cells, and a TAP-tagged MS2 has now been obtained for this work to proceed in the future. Other work in the laboratory has revealed that SRP proteins are not associated with the pre-SRP that accumulates in yeast cells over-expressing La (Jeremy Brown unpublished). That GFP-MS2 can bind to it indicates that the RNA is not sequestered from all proteins, and it is likely that the RNA is at a very early stage on the biogenesis pathway. Identification of proteins that are associated with it will likely be very informative on early steps in SRP, and possibly other RNP assembly.

Value of the Studentship to the Student:

This summer studentship has given me an opportunity to have a first-hand experience of how research is carried out. I gained many valuable and essential laboratory skills, as well as learning that the outcome of an experiment can be affected by a very small difference or mistake. I have become much more confidence as a scientist and this project has confirmed my desire to continue my career in research. I have had a wonderful time in the laboratory, and am very grateful for helps and encouragements from Dr Jeremy Brown and his PhD students during my time there.

Value of the Studentship to the Lab:

The work carried out, though not what was initially aimed for, has been important, and images obtained by Kittiphat will be used almost immediately in a publication that we intend to submit this year. Kittiphat will be acknowledged appropriately for his contribution.

References

1. Leung, E. and Brown JD. (2010) Biogenesis of the signal recognition particle. Biochemical Society Transactions. 38:1093–1098

2. Srisawat C., Engelke DR. (2001) Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins. RNA. 7(4):632-41

3. Gavin AC, Bösche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Höfert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature. 10;415(6868):141-7.

Report on Biochemical Society Summer Vacation Studentship 2010 Student: Kong Ho Tang @ University of Oxford Hosting Laboratory: Department of Biochemistry,

University of Oxford South Parks Road Oxford OX1 3QU

Project Proposer & Supervisor: Prof. Jane Mellor Bench Supervisor: Dr. Ivan Boubriak

Research Project Title: A study of the domains of Spt7 that influence longevity in the yeast Saccharomyces cerevisiae.

Background information to the project

Approximately 10% of genes that are transcribed in budding yeast are dependent on the presence of a protein called Spt7, which is an important integral component of a multi-subunit complex called the SAGA complex. The SAGA complex plays key roles in balancing growth and longevity in S. cerevisiae and the complex is regulated by Spt7 and Sgf73, both of which are acetylated by Gcn5, a lysine acetyltransferase that is also present within the SAGA complex. While Spt7 represses the Gcn5 activity, Sgf73 activates the acetyltransferase activity of Gcn5.

Spt7 is acetylated at the C-terminal region and an acetyl-lysine interaction domain surrounded by a bromodomain near the N-terminal region. Both these domains may interact, as suggested by previous experiments where separate expression of the C-terminal and N-terminal regions have both resulted in the suppression of spt7∆.

Aims of my project

The primary goal of my project is to understand how Spt7 regulates the activity of the SAGA complex, by testing whether the bromodomain of Spt7 interacts with the acetylated C-terminal region. The biochemical characterization of Spt7 and its associated Gcn5 acetyltransferase involves a series of experiments:

1. Creation of FLAG-tagged/His6-tagged C-terminal region of SPT7 (wild type and various deletion mutant forms) and the GCN5 acetyltransferase in E.coli. (The TAP-tagged N-terminal Spt7 constructs are previously prepared in the lab)

2. Expression and purification of the two Spt7 domain constructs and Gcn5 using affinity chromatography.

3. In vitro acetylation of the two domains using the purified Gcn5.

4. Interactions assay for the ability of the two domains to interact in a pull-down experiment using anchored TAP-tagged Spt7 N-terminal domain & examine the potential interaction partners with the FLAG-tagged Spt7 C-terminal domain constructs.

There are no major departures from the original proposal, however the original aim of the studentship could not be realized. Most of the Spt7 constructs were expressed near to the final stages of the summer studentship. In spite of this success, there were problems with PCR amplifying the Gcn5 acetyltransferase, which although was later resolved, the enzyme was not expressed due to time constraints. Consequently, all the subsequent downstream experiments such as in vitro acetylation and interaction assays could not be performed, which was a slight disappointment.

Account of Experimental Procedures & Results

1. Creation of protein constructs for C-terminal Spt7 domains and Gcn5 (Wildtype and mutated versions). This entails:

i) Designing PCR primers to amplify the C-terminal regions of Spt7, along with the Gcn5 acetyltransferase. Various additional PCR primers were also designed to introduce deletion mutations in both the N and C-terminal Spt7 constructs and Gcn5 via PCR-mediated mutagenesis. The PCR amplification of Spt7 and Gcn5 genes were successful, resulting in 6 different plasmids (4 for Spt7 C-terminal constructs and 2 for Gcn5 WT and Gcn5 with bromodomain deletion).

ii) Ligation of PCR insert to entry cloning vector (e.g. pJET1.2 from Fermentas) and subsequent bacterial transformation (cloning and sub-cloning) into chemically competent bacterial cells and subsequently into expression bacterial cells (T7 E.coli); employing techniques such as: Plasmid DNA Mini-prep; Agarose Gel Electrophoresis; Gel extraction; Standard sterile microbiology techniques.

iii) Verification of new recombinant vector via restriction enzyme digestion of plasmid DNA miniprep and then subsequent DNA sequencing using T7 promoter sequencing primers.

iv) Bioinformatics of DNA sequencing data.

v) Creation of glycerol stocks for successful recombinant clones.

Personal value of this studentship

I have thoroughly enjoyed working in a professional research environment. The interdisplinary nature of my summer studentship has provided me a vast array of new insights, experience and learning opportunities in biochemical research. The studentship also allowed me to go beyond the limits of following protocols (as in most undergraduate lab practicals), but to have some first-hand experience and more academic freedom as real research scientists: to design experiments that incorporates a much wider range of practical techniques (from designing PCR primers for amplifying target genes to the optimization of the final protein of interests), designing appropriate control experiments, documentation of all the experimental protocols performed and results, time management skills, trouble-shooting skills when problems arises with experiments, and not to mention interpersonal skills with other members of the lab. Overall, I believe that these experiences during my studentship have all contributed a new dimension of my perspective of what it would be like to work in a professional biochemical research laboratory and this studentship has definitely inspired my decision towards a PhD and beyond in the biochemical field.

Future directions in which the project can be taken

1. Resume work on the optimization of expression and purification of the Spt7 constructs and Gcn5 protein to increase their yield (in E.coli and subsequently in budding yeast).

2. Transform budding yeast with these constructs

engineered to express Spt7 N-terminal and C-terminal constructs in a spt7 null background.

3. Site-directed mutagenesis on the key residues

of the bromodomain and express these construct to assess the effect of these mutations for interactions with the Spt7- C-terminal region.

Research value of this studentship to the lab

Although the original aim of the studentship was yet to be accomplished and yielded limited data, the endowment of my preliminary research efforts in cloning the various Spt7 constructs and Gcn5 acetyl-transferase recombinant cells (wildtype and mutants) that I have made during this studentship will undoubtedly accelerate the pace of research output for this lab. All these endowment will be analyzed in further downstream Spt7 characterization experiments to continue addressing the same question: how Spt7 is involved in the regulation of the SAGA complex in budding yeast, which is fundamentally linked to various pathways correlated with longevity in budding yeast and remains a core epigenetic research interest in this lab.

Conclusion

Wildtype Spt7 constructs, Gcn5 acetylases and their mutant variants were successfully cloned and ligated into pIVEX2.3 bacterial expression vector (Roche Applied Science). These recombinant DNA were then transformed into expression cells

Both N-terminal and C-terminal Spt7 protein constructs were over-expressed in E. coli Top10 (invitrogen) or GC5 (fermentas) chemically competent cells, sub-cloned into expression E. coli cells, and then purified successfully using a column with affinity to the His-tag, as well as analysing soluble and insoluble fractions.

2. Expression and Purification of the C-terminal Spt7 constructs using His-tag

Two of the recombinant His-tag Spt7 C-terminal constructs was purified from an IPTG-induced bulk cell culture of E. coli cell lysate using an affinity chromatography column to the His-tag. The fractions are eluted with 40mM, 60mM and 200mM Immidazole.

The protein constructs eluted from the column were analysed by SDS-PAGE and Western blots, which confirmed good purification of all recombinant C-terminal End Spt7 proteins. Purified proteins kept at -80 oC for future downstream analysis.

Due to the bands' intensity, further optimization of induction conditions for various recombinant expression bacterial cell lines are required to yield a sufficient amount of protein for use in downstream pull-down assays of the Spt7 N-terminal and C-terminal constructs.

Western Blot to detect His6-tag proteins in purified fractions from His6-tag affinity chromatography column.

The Western blot of the purified fraction had generated an interesting result: although there were no bands at 40kDa (the expected size for our Spt7 C-terminal constructs), instead there are intense bands near the 80kDa regions. This result is also observed with some of our other Spt7 C-terminal constructs. These observations may suggest that the Spt7 C-terminal region may homodimerize and this may have further in vivo functional implications. However, more rigorous experimental evidence will be required prior any valid conclusions can be drawn.

Student: Liuhao Wu Supervisors: Dr Klaus Okkenhaug, Verity Dale Institution for placement: Cambridge University


Project Title: How Listeria monocytogenes exploit PI3K during infection of macrophages. Aims

1. Culture macrophage from bone marrow derived monocytes

2. Determine if PI3K-deficient (p110δD910A) macrophages are resistant to infection with Listeria monocytogenes.

3. Investigate the effects of PI3K inhibitors on macrophages infected with Listeria monocytogenes.

4. Exploring ways of imaging intracellular Listeria monocytogenes in macrophages.

Project Background

Listeria monocytogenes is a gram positive food-born pathogen that causes listeriosis. Its detrimental effects on the host allows it to be classified under Biohazard Level 2. Listeria sp. infect macrophages and reproduce in the cell by escaping the phagosome. Our main goal is to understand how Listeria interacts with macrophages, in particular how the catalytic subunit p110δ of Class IA Phosphatidylinositol 3-kinases (PI3Ks) contributes to bacteria invasion. PI3Ks are intracellular lipid kinases that phosphorylate PIP2 into PIP3.

PIP3 then recruits proteins containing a PH domain which leads to their activation and initiation of signalling pathways, causing cellular functions such as proliferation and motility.

Phagocytosis of Listeria monocytogenes by

macrophages involves PI3Ks and previous experiments showed that mice harbouring a mutation of PI3K (p110δD910A) contained less CFU (colony forming units) of Listeria 4 hours post infection than wild type mice (WT). This project investigates the role of p110δ in Listeria infected macrophages. The work will complement the understanding of how pathogens invade the immune system and help the development of new treatments. Departures from original proposal

Initial results for p110δD910A macrophages infected with Listeria were inconclusive and due to time restraint, repeats were not carried out. As a result the data are not included in the report. Other than that, there were no major deviations from the initial plan.

Bone marrow derived macrophage culture

Part of the project

Figure 3. FACS staining Macrophages stained F4/80 and CD11b positive

Figure 2. PI3K activation mechanisms Antigens on Listeria activate macrophage receptors. This triggers a chain reaction that results in PI3K activation. PIP2 is then phosphorylated to PIP3. This recruits proteins that are involved cell activities.

Figure 1. Listeria infection Listeria is able to survive in macrophages unlike many other bacteria

required me to culture macrophages; this was done under sterile conditions. Mouse bone marrow was acquired from the femur and cultured in macrophage medium containing L-cells. After 3 days of incubation at 37°C, the non-adherent monocytes were collected and diluted in fresh macrophage medium. These were incubated further for 3 days to allow the monocytes to differentiate into macrophages.

The macrophages were then removed and transferred into a 24 well plate. FACs was carried to confirm the phenotype of the cells. Anti-mouse F4/80, CD11b and CD11c antibody were used to identify macrophage. Imaging intracellular Listeria monocytogenes

Numerous methods were tried to allow the Listeria to be detectable under a microscope. Initially we tried to make fluorescent Listeria by introducing plasmid containing RFP (red fluorescent protein). Next we tried identifying the bacteria by using Listeria specific antibody tagged to a fluorophore. Both methods gave poor results. In the end we decided to use CFSE (Carboxyfluorescein succinimidyl ester) labelled Listeria. This will allow us to visually determine the effect of PI3K inhibition.

The process involved incubating the bacteria in CFSE solution for 15minutes. After incubation, we infected the macrophages with the labelled bacteria. 1 hour post-infection, extracellular bacteria were killed using gentamicin. We then analysed the macrophage under a fluorescent microscope. The intracellular Listeria could be seen within the macrophages. This method was established and can now be used to assess any differences between macrophages with inactive and active p110δ.

Drug induced inhibition of PI3K

Macrophages were cultured from bone marrow cells under sterile conditions. They were then pre-treated with 3µM of IC87114 (selective p110δ PI3K inhibitor) for 1 hour. After which, the macrophages were infected with Listeria monocytogenes (MOI of 2). At 1h and 4h post-infection, extracellular bacteria were killed by adding gentamicin. The macrophages were lysed using distilled water to release the phagocytosed intracellular bacteria. The number of bacteria was analysed by counting the number of bacterial colonies on an agar plate.

Appropriate controls were done to ensure the data acquired are valid (data not included).

The result showed that the number of intracellular bacteria increased over time. This is probably contributed by both an increased bacterial entry and replication over time. In addition, the number of bacteria in IC87114 treated macrophages was significantly less than untreated cells. We hypothesise that the inhibition of PI3K reduced the

phagocytic ability of the macrophages. However it is also possible that PI3K inhibition caused an increase in bacterial killing.

Future directions in which the project can be taken

How PI3K deficiency affects intracellular spreading of Listeria can be analysed by infecting the macrophage with fluorescent tagged Listeria. We should hopefully see actin polymerisation and bacteria movement between the macrophages. In addition, the ability to visualise the bacteria will allow us to determine if the decreased number of bacteria in IC87114 treated cells are due to a decrease in entry or an increase in killing of intracellular Listeria.

Value of the studentship to the student

I found this project extremely engaging and educational. The project has complemented my existing understanding of the immune system and taught me many skills such as presentation, communication and teamwork. In addition, I was introduced to new laboratory techniques and safety protocols which will be invaluable to me in future projects. Despite the enjoyment, there were challenging times when obstacles were met. However this further developed my problem solving skills; refining protocols and planning experiments. This experience will definitely help my laboratory project in pathology next year.

Value of the Studentship to the Lab

Liuhao is a conscientious and diligent student and we have high confidence in the experiments he carried out. Some of the techniques were new to the lab and turned out to be more challenging than initially anticipated. This did not discourage Liuhao and he thought independently about ways to improve the published protocols to obtain more

Figure 5. Colony numbers in drug treated and untreated macrophage The number of colony forming unit (CFU) was counted on the agar plate after 1h and 4 h post-infection. The data shows that IC87114 treated macrophages supported less intracellular bacteria than WT at 1h and 4h. The number of colonies also increased over time.

Figure 4. Fluorescent Listeria CFSE labelled Listeria is stained green within the macrophage

*P<0.05

reliable and reproducible results. As a consequence, he has helped kick-start a research area that the lab will continue to invest in.

Biochemical Society Studentship Lab Report Student: Luke C Dabin Supervisor: Dr Tim Dafforn, Biosciences, University of Birmingham

How does the protein ZipA mediate the interaction between the FtsZ fibre and the membrane during bacterial cell division? Background: ZipA is an actin homologue involved in the formation of the divisome – a protein complex responsible for drawing the membrane inwards during bacterial cell division. ZipA plays two crucial roles in this process: it forms the link between the tubulin-like FtsZ cytoskeleton and the cell membrane, as well as providing a scaffold for recruitment of other divisome proteins. Dr. Dafforn’s lab uses a novel biotechnique to excise membrane discs from bacterial cells, complete with associated membrane proteins. This allows study of the protein in situ in the membrane without the use of detergents. This project was designed to investigate the properties of ZipA within the membrane and its interaction with FtsZ.

Figure 1. Order of divisome protein recruitment during division ZipA is among the first of the components to be recruited, and is required for formation of the Z-ring cytoskeleton. (Margolin W (2005) FtsZ and the division of prokaryotic cells and organelles, Nature Reviews Molecular Cell Biology 6, 862-871)

Aims: 1. Produce His-tagged ZipA via over-expression of zipA gene in E. coli, monitoring presence and purity of

protein in bacteria by SDS-PAGE and Western Blotting. 2. Purify ZipA from lysed bacterial membranes via SMALP (SMA-Lipid Particle) technique, via affinity

chromatography and size-exclusion chromatography. 3. Obtain ZipA sample of high purity and concentration for subsequent analysis, such as Circular

Dichroism (CD), analytical ultracentrifugation (AUC), X-Ray crystallography etc. 4. Reconstitute ZipA proteoliposome from SMALPs and observe GTP-FtsZ-ZipA induced membrane

morphology change via confocal microscopy. Alterations to original proposal: FtsA was studied alongside ZipA, FtsA being a less well-characterised protein with a similar role and structure to ZipA. Moreover difficulties during purification meant the later aims were untenable in the allotted time period. However, this allowed the purification protocol to be optimised and as a result efficiency was greatly increased. Incubation of E. coli & Harvesting of ZipA/FtsA Expression of plasmid-contained zipA/ftsA genes in 800ml cultures of BL21(DE3) pre-transformed E. coli was induced with 0.1mM IPTG after an optical density of 0.6-0.8 was reached. Timescale sampling showed that after 2 hours Western Blot signal was at maximum, whereas leaving the cultures to induce overnight at 25oC produced a greater final mass with minimal protein degradation. Purification was also optimised, 60g lysed E. coli yielding a 3 mgml-1 ZipA solution. Total harvested ZipA bacterial mass was over 120g, with typical centrifuged pellets weighing 16-20g if induced for 3 hours, and 25-30g if left overnight.

Biochemical Society Studentship Lab Report Student: Luke C Dabin Supervisor: Dr Tim Dafforn, Biosciences, University of Birmingham

Analysis of ZipA ZipA SMALPs were analysed using CD and AUC. Whilst the AUC results were poor due to over-dilution of the sample, the CD results were more promising. The data indicates that ZipA is correctly folded within the SMALPs – CD can also be used to monitor conformational changes to the FtsZ cytoskeleton induced by ZipA in later experiments.

Future experiments: ZipA crystals are currently being seeded for further analysis. The lack of FtsA in the lysed membranes indicates it may be contained within the cell cytoplasm, but attempts to purify this fraction resulted in possible leaching of Nickel ions by E. coli metallophores, an idea supported by the loss of colour in the resin after the first elution. FeSo4 can be added to the SMALP-Resin binding mixture to outcompete these metallophores and permit binding. Value of studentship to student: This placement has afforded me experience that will greatly complement my imminent 3rd year project on X-Ray crystallography. The training in routines and scientific practice will be a huge benefit to me in the lab. Discussions with Dr. Dafforn and Dr. Jamshad during my project helped me affirm my desire to pursue research as a career and I intend to apply to a postgraduate course relating to clinical biochemistry. Value of studentship to laboratory: The work I have undertaken has culminated in an effective and simple protocol to produce and extract ZipA from E. coli, as well as excluded several methods of harvesting FtsA. These proteins are key elements of other projects in the lab. Academics from Warwick University have visited the Dafforn lab and been trained in SMALPing technique by me; I later purified and analysed membrane protein samples from the visiting lab. In this way both Warwick and Birmingham universities have benefited.

M N 2 1 0 N 2 1 0 Figure 2. Western Blot of FtsA induction Sample times: 0h, 1h, 2h and the next day (from right to left)

Figure 3. Coomassie stained gel of affinity column elutions Binding of ZipA-SMALPS to the Nickel resin was improved over the project course – initially all tagged protein was present in the flowthrough. At this stage it is clearly visible in fractions corresponding to 100-300mM imidazole elutions. However, at the end of the project FtsA-SMALPs were still not binding to the resin.

Figure 4. CD spectrum of ZipA SMALPs

Peptide groups absorb between 190-250nm, positive/negative CD transitions at 200nm and 225nm indicate the presence of α-helices.

200nm 225nm

Student: Manuela Lehmann Supervisor: Heinz Peter Nasheuer Biochemical Society Summer Vacation Studentship 2010 – Project Report Student: Manuela Lehmann

Lab: Department of Biochemistry, National

University of Ireland, Galway, University Road, Galway, Ireland

Supervisor: Dr. Heinz-Peter Nasheuer, Head of

School of Natural Sciences and Senior Lecturer

PhD supervisor: Irina Tikhanovich

Title of Research: RNA binding activities of human

primase and BKV large T-Antigen

Introduction: DNA polymerases are not able to start the DNA synthesis de novo and need a specific enzyme, primase, to synthesize short RNA primers for the initiation of the DNA replication (1). In eukaryotes the DNA polymerase α use these short templates to elongate the new synthesized DNA strand (2). Human primase consists of the subunits p48 and p58 and forms a four-subunit enzyme complex with DNA polymerase α. According to earlier studies, p48 has a primase activity which becomes stabilized in the heterodimer complex with p58 (1, 2). In this project we wanted to study the binding activity of the complex p58-p48 to RNA dependent on different conditions. For comparison, the binding activity of BK virus large T-Anti-gen (BKV TAg) was tested under the same conditions. TAg is an essential enzymatic viral protein for DNA replication of Polyomaviruses such as simian virus 40 (SV40), JC virus and BKV. The protein is a hexamer and helps to unwind the dsDNA by binding within the core origin of the virus DNA. (3)

Description of work: To test the binding activity of human primase p58-p48 complex to RNA or DNA in vitro Electrophoretic Mobility Shift Assays (EMSAs) were carried out. Human primase was purified as described before with slight changes (2). In brief, to express human primase BL21 (DE3) LysS cells were transformed with pET 11 p48 His p58 vector and selected on LB ampicillin agar plates. One single colony was chosen and grown overnight in 200 mL LB-AMP medium. The culture was diluted 1/50 in 500 mL fresh LB-AMP medium and further grown at 37°C until the OD (Optical Density) reached 0.5-0.6 at 600nm. The expression of primase complex was induced by adjusting the medium to 1 mM IPTG. After 4h, the cells were harvested, lysed and sonicated to release the primase complex, which was purified by using a nickel affinity chromatography. The purity and identity was defined by SDS-PAGE followed by Coomasie Brilliant Blue staining or western blot analysis, respectively. The amount of primase was determined by Bradford assay.

For the binding assays multiple RNAs and DNAs were used. RNA was synthesized in vitro using plasmids containing short human and mouse Y RNAs (4) which were amplified by PCR and purified by gel electrophoresis. The amplified DNA was used as a template for transcription in vitro and the in vitro transcribed RNA was precipitated by ethanol. In addition, the RNA of clone 62 (GC-rich RNA) and a short DNA (EP-ER, 65 nucleotides) were used for the EMSA. To test at which amount of primase the binding starts different concentrations of human primase were used. In brief, increasing amounts of primase (0, 0.008, 0.017, 0.035, 0.07, 0.14 µg) were added to a constant amount of RNA (1 µg for hY RNA) in 6 samples containing EDTA-buffer. These mixtures were incubated at 37°C for 15 min, then loaded on a 2% agarose gel, separated by electrophoresis and stained with Ethidiumbromid. Beside the binding activity of primase, BKV TAg to the same RNAs and DNA were analyzed. BKV TAg was expressed and purified from insect cells as outlined before (3). In brief, High Five insect cells were infected with baculovirus encoding TAg. The infected cells were harvested and BKV TAg was purified by using immobilized Talon affinity chromatography. The purity of BKV TAg was verified by SDS-PAGE and Coomassie staining. In addition, the impact of ATP to the binding activity of TAg to RNA or DNA was studied. Therefore, the same binding assays like described before were carried out and different amounts of ATP (0; 4 mM) were added. Moreover, the C-terminal domain of the primase subunit 58CT was purified under the same conditions like the primase complex p58-p48 described before.

Results: In all EMSAs human primase complex p58-p48 showed high affinity to bind to RNA. In case of clone 62 RNA the formation of stable primase – RNA complexes start at a concentration of 0.035 µg human primase to 0.06 µg RNA. Since the concentration of primase reach 0.07 µg the entire RNA were bound in complexes (Figure 1).

Figure 1: Binding activity of human primase complex p58-p48. EMSA using 0.06 µg RNA of clone 62 and indicated amounts of human primase complex p58-p48 (0, 0.008, 0.017, 0.035, 0.07, 0.14 µg).

Using 0.54 µg RNA of the same clone 62 but transcribed in the opposite direction, 0.017 µg human primase was enough to build stable complexes. To bind the entire RNA, a concentration of 0.035 µg primase was necessary.

Mouse Y RNA, possesses nearly the same length as the human Y RNAs, were also tested for possible complex building with primase. In this EMSA, 1 µg mY RNA were used as substrate and 0.14 µg of primase were essential for visible complex formation. Testing the same RNA substrates used for binding with primase BKV TAg showed also an affinity to bind RNA. In case of clone 62 the establishment of TAg – RNA complexes started at a concentration of 0.15 µg TAg to 0.06 µg RNA (Figure 2).

Figure 2: Binding activity of BKV T-Antigen. Electrophoretic mobility shift assay using 0.06 µg RNA of clone 62 and indicated amounts of TAg (0, 0.0375, 0.075, 0.15, 0.3, 0.6 µg).

BKV TAg bound RNA of clone 62 with a concentration of 0.3 µg for noticeable complexes on the gel. For both human and mouse Y RNAs (~ 1 µg) the complexes were detectible on the gel at an amount of 0.6 µg BKV TAg. When ATP (4 mM) was added to the assay of BKV TAg and RNA, the complex formation could only be determined with higher amounts of TAg than without ATP on the gel (Figure 3).

Figure 3: Impact of ATP on the binding activity of BKV T-Antigen. EMSA using 0.1 µg RNA of clone 62 and indicated amounts of TAg (0, 0.0375, 0.075, 0.15, 0.3, 0.6 µg), in the left panel EMSA was performed without ATP and in the right panel 4 mM ATP was added to the substrate.

An EMSA with short ssDNA (EP-ER, 0.39 µg), the same amounts of TAg used in Figure 3 and ATP (0, 4 mM) was also carried out. TAg bound at a quantity of 0.3 µg to the DNA when there is no ATP added to the assay. The DNA and TAg formed visible complexes at a concentration of 0.6 µg TAg.

Discussion: Whereas it is known that TAg binds to DNA it has not previously been reported that TAg binds to RNA. We demonstrated that TAg is also able to form stable complexes with different RNAs tested. Thereby, a higher concentration of BKV TAg is necessary to bind the smaller Y RNAs than the larger RNA of clone 62. Remarkably, TAg binding is reduced since 4 mM ATP is added to the assay as shown in Figure 3.

As we tested DNA and a variety of RNA, the same results shown up for TAg binding activity. Human primase complex p58-p48 was also shown to bind RNA with high affinity. Notable, human primase binds to all verified RNAs with much higher affinity than BKV T-Antigen. These new findings were very exciting and therefore the focus of my work shifted to the RNA binding instead primase-DNA interactions.

Acknowledgements: I would like to express my sincere gratitude to Dr. Heinz-Peter Nasheuer for the given opportunity to get invaluable lab experience and help with the report. Especially I would like to thank Irina Tikhanovich for all her patient guidance, encouragement and her assistance on the bench. Also I want to thank Ronan Broderick and Aoife Corduff for their help and kindness.

References: 1. Frick, D. N., and C. C. Richardson. 2001. DNA

primases. Annu. Rev. Biochem. 7039-80.

2. Schneider, A., Smith, R.W.P., Kautz, A.R., Weisshart,

K., Grosse, F. and Nasheuer, H.P. 1998. Primase activity of human DNA polymerase α-primase. Divalent cations stabilize the enzyme activity of the p48 subunit. J Biol Chem, 273:21608-21615.

3. Mahon, C., B. Liang, I. Tikhanovich, J. R. Abend, M.

J. Imperiale, H. P. Nasheuer, and W. R. Folk. 2009. Restriction of human polyomavirus BK virus DNA replication in murine cells and extracts. J. Virol. 83:5708-5717.

4. Krude T, Christov CP, Hyrien O, Marheineke K. 2009.

Y RNA functions at the initiation step of mammalian chromosomal DNA replication. J Cell Sci. 122:2836-2845.

Value of studentship to the student: As my first experience of working in a laboratory for such a long time, the studentship experience was significant in increasing my understanding of how research is carried out and how important small details are for the success or failure of an experiment. Before I attend the internship I was unaware of the problem-solving involved in optimizing a protocol, and now I feel that I have a more detailed understanding of laboratory science. However, it was invaluable as I learnt that ups and downs are quite common in scientific research. During my internship I found both the challenge of performing experiments and the satisfaction when you get good results enormously rewarding.

Value of studentship to the lab: It was a pleasure to interact with Manuela. She worked very hard and produced various interesting findings (even more than she could present here). Some of her results I will include in my talk at the conference “Replication Meets Repair” in Jena, September 2010, where she is named as a coworker on the submitted abstract.

Student: Maria Andryieuskaya Supervisor: Tracey Melvin Institution for placement: University of Southampton

Optical trapping of embryonic stem cells.

Optoelectronics Research Centre, University of Southampton.

Summer Project 2010. Introduction When the proposal for the Biochemistry Society was written, the objective was to build a system to purify differentiated and undifferentiated embryonic stem cells (ESCs) by optical methods. But since the proposal submission the team have discovered that their optical approach allows for a more detailed assessment of ESCs and they concluded that there are separate, different sub-populations within the differentiated and undifferentiated samples. This was an exciting finding and thus the project aims changed slightly and the project involved the creation of cell arrays for the assessment of individual cells optically and then for later biochemical characterisation by immuno-staining or qPCR approaches. This approach would then allow the various sub-populations to be characterised at the single cell level.

Optical trapping methods provide an ideal tool for non-invasively investigating eukaryotic cells in suspension. The method already developed by the team was to monitor the displacement of the cell as it moves in the transversal direction into a Gaussian beam. Assuming the transversal force is predetermined by structure of the eukaryotic cell, nucleus/cytoplasm size and nucleus position in the centre of a cell, non-uniform refractive index (RI) a theoretical model to predict the effect of a Gaussian beam was developed. Fitting the experimental data the critical parameters governing cell displacement were determined to obtain values of RIs and classify cells.

In this report I present results obtained during the period of the summer project that prove applicability of the model. We list values of RIs of the germ teratoma cells NT1, which were easier cells to learn to do cell culture techniques than ESCs. And to work with and characterize cell individually a new device – an array of micro-cavities - was fabricated such that cells were placed in individual wells and the cells could be interrogated optically through the lower glass face. Materials and Methods Optical studies using cells in suspension Optical tweezers were created with a ytterbium fiber laser (IPG Photonics, 5W, λ=1064 nm). A laser beam was reflected through 900 vertically by a mirror up through an antireflection coated aspheric lens (Thorlabs C330C, N.A.=68) into the base of a chamber, providing an effective N.A.=0.4. The typical laser power used was 45-70 mW. Polystyrene beads 20 µm and 15 µm with the refractive index 1.585, the terratoma cells NT1 were used for optical trapping. Cells were grown in plastic flasks in DMEM (Gibco) supplemented with 10% foetal bovine serum, 1% Penicillin-Streptomycin at 370C in an 5% CO2 atmosphere. At confluence the cells were disadhered with Trypsine (Gibco) for 5 min at 370C, the

supernatant was collected, centrifuged at 3000 rpm for 3 min and removed. Pellet with cells was resuspended in 3 ml of DMEM and added to TrypLE Express (Gibco, RI=1.335, viscosity 1.0506*10-3 Pa*sec) in 1:5 ratio. A suspension of the cells under investigation was placed in a chamber formed with a polystyrene coverslip (Agar Scientific) modified with Pluronic F127 to prevent adhesion, a PDMS frame 1 mm deep and a polystyrene cover slip. Visualisation was realised by a CCD camera (Hamamatsu ORCA) 30 fps. Masks for arrays of microwells created were designed in CorelDraw and printed on a transparency with the highest possible resolution. Two types of masks were used: dots 150 µm in diameter/ 100 µm separating gap and dots 200 µm/ 200 µm separating gap. Fabrication of cell microarrays A lithographic mask was designed using the software L-Edit. Making the photolithography in order to obtain a wells’ depth of more then 20 µm a negative photoresist SU8-25 (MicroChem) was spun on a glass substrate 50*50 mm using a G3P -SpinCoat for 30 sec, baked on a hotplate 1 min at 650C and 4 min at 950C, exposed for 11 sec with a lamp 16.5 mW/cm2, postbaked on a hotplate 1 min at 650C and 3 min at 950C, developed for 5 min in EC developer. The final structure is shown in Figure 2. Results and Discussion A Gaussian laser beam, created with a laser focused through a aspheric lens, was used (Figure 1).

Figure 1. Experimental setup and Movement of a eukaryotic cell in the presence of a single-beam optical trap. A laser beam λ=1064 nm is reflected through 900 vertically by a mirror up through an antireflection coated aspheric lens N.A.=68 into the base of a chamber, providing an effective N.A. =0.4. The chamber holding the cell is moved to the right and to the left with a motorized translation stage. Through an objective ×10 of a DIC microscope the light is directed to the beam-splitter and after it to CCD camera and oculars of the microscope. Differential interference contrast (DIC) microscopy images of a terratoma cell (NT1) suspended in medium of refractive index n=1.33478. The location of the laser beam is identified by the white spot. The experimental procedure illustrates following trapping of the cell in the optical tweezer: (a, b, c) Within 2.5, 5.0, 7.5 sec the cell is drawn into the laser beam until it stops in the centre of the nucleus.

A polystyrene sphere or cell in suspension were trapped and held in the laser beam and levitated ~7-10 µm above the surface. The repetitive displacement of the

translation stage with the chamber on the left and on the right of the laser beam occurring, the object tended to return in the laser beam focus. A sequence of 3000 frames was recorded per measurement, after that instantaneous drag force was evaluated as a function of the distance of the laser beam to the centre of the object using a program written within IgorPro 6.12. A lot of parameters turned out to predetermine an effective drag force, so based on theoretical model of 3D Rayleigh scattering of light on ellipsoidal micro-sized objects within a nonuniform refractive index the longest axis of ellipsoidal object, the eccentricity, the radius of a nucleus, the waist and the power of the laser beam, the viscosity and the refractive index of the medium, the asymmetric position of a nucleus, the refractive indices of the cytoplasm and the nucleus were considered. In MatLab 2008 the correspondent mathematical model was realized. To confirm applicability of the model to micron-sized objects 62 polystyrene beads (RI=1.585, eccentricity =1) of 13-21 µm were trapped and the correspondence of the particle size on the image and substituted in the model one was 84%. The curve shown in Figure 2 performs theoretical and experimental data of the effective drag force subject to time passed after displacement of the stage. The typical values of the drag force were 2-3 pN. Thus, using the model the refractive indices of the cytoplasm n1= 1.337÷1.342 and nucleus n2= 1.345÷1.351 of the 35 cells NT1 were determined at the power used 45-70mW. Also there was not found any correlation between cell and nucleus sizes and refractive indices, but it could be stated that the refractive index of the nucleus was higher then of the cytoplasm. It was observed how cells oriented with their longest axis along the optical axis of the beam and two types of cells in the suspension with symmetric and asymmetric position of a nucleus to the centre of a cell.

Since it is possible to to distinguish different cells by their refractive index it was made sense to characterise these optically first and then fix them for individual biochemical characterisation. Arrays of micro cavities were assumed to provide a platform for characterizing single cells firstly optically and later by biochemical means such as qPCR or immunocytochemistry. Therefore several types of 15*15 mm arrays of microwells were created in the ORC cleanrooms by photolithography with a negative photoresist SU8-25.

As the average diameter of cells is ~ 18 µm, one array consists of 1395 cavities 200 µm in diameter with separation 200 µm and the second one – 3456 cavities 150 µm in diameter with separation 100 µm. In order to define a position while experiment is carrying out in the top right corner of the square 5*5 wells one cavity was changed for letter and number (Figure 3). The depth of wells was 20-25 µm. Cells in the suspension were added to the arrays the calculated density allowed cells to be collected in each cavity separately. After that arrays were covered by modified glass cover slips to prevent drying and optical trapping studies done.

Conclusions Optical trapping and studies to establish the force exerted by a Gaussian beam provides a non-invasive technique to characterize mammalian cells in the suspension as a function of the refractive indices of the cytoplasm and nucleus. By performing these studies on individual cells it will be possible to later characterise the same cell by biochemical means. The team in the ORC will now use these arrays with ESCs so that the different classes of differentiated and undifferentiated cells can be evaluated optically and then by biochemical methods.

Acknowledgments I am very grateful to the Biochemical Society for the funding, the ORC at the University of Southampton, Dr Tracy Melvin – my supervisor – and her team members for a possibility to gain invaluable experience and learn so many techniques new for me.

Photograph taken in the laser/cell biology laboratory

Student: Michael Whitehead Supervisor: Susan Broughton Institution for placement: Lancaster University

How insulin/IGF-like signalling affects ageing using Drosophila melanogaster as a model

organism Introduction

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved signalling pathway regulating lifespan. Although lifespan extended reductions in IIS improve some measures of health in model organisms and contribute to healthy ageing, little is known about the role of this pathway in ageing of the brain. In Drosophila, median neurosecretory cells (mNSC) in a region of the brain called the pars intercerebralis produce three of the seven Drosophila insulin like peptides (dilp2, 3 and 5). These cells are functionally equivalent to the β-cells in the islets of langerhans (in pancreas) in their control of metabolic homeostasis. When these mNSC are ablated using a protein called reaper (pro-apoptic) flies have an extended lifespan (Broughton et al, 2005). Furthermore my supervisor Dr Susan Broughton has shown that the expression of dilp4, an insulin-like peptide seen in neurons throughout the brain but not in the mNSCs (Gronke et al, 2010) is increased in these long lived ablated flies (unpublished data). Also, she has found that when a genotype is produced with increased dilp4 expression specifically in the central nervous system (CNS), with no other dilp alterations, flies’ lifespan is extended to a small extent (unpublished data). Hence these data raise questions about the role of dilp4 (and IIS) in ageing of the brain and the whole organism, and its potential role in behavioural and cognitive senescence. Project

The plan at the start of the summer was to use a phototaxis assay of learning to determine the cognitive senescence phenotype of genotypes with increased and decreased insulin signalling in the brain. Consequently to determine the role that IIS and dilp4 have in age-specific neuronal function and behaviour. Sadly I decided it wasn’t possible to do this assay as it was simply taking far too much time, so I wouldn’t have got enough data for any differences to be statistically significant.

Instead I used a negative geotaxis assay to look at how locomotor behaviour decreases with age in control flies compared to mNSC-ablated flies and those with increased or decreased IIS in the CNS. This locomotor behaviour is controlled by the brain and the normal decline of it is reduced in long-lived flies with reduced IIS (Martin & Grotewiel, 2006). However, it is not known whether delayed ageing of the brain or delayed ageing of other tissues, such as muscle, is the predominant factor in the improved age-specific performance of negative geotaxis behaviour in these flies. Hence the aim of the experiment was to determine the role of IIS in brain ageing and to what extent IIS in the CNS is involved in age-related decline of the negative geotaxis behaviour.

The GAL/UAS system is used to direct the expression of genes to specific tissues. To do this two flies have to be crossed, one with a tissue specific promoter controlling GAL4 expression and the other with an upstream activation site (UAS) for GAL4 which induces the expression of the desired gene (GAL4 is from yeast so cannot be found in Drosophila). The progeny from this cross can then be tested for expression of the desired gene using quantitative PCR. ElavGAL4 was used to drive the expression of dilp4 (elavGAL/UAS-dilp4) which increases IIS in the CNS. This system was also used to express UAS-InRDN (elavGAL/UAS-InRDN) which is a dominant negative insulin receptor which decreases IIS in the CNS. A similar system was used to produce the ablated flies (d2GAL/UAS-rpr), in this case d2GAL drives expression of UAS-reaper in the Dilp producing cells in the fly brain. Two controls for the ablated genotype were also used (d2GAL/+ and UAS-rpr/+). Results

Figure.1 shows that the decline of the ablated flies’ locomotor behaviour in the negative geotaxis assay was attenuated compared to the controls. However, the normal decline of this behaviour in the genotypes with increased or decreased IIS specifically in the CNS was not altered. A statistical analysis shows that the ablated flies had a statistically significant difference in the performance index compared to all other genotypes from age 49 onwards. These data show that although reduced IIS throughout the fly improves age-specific locomotor behaviour, altered IIS in the CNS does not, suggesting that altered IIS in the brains of ablated flies is not the main factor contributing to their attenuated decline of negative geotaxis behaviour. It is likely that tissues other than the brain, such as muscle, are probably contributing to the decrease in age-related decline of locomotor behaviour in the ablated flies.

Future experiments will build on this research by examining the role of IIS in the brain for senescence of exploratory walking (which assay I helped setup), learning (phototaxis assay) and sleep behaviours. In parallel, other work will try to identify other tissues involved IIS-related behavioural senescence. Later, tissue specific signalling/molecular mechanisms involved can be identified by, for example, tissue specific microarray expression analysis. Figure 1

Benefits of the project to me and to the lab

I have learnt what it is like to do research for real and it doesn’t always go to plan which means that you have to be extremely flexible by adapting to these changes in circumstance. Throughout the project I have been to lab meetings and a number of journal clubs (although I didn’t present any) with graduate students at the university. Consequently I have had an invaluable experience as to what a PhD would be like and I would recommend this to anyone because it isn’t exactly what you expect and is completely different to undergraduate work during your degree! Furthermore I have gained a whole host of skills which will make it easier when I come to apply for a PhD which after this experience I am definitely intending to do. This experience will also make my dissertation project for my degree much easier than otherwise.

Figure.1 - Negative geotaxsis assay to show how insulin/IGF-like signalling affects locomotor behaviour with age. The PI is calculated from the climbing speed of the flies, i.e how many flies in a group of 15 reach a certain point within 15 seconds. Genotypes are: d2GAL/UAS-rpr (mNSC ablated), UAS-rpr/+, d2GAL/+, elavGAL/+, elavGAL/UAS-InRDN, elavGAL/UAS-dilp4. Statistical analysis. Planned comparisons of mean PIs were performed by t-test to compare the difference in the performance index (PI) between the ablated flies and its controls at each time/age point. For each genotype, the data is shown as mean PI +/- SEM, N=3 and * indicates significant differences from the controls (p<0.05).

*

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Sue included the data I generated over the summer in a poster she presented recently at the Neurofly 2010 so I think my work was very beneficial to the lab. I have also helped her to set up an assay videoing flies in an arena to measure exploratory walking.

References Broughton.S.J., Piper,M.D., Ikeya,T., Bass,T.M., Jacobson,J., Driege,Y., Martinez,p., Hafen,E., Withers,D.J., Leevers,S.J and Partrisge,L (2005) Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands. Proc.Natl.Acad.Sci.U.S.A,102, 3105-3110. Gronke,S, Clarke D-F., Broughton SJ, Andrews TD and partridge L. (2010) Molecular evolution and functional characterisation of Drosophila insulin – like peptides. Plos genetics 26:6(2) e1000857 Martin,I. and Groteweil,M.S (2006) Distinct genetic influences on locomotor senescence in Drosophila revealed by a series of metrical analyses. Exp.Gerontol., 41, 877-881.

EXPRESSION, PURIFICATION AND NMR STUDIES OF DELETION MUTANTS OF LA-RELATED PROTEIN 7 (LARP7)

Michał Barski

Department of Biochemistry Randall Division of Molecular Biophysics, King’s College London

Supervisor: Dr. Maria R (Sasi) Conte

BACKGROUND: LARP7 belongs to a family of La-related proteins (LARPs) containing a characteristic RNA binding unit called the “La module”, initially discovered in the La protein (Figure 1). Structural characterisation of the La protein, referred to as genuine La, has only recently been revealed1-3 but little is known about LARPs. Genuine La binds 3’ UUU-OH RNAs (mainly RNA Polymerase III transcripts) protecting them from exonucleolytic degradation. La was also shown to have RNA chaperone activity and to control translation for some mRNAs4. LARP7 was found to interact with 3’ UUU-OH 7SK RNA, forming a stable 7SK ribonucleoprotein (RNP) complex that binds to the positive transcription elongation factor b (P-TEFb) thereby down-regulating its activity5-8. As P-TEFb controls the transcription of many genes, understanding the structure and interactions of LARP7 may provide invaluable insight into its roles in vertebrate embryogenesis and tumourigenesis.

AIM: The aim of the project was to advance our molecular understanding of human LARP7, by generating deletion mutants of LARP7 for biochemical and biophysical analysis. The overall aim of these experiments was to learn the LARP7 structure and properties as well as to understand the structure-functional differences between various members within the LARP family. In this preliminary part of research it was particularly important to ascertain:

• Correct amino acid boundaries of each of the domains within the protein (map of the protein) • Affinity of the La module of LARP7 towards 3’ UUU-OH RNA • Preliminary information about secondary and tertiary structure

Figure 1: Domain organisation of genuine human La protein and human LARP7

APPROACH: According to amino acid sequence analysis9, human LARP7 consists of three domains: the La motif, the RNA-recognition motif (RRM) 1 and the RRM2 (Figure 1). Since in the genuine La the first two domains work together to bind RNA, they are collectively referred to as the La module. Three different deletion mutants of human LARP7 were studied. These had been subcloned into a pET-DUET1 and/or pQE2 expression vector, with a removable His-tag attached at the N-terminus, and expressed in relevant strains of E.coli. A large part of the work of this project was to establish a protocol for production and purification of these deletion mutants from scratch in order to obtain large quantities of stable, folded and soluble proteins.

RESULTS

RRM2: The deletion mutant encompassing LARP7 RRM2 (encompassing residues 453-565) was expressed in E.coli Rosetta cells and successfully purified by FPLC on a Nickel affinity column followed by a heparin column. From a litre of culture we obtained almost 30 milligrams of a stable purified protein, which enabled us to carry out NMR experiments on this protein. Figure 2: 1H proton NMR spectrum of the RRM2 of human LARP7 recorded at 11.75 T and 25º C

in buffer containing: 20 mM Tris, 100 mM KCl, 0.2 mM EDTA, 1 mM DTT; pH 7.25

The 1-D 1H NMR spectrum confirmed that the protein was well folded: figure 2 shows a good dispersion of proton signals, especially in the 0-2 ppm region (which represents mainly protons from methyl groups) and 6-10 ppm (amino groups). This conspicuously shows that each of the protons in this region exists in a unique chemical environment, which happens only in a highly ordered structure of a folded protein. These preliminary results indicate that the RRM2 of LARP7 could be structurally characterised by NMR.

La module: The expression and purification of this deletion mutant (residues 1-217) was not as straightforward as expression levels were low and the protein appeared in the insoluble fraction of the cell. Although this mutant was subcloned in both pET-DUET1 and pQE2 and several experimental conditions (temperature, induction time, IPTG concentration) were tried, we did not manage to obtain soluble protein. An attempt to obtain protein from the insoluble fraction was made using the Sarkosyl method of extraction10 but no folded protein was obtained.

La motif: This deletion mutant (residue 1-112) was expressed in reasonable quantities at 37oC in Rosetta cells, but it was only present in the insoluble fraction of the cell. Again the Sarkosyl method was attempted but did not provide folded protein. We also tried to refold the protein using a refolding procedure in decreasing concentrations of urea but this was also unsuccessful.

RRM1: Expression of the RRM1 of LARP7 (residues 112-217) was accompanied by coexpression of a foreign protein which was impossible to identify and separate during the timescale of this project.

FUTURE DIRECTIONS: Further work is necessary to improve expression and solubility of the La motif, RRM1 and La module of LARP7, perhaps using different expression vectors (different tags, E.coli strains etc.). The structure analysis of the RRM2 of LARP7 can commence straightaway.

ANY DEPARTURES FROM THE ORIGINAL PROPOSAL: The cloning of LARP7 deletion mutants had already been obtained in the lab before the start of this project. This meant that time could be fully dedicated to expression and purification of proteins and to carry out preliminary NMR analysis of purified LARP7 RRM2.

VALUE OF THE STUDENTSHIP: The studentship had a tremendous influence on the way I think about science and research. I reckon that both my supervisors as well as working in the lab itself taught me how to deal with scientific problems and opened my eyes to a whole range of issues in science that I would have otherwise never thought about. I also learned how to operate many important instruments and lab equipment, which is also a crucial skill. Overall, I can say that this studentship has raised my interest even more in pursuing a lab-based and science-oriented career in the near future.

VALUE OF STUDENTSHIP TO THE LAB: Michał has been an excellent student who has contributed actively and positively to the life of the lab in general and the LARP7 project in particular.

References

1. Jacks A, Babon J, Kelly G, Manolaridis I, Cary PD, Curry S, Conte MR. Structure of the C-terminal domain of human La protein reveals a novel RNA recognition motif coupled to a helical nuclear retention element. Structure, 11: 833-43. (2003)

2. Alfano C, Sanfelice D, Babon J, Kelly G, Jacks A, Curry S, Conte MR. Structural analysis of cooperative RNA binding by the La motif and central RRM domain of human La protein. Nat Struct Mol Biol, 11: 323-9. (2004)

3. Kotik-Kogan O, Valentine ER, Sanfelice D, Conte MR, Curry S. Structural analysis reveals conformational plasticity in the recognition of RNA 3' ends by the human La protein. Structure, 16: 852-62. (2008)

4. Wolin SL, Cedervall T. The La protein. Annu Rev Biochem, 71: 375-403. (2002) 5. Krueger BJ, Jeronimo C, Roy BB, Bouchard A, Barrandon C, Byers SA, Searcey CE, Cooper JJ, Bensaude O,

Cohen EA, Coulombe B, Price DH. LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res, 36: 2219-29. (2008)

6. He N, Jahchan NS, Hong E, Li Q, Bayfield MA, Maraia RJ, Luo K, Zhou Q. A La-related protein modulates 7SK snRNP integrity to suppress P-TEFb-dependent transcriptional elongation and tumorigenesis. Mol. Cell, 29: 588-599. (2008)

7. Markert A, Grimm M, Martinez J, Wiesner J, Meyerhans A, Meyuhas O, Sickmann A, Fischer U. The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and effects transcription of cellular and viral polymerase II genes. EMBO reports, 9: 569-575. (2008)

8. Xue Y, Yang Z, Chen R, Zhou Q. A capping-independent function of MePCE in stabilizing 7SK snRNA and facilitating the assembly of 7SK snRNP. Nucleic Acids Research, 38: 360-369. (2010)

9. Bousquet-Antonelli C, Deragon JM, A comprehensive analysis of the La-motif protein superfamily. RNA, 15: 750-64. (2009)

10. Tao H, Liu W, Simmons BN, Harris HK, Cox TC, Massiah MA. Purifying natively folded proteins from inclusion bodies using sarkozyl, Triton X-100 and CHAPS. BioTechniques, 48: 61-64. (2010)

Student: Natalia Grundwald Supervisor: Dr Marcus Adrian Nicholas Rattray Biochemical Society studentship report 2010 Understanding glutamate transporter internalisation under pathophysiological conditions Background Pathologies of glutamate levels are found to be involved in an increasing number of neurodegenerative disorders like Amyotrophic Lateral Sclerosis. It is thought that high concentrations of glutamate in extracellular space can cause neurotoxicity. One of the main means of controlling glutamate levels in those regions is Glutamate Transporter 1 (GLT-1) present on astrocyte membranes. For that reason ability to control levels of GLT-1 would significantly increase chances of fighting disorders caused by glutamate induced excitotoxicity. Glutamate transporters are multimembrane-spanning proteins that form as non-covalently bonded homomultimers of 3 subunits and belong to the ubiquitous solute carrier 1 (SLC1) family of secondary solute transporters. It has been shown that GLT-1 mostly migrates on SDS-PAGE as either a ~75kDa monomeric band, representative of mature protein, or its multimers (trimers). Previous research has shown that GLT-1 is most likely internalised through clathrin coated vesicles since compounds inducing that path seem to stimulate down regulation of the transporter and compounds blocking this effect seem to stop it. There is no significant evidence, so far, for or against clathrin independent pathways. In this project the internalisation was induced by phorbol ester (PMA) and I showed that three compounds blocked it most significantly: Sucrose (200mM), Ammonia (5-50mM) and MG-101 (2nm-20μM). Sucrose works through changing cellular osmolality, while ammonia has a known effect on the lysosomal function. MG-101 inhibits calpain, an enzyme that possibly has a role in limiting the degradation of GLT-1. Aims The project had 2 main aims: to prove that pathophysiological stimuli cause downregulation and degradation of GLT-1 and to elicit the exact route of intracellular trafficking of GLT-1. Description of work carried out and results Three main methods for following the fate of GLT-1 transporter under different conditions were used. A novel cell based assay was used for primary screening of compounds to block internalisation and degradation, Western Blotting was used for researching further evidence of protection against degradation and immunocytochemistry was used to visualize the effects. I learned a range of supportive techniques, like maintaining a stable cell line, growing plasmids, transfections of primary cells and fluorescence microscopy. Due to time constraints a stable cell line of HEK293a cells expressing V5 tagged GLT-1 was mainly used. However immunocytochemistry was done on rat cortical astrocytes. For screening of compounds that could possibly block internalisation and degradation of GLT-1, a cell based assay in 96-well plates on the stable cell line was used with luminescence used to detect amount of degradation following PMA stimulation. In the early phase of the project the experiments concentrated on compounds that have been previously shown to have some blocking effect by the host lab. From those compounds MG-101 seemed to be most promising, showing a concentration-dependent reduction of PMA-induced GLT-1 degradation. In the subsequent part of the experiment some compounds, found through my literature research, were used and sucrose and ammonia both shown evidence of protective function against PMA induced degradation of GLT-1. All three compounds have been used in Western blotting experiments. During the Western Blotting procedures very specific antibodies against V5-tag have been used resulting in clean bands with low

background. The experiment was repeated 3 times on 9% gel to achieve good resolution for both monomers and trimers of GLT-1. When stimulated with PMA the bands have shown a decrease in density of trimeric, mature bands in comparison to control. Each of the 3 compounds used: MG-101, Sucrose and Ammonia blocked the effect of PMA. The immunocytochemistry experiment used primary astrocytes transfected with plasmids expressing wild type SOD-1 gene tagged with green fluorescent protein (GFP), a GFP-tagged G93A mutation of SOD-1 and/or GLT-1 with V5 tag. The aim was to co-transfect the cells with SOD-1 and V5-GLT-1 in order to create environment allowing visualization of the impact the mutant SOD-1 will have on the internalisation of GLT-1. The initial experiment has shown that there was very good transfection efficiency for V5-GLT-1 (red, Fig. 1) using concentrations advised by the manufacturer of Lipofectamine 2000; however the GFP SOD-1 was co-transfected with lower efficiency (Fig.2). The experiment needed to be optimized for co-transfection in these cells with those genes. To that end another experiment was devised and as a result more suitable concentrations of reagents were found (Fig. 3). As a result of the studentship more data supporting the internalisation of GLT-1 through a clathrin dependent vesicle pathway has been gathered, as the compounds used had been shown previously to inhibit lysosome and clathrin coated vesicle formation. The effect that PMA has in downregulation of the GLT-1 has also been clearly shown. Future directions Further research is necessary to exactly elicit the pathway either through immunocytochemistry and through the use of more compounds and GLT-1 constructs to block certain points in the pathways linking clathrin coated vesicle internalisation to lysosomal degradation. Additional internalisation pathways may play a significant role in the process it would be useful to experiment with compounds known to block alternative internalisation and check whether they have any effect. Departures from original It was not possible to comply with certain points of the original proposal due to time constraints (range of compounds tested) or unexpected problems (cotransfection issues), however a range of compounds was used and cotransfection process was optimised. In addition, Western Blotting has been added to the project to confirm the cell-based assay findings. Value of studentship to lab For the lab the studentship has been valuable as the student was able to show that certain compounds, like MG-101, have potential in protecting against PMA induced GLT-1 down regulation and degradation. The work has demonstrated proof of principle of an entirely novel assay system for monitoring the regulation of GLT-1 protein, and some of the data produced will lead to publication. Value of studentship to student The summer studentship was important for me in many ways. It allowed me to work in a true academic environment and experience the relationship and respect for knowledge and opinions of team members present in the host lab. In terms of research skills I was able to try out and learn a broad range of techniques used in modern biomedical lab as described above and some soft skills like keeping a proper lab book and managing efficiently multiple experiments at a time. I have learned a lot about what it really means to be a researcher, while being allowed to test my own hypothesis surrounded by people willing to help out and guide me even through some beginner’s mistakes that I have made. Now that I have first-hand experience of research environment, I definitely know that I want to study for a PhD.

Student: Olivia Henry Supervisor: Finn Werner Institution for placement: University College London

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A Protein Interaction Study The premise of my Summer Studentship in Dr Finn Werner’s laboratory was the study of the interaction between proteins involved in transcription, namely the transcription factors Spt4/5, TFE and S10. Spt4/5 is a transcription factor which associates with RNAP, stimulating elongation by coordinating a conformational change in RNAP. TFE is a transcription factor, involved in initiation, which plays a role in initiating the melting of the DNA double helix. Interestingly, despite the fact that Spt4/5 and TFE fulfil very different functional roles, both transcription factors bind to the coiled coil clamp tip region of the RNAP. Therefore it seems likely that an interaction might exist between the two proteins as transcription shifts from initiation to elongation; however, this interaction has only been inferred and as yet there is no direct evidence. Therefore, one of the primary aims of this investigation was to attempt to directly determine whether there is a true interaction between TFE and Spt4/5. In addition I examined S10, an anti-termination factor, since theoretically speaking, a complex formation between S10 and Spt4/5 also seemed highly plausible. In order to examine these interactions I first had to express and purify the following archaeal proteins using a bacterial expression system: GST-Spt4/5, GST-Spt4/5-NGN domain only, His-TFE, S10 as well as two mutant forms of Spt5 which I subsequently spin-labelled. In order to purify these proteins for the interaction studies, I implemented a plethora of techniques which worked to exploit the different features of the recombinant proteins: FPLC (separated proteins on basis of GST and His tags), centrifugation, enzymatic treatment, size exclusion chromatography, heat inactivation, and dialysis. I also used a number of preparative techniques including use of concentrators and ammonium sulphate precipitation. My interaction studies took two distinct forms. Implementing a biochemical approach, I used a co-purification technique to assess both the likelihood of an interaction and to determine whether the protein complex could be purified in any meaningful amount. Secondly I implemented CW-EPR to assess the presence of any interaction in biophysical terms. In this report I have briefly discussed the findings of these interaction studies. The possible outcomes of a co-purification of Spt4/5 NGN and TFE:

Extent of binding Flow through fractions “Wash” fractions Elution fractions Complete Binding NO PROTEIN NO PROTEIN Spt4/5 NGN + TFE Weak binding NO PROTEIN TFE – weak interaction

disrupted Spt4/5 NGN + TFE

No binding TFE (Some TFE due to non specific binding to the column)

Spt4/5 NGN

The results of my co-purification of Spt4/5 and TFE are as follows:

I also stained the TFE + Spt4/5 NGN gel using silver staining since this technique is able to detect much smaller amount of protein than Coomassie staining. Both these gels indicated that there is no interaction between TFE and Spt4/5 NGN under these conditions. I repeated this analysis for Spt4/5 + S10, and found that there was no interaction between these two transcription factors under these conditions.


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The use of CW-EPR allowed me to examine binding between a spin labelled Spt4/5 and TFE/S10 in a more quantitative way than the co-purification studies and thereby enabled be to test my conclusion of there being no interaction in either case.

If binding exists, tumbling of the Spt4/5 + TFE/S10 complex should slow down relative to the Spt4/5 alone and thus the peaks of the TFE/S10 spectra should appear broader, compared to the control spectra. Whilst there is some variation in amplitude between the three spectra (see above) they largely overlay indicating that the overall tumbling has not been affected. Thus for both TFE and S10 I can conclude that, under these conditions, there is no specific binding with Spt4/5 with sufficiently high affinity to affect the overall tumbling of the Spt4/5 complex. I repeated this experiment using an alternative Spt4/5 mutant (with different spin labels) and found that again overall tumbling was not affected. These conclusions support the results of my co-purification analysis, further corroborating that under these conditions Spt4/5 does not form a complex with either TFE or S10. In an additional feature of the project, I was able to assist in the DEER measurement and analysis of Spt4/5. DEER analysis can be used to measure distances between spin labels. Therefore by measuring the distance between two spin labels located at different positions in a Spt4/5 complex it was possible to determine the orientation of the Spt4 and Spt5 relative to each other, thereby contributing to the effort of building a solution proximity map of Spt4/5. In concluding, I would like to highlight the particular relevance of my investigation of the potential interaction between Spt4/5 and TFE in dissecting the mechanism of transcription. Since TFE is involved in initiation and Spt4/5 is involved in elongation, and both bind to the same coiled coil clamp tip domain on RNAP, it follows that in switching from initiation to elongation there must be an exchange between the two. This exchange process is likely to take one of two forms – replacement or recruitment. In a replacement mechanism, as the name suggests, TFE may be replaced by Spt4/5 on transition from initiating to elongating complex. In this case it is likely that there is no interaction between the two. Alternatively, a recruitment mechanism may exist in which TFE works to recruit Spt4/5 to the coiled coil clamp tip. If an interaction exists between the two then this mechanism of recruitment seems more likely. Since I found that no interaction exists it seems likely that a replacement mechanism between Spt4/5 and TFE exists under these conditions. My studentship in Finn Werner’s lab this summer has proved to be invaluable, as it enabled me to gain first-hand experience of what research is really like. Academically speaking, the studentship facilitated me to develop my understanding of the transcriptional mechanism as well as enabling me to acquire and develop practical skills in the context of the lab. Moreover, I felt a real sense of achievement in being able to see a project through from beginning to end. On a more personal note, I really enjoyed the time spent with the other


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members of the lab and I am extremely grateful for all their help and insight. Therefore, my thanks go to Dr Finn Werner, Dr Dina Grohmann and Daniel Klose, and to the Biochemical Society for so generously funding my studentship.

Biochemical Society Studentship Report, Summer 2009 Assessing the role of Zimp7 & Zimp10 in the DNA Damage Response Student: Philip Burn Supervisor: Dr. Yaron Galanty (Steve Jackson Laboratory) Background Recent work at the host laboratory has indicated a role for the SUMO E3 ligases PIAS1 and PIAS4 in the DNA Damage Response (DDR), where they accumulate at sites of DNA double strand breaks (DSBs) and mediate the recruitment of other DDR factors including 53BP1, BRCA1 and RNF168 through SUMO conjugation. Zimp7 and Zimp10 are PIAS-like proteins, specifically sharing a conserved zinc-binding SP-RING/Miz domain, which is responsible for the E3 ligase activity in PIAS proteins. In addition published data showing Zimp interactions with p53 and components of the SWI/SNF chromatin remodelling complexes as well as their presence at replication foci suggest the Zimp proteins are strong candidates for a role in the DDR. Project aims

1. Generate GFP tagged constructs of both the Zimp7 and Zimp10 genes. 2. Begin the process of generating cell lines that stably express GFP tagged Zimp7 and Zimp10 genes. 3. Determine whether Zimp7 and Zimp10 have a role in the DDR by investigating whether the proteins

accumulate at DSBs. 4. Investigate potential modifications of Zimp7 and Zimp10 in response to DNA damage.

Departures from the original proposal In the original proposal, the focus of the project was the proteins PIAS2 and PIAS3, with Zimp7 and Zimp10 considered as a route for investigation if there was enough time at the end of the project. However, the focus was shifted to the Zimp proteins; this was due to the fact that there is only a very limited amount of published data concerning these proteins, in particular there is none relating to a potential DDR role. Therefore, any discoveries made would be of greater significance. Results Generation of GFP tagged Zimp7 and Zimp10 clones The Zimp7 and Zimp10 genes were sub-cloned from the pcDNA vector into the pEGFP-C1 vector via amplification of the open reading frames with the Phusion PCR polymerase. The GFP tagged constructs were then transiently transfected into both U2OS and HEK293 human cell lines in order to confirm that the proteins expressed were both full length and localised to the correct compartment of the cell. The transfected HEK293 cells were harvested for Western blot analysis and probed with anti-GFP antibody – these results (Fig. 1a) indicate that the expressed proteins were approximately of the correct predicted size. The transfected U2OS cells were imaged using confocal fluorescent microscopy, and this confirmed that the proteins localised to the nucleus (Fig. 1b). Zimp7 and Zimp10 display slower migration on SDS-PAGE following exposure to mimosine HEK293 cells transfected with the GFP-Zimp7/10 constructs were exposed to the following DNA damaging agents; ionising radiation (IR), camptothecin (CPT) and mimosine (Mim). The cells were harvested for a Western blot, and probed with GFP antibody. This revealed a shift in size of the Zimp proteins in response to mimosine treatment, thus indicating a potential modification of the proteins (Fig. 2a). The presence of DNA damage was confirmed by probing for phospho-Chk1 (pS345) and phospho-KAP1 (pS824) (Fig. 2b), both hallmarks of DNA damage. An immunoprecipitation experiment was carried out, harvesting transiently transfected Hek293 cells and pulling down the Zimp proteins with anti-GFP beads. However the results of this were inconclusive and due to time restrictions it was not possible to repeat the experiment.

Biochemical Society Studentship Report, Summer 2009 Assessing the role of Zimp7 & Zimp10 in the DNA Damage Response Student: Philip Burn Supervisor: Dr. Yaron Galanty (Steve Jackson Laboratory) Generation of U2OS based cell lines stably expressing GFP tagged Zimp constructs U2OS cells were transfected with the GFP-Zimp constructs, spread evenly over 15 cm dishes and exposed to the selection marker G418 (resistance to G418 is conferred by the pEGFP plasmid). After 2 weeks, monoclonal colonies had formed which were collected and transferred to a 24 well plate based on whether they were GFP positive or negative. Time constraints prevented the monoclonal populations from being taken any further; the host laboratory will continue characterizing these cell lines. However, a polyclonal population was simultaneously cultured. This population was sorted via flow cytometry to enrich the GFP positive population and then allowed to continue to grow until there were enough cells to be used in experiments. Zimp7 and Zimp10 are recruited to sites of DNA damage only in a sub-population of cells The polyclonal populations stably expressing either GFP-Zimp7 or GFP-Zimp10 were synchronised by

thymidine block and released 3 hours before imaging to increase the S phase population (it was suspected that the proteins acted in this cell cycle stage due to an initial experiment involving transiently transfected cells – Fig. 3a). The cells were also incubated with BrdU, a synthetic thymidine analogue that sensitises the DNA to laser damage. The cells were damaged by laser micro-irradiation through the nucleus, and subsequently imaged at various time points following the damage. Accumulation of the proteins at DSB sites was observed in some of the cells from the asynchronous population, whilst in the synchronised cells, only one or two cells showed very weak accumulation and the rest were negative (Fig. 3b) – this suggests that whilst the proteins are regulated in a cell cycle dependent manner, they could be inactive during S phase (at least early S phase) after DNA damage. The enrichment of cells in S-phase was confirmed by FACS analysis (Fig. 3c). The above observation is not sufficient to obtain a firm conclusion and a laser micro-irradiated asynchronous population co-stained for Zimp7 or Zimp10 together with cell cycle markers such as PCNA, Cyclin-A and others is needed.

Further investigation There are many avenues of investigation to continue research into the function of these two proteins in the DDR. Further synchronisation experiments as well as co-staining for Zimp7 or Zimp10 together with cell cycle markers such as PCNA, Cyclin-A and others is needed to determine the exact cell-cycle stage in which the proteins are able to respond to DNA damage. Also, the modification in response to mimosine can be further investigated by repeating the IP and probing for common modifications such as SQ/TQ phosphorylation (by the ATM/ATR kinases), SUMOylation or cell cycle dependent modifications – to determine whether the modification is in response to the cell cycle or the DNA damage. Other lines of investigation would be to determine the function of the proteins, for instance by knocking down the Zimps with siRNAs and assessing the resulting phenotype and also co-IP experiments to investigate any possible interactions of the Zimps with DDR proteins. It will also be important to determine the proteins required for Zimps accrual at DSB. Value of the studentship The studentship has been of immense value to me as it has been the first extended period of time where I have been able to work in a lab. I have been able to get firsthand experience of many techniques that are required to conduct research such as; cloning, PCR, electrophoresis, SDS-PAGE, tissue culture, flow cytometry, fluorescent imaging, Western blot and many more. It has also given me experience of problem solving and connecting isolated techniques into an order that allows for the study of molecular processes and events. For the host lab, the studentship has been the initial steps of a new project investigating the role of the Zimp proteins in the DDR. Researchers in the lab will be able to build on the primary results obtained during the studentship, investigating the properties outlined earlier in order to gain further insight into the function of the Zimps in the DDR.

Student: Rachel Slaughter Supervisor: Simon Morley Institution for placement: University of Sussex During muscle cell regeneration, critical information stored in the DNA has to be decoded to produce a wide variety of essential proteins. The general transfer of information from DNA to protein is carried out by mRNA which is a copy of the DNA sequence. This mRNA has to be decoded into protein by a complex process known as translation which requires ribosomes and translation initiation factors (eIFs) that interact together to help make the proteins required. One protein, 4E-BP1, prevents the interaction of its partner, eIF4E, with the scaffold protein, eIF4G, and stops the recruitment of mRNA to the ribosome and halts protein synthesis. When protein synthesis is needed, the cell signals to 4E-BP1 to release the eIF4E/mRNA from the 4E-BP1/eIF4E/mRNA complex to let it work. The cell does this by marking the 4E-BP1 with phosphate groups in a process known as phosphorylation. My work investigated the pathways cells use to signal this event. During the tenure of this summer placement, I was able to master culturing mouse pre-muscle cells and preparing extracts from these to investigate cell signalling using SDS-PAGE and Western blotting. To analyse the importance of signalling to 4E-BP1 in controlling protein synthesis, I used a variety of cell-permeable inhibitors of different signalling pathways. The doses and times for treatment had to be optimised and their efficacy proven using an array of phospho-specific antisera. In addition I was able to compare and contrast the effects of selected inhibitors on cell growing in log phase with those exiting the cell cycle and undergoing myogenesis. The overall outcome of this work was that I have become competent in using such techniques to investigate intracellular signalling to 4E-BP1 in cells undergoing log phase growth and those induced to differentiate into muscle cells. My main finding was that a number of drugs which targeted the mTOR pathways were good at blocking this signalling to 4E-BP1, yet they performed at different levels of efficiency in these cells. This work will form the basis of an approach to investigate novel modifications of 4E-BP1 observed during myogenic differentiation. The placement was fantastic and gave me a wonderful insight into how research works. I have learnt many new skills and gained valuable experience of working in a lab, which will come in extremely handy for the project I will undertake for the final year of my Molecular Medicine degree. The skills I have learned include:

• Cell culture and harvesting • SDS-PAGE and Western blotting • Autoradiography • Bradford assays for determining protein concentration in extracts • Time-management • Working as part of a team but also having to use my initiative and work independently on

my project I experienced a situation where the cells became contaminated with yeast. This was a good learning experience for me as it taught me how to deal with such situations and get my research back on track again. At one point during my time in the lab, one of the drugs I used killed off the

cells treated with it. This was unexpected, and it taught me that research is unpredictable and not to be disheartened if things do not go as planned. I enjoyed the placement immensely: it has further influenced my interest into carrying out a PhD and continuing my progression as a biochemical research scientist. I would recommend the placement to any future students considering it, as the experience has been invaluable to me and will go a long way to aiding my third year dissertation project and to any future research that I endeavour to carry out.

Molecular Role of C-Reactive Protein and Complement Factor H in Causing Deposits That Lead To Age Related Macular Degeneration In the Eye. Student: Ramanjit Kaur Arora Supervisor: Professor S J Perkins Ami Miller Background: Complement Factor H is a 150 kDa regulatory protein which plays a very important role in the regulation of the alternate pathway of complement activation. Factor H consists of 20 short consensus repeats each made up of 60 amino acids. Age related macular degeneration which is a major cause of blindness among the elderly in the western world is caused due to deposits called drusen formed in the retina. This is due to polymorphism that exists in complement factor H. The presence of a histidine residue in place of a tyrosine residue at position 402 increases the risk of developing age related macular degeneration. Since this condition affects the macula, which is present in the retina, it affects central vision. C reactive protein which is an acute phase protein produced by the liver and is also a ligand for Complement Factor H. The concentration of C reactive protein increases in response to an inflammatory process in the body. It activates the classical complement pathway and helps in preventing the spread of infection through the body. This project aims at studying the interaction between C reactive protein and the allotypes of complement Factor H. Aims: The aims of this project were to purify the two allotypes of Factor H from plasma using various chromatographic techniques and to determine the molecular role of C-reactive protein and Complement Factor H in causing Age Related Macular Degeneration in the Eye. Materials and Methods: Genotyped plasma stocks containing Histidine 402 and Tyrosine 402 allotypes of Complement Factor H were provided. The two allotypes of Factor H were purified from plasma using various chromatographic techniques such as affinity and size exclusion chromatography. In the first step of the purification procedure, dialysed plasma was passed through a guard column immobilised with non-immune IgG antibody. This allowed the removal of various proteins such as fibronectin and rheumatoid factors. Purified plasma obtained from the IgG column was then subjected to another guard column with lysine immobilised on sepharose. Passing the plasma through this column allowed the removal of proteins such as plasminogen and plasmin. Plasma obtained from the lysine-sepharose column was allowed to flow through the MRC 0X23 column from which complement Factor H was eluted. Complement Factor H obtained from the MRC 0X23 was passed through Hi Trap Protein G HP column to remove any IgG contaminants present. The sample containing Complement Factor H was then concentrated using Centrifugal Filter Devices. Since

Factor H has a molecular weight of 150 kDa, it is unable to pass through the membrane present in these centrifugal devices, while other proteins present in the sample that have a molecular weight of less than 50 kDa flow through. This procedure was used to help concentrate the Factor H solution. The concentrated Factor H solution was passed through the Superose 6 column which separates proteins according to size and helps to remove serum albumin which is also present at a very high concentration in plasma. Thus, finally purified Factor H solution obtained after the Superose 6 column was again concentrated using centrifugal devices. The same procedure was carried with the four preparations done, two with each of the allotypes. Results and Discussion Low concentrations of Factor H were obtained after the purification procedure. Thus, the purification procedures were carried out in duplicates, i.e. four purification preparations were carried out in which two samples of each allotype of complement Factor H were purified. Future Plans: Samples of the two allotypes of complement factor H that were purified would be used to to carry out further experiments. Thus, I would be returning to the laboratory in October to carry out Surface Plasmon Resonance which would allow me to study the binding affinity between C reactive protein and Factor H. Studying these interactions with the two different allotypes would help in the better understanding of their relevance to age related macular degeneration. Departure from the original proposal: The original proposal suggested that two preparations would be carried out in which the two homozygous forms Tyrosine 402 and Histidine 402 would be purified. However, since low yields of the two homozygous forms were obtained, the purification procedure was carried out in duplicates. Value of the studentship to the student: This internship was an invaluable experience for me. I had the opportunity to have hands on experience on the various chromatographic apparatus such as the various guard columns and the AKTA instruments which are greatly used in protein purification procedures. I had already learnt about the techniques during my course and this provided me with the chance of putting the theory that I had learnt into practice. This internship has greatly boosted my confidence of working independently in the laboratory which would greatly help me in my final year project. Value of the studentship to the laboratory: This internship has provided the laboratory with purified complement factor H which would be used for carrying X ray data collection and would also be greatly used in carrying out further research.


Student: Robyn Foster (University of St Andrews) Date: 1st June – 6th August Supervisor: Dr Christopher Law (Queen’s University Belfast)

Project Title: Hetero- and homologous overexpression of two integral membrane proteins in Escherichia coli

Background Overexpression of membrane proteins in the amounts required for structural studies is notorious for its challenges and unpredictable nature. There is a large number of bottlenecks that can affect overexpression, such as differing protein production techniques in the host (in heterologous expression), failure to modify the protein after translation, lack of space in the membrane and low protein folding capacity of the host for the extra protein being produced1. Optimisation of overexpression conditions often alleviates these problems, and specialised hosts have been developed with highly improved abilities to overexpress proteins. Some examples include those derived from the Escherichia coli BL21(DE3) strain which are designed to cope with the potentially toxic effects of protein overexpression1, and the Rosetta 2 strain which helps enhance the expression of proteins that contain codons rarely used in E. coli2. Understanding and rationalising the approach to the procedure is the key to a successful experiment. In my project, different overexpression conditions were investigated in order to optimise protein production including varying factors, such as overexpression strain, growth medium, temperature, growth time, inducer concentration and induction time.

Aims To heterologously overexpress BmtA (a membrane-spanning protein involved in virulence of a Lyme disease-causing spirochaete3), and homologously overexpress MdtM (an E. coli inner membrane multidrug resistance protein4) in E. coli cells to give sufficient quantities for structural studies.

Deviations from original proposal The original proposal was to overexpress only BmtA, but because there were few positive results my supervisor allowed me to work on a second protein to maximise my chances of success in the project. There was not enough time to scale-up the successful experiments to allow a full purification and biochemical characterisation of each protein; however MdtM was semi-purified using immobilised metal affinity chromatography (IMAC).

Methods The wild-type bmtA gene with a C-terminal His6 tag was ligated into a pET26b(+) vector, and transformed into E. coli Rosetta2 cells. The transformed cells were grown overnight on Luria-Bertani (LB) agar plates supplemented with the appropriate antibiotics for selection. A single colony from the plate was used to inoculate a flask containing Terrific Broth (TB). The culture was allowed to grow until an OD at 600nm of 1.20 was reached. At this point, the cells were induced using isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown for a further 4 hours before sampling. Samples of the whole cells were then run on a 10% SDS-PAGE gel. A slightly different method was carried out for MdtM. The wild-type mdtM gene was ligated into a modified pBAD vector to give a protein product that contained a C-terminal His6 tag. The plasmid was transformed into E. coli BL21-AI cells. Transformed cells were grown overnight on LB agar plates supplemented with the appropriate antibiotics for selection. A single colony from the plate was used to inoculate 6 flasks containing LB broth until an OD at 600nm of 0.60 was reached. At this point, the cells were induced using arabinose and grown for 4 hours. The cultures were harvested and centrifuged, and the resulting supernatant was decanted. A buffer containing DNase and protease inhibitors was used to resuspend the cells, and the sample was sonicated to break the cells. This was again centrifuged, and supernatant samples were run on a 10% SDS-PAGE gel. Both proteins of interest were detected by Western blot using a probe for the His6 tags.

Results A large number of experiments were carried out with mixed results. Below are the most promising results from many weeks of experimentation during my time in the lab.

Future research With further research, these proteins can most definitely be expressed and purified in sufficient quantities for structural analysis and functional studies. This, in the case of BmtA, will allow a full functional and structural analysis and may even lead to the development of novel drugs for the treatment of Lyme disease.

Value of project to student Having completed my summer project, I certainly feel more confident in the laboratory environment. I have learned such a wide variety of fundamental techniques that I will be sure to put them into practice again in my Honours years. The responsibilities I was given in the lab to design and carry out my own experiments have taught me the value of proper laboratory procedure, time management and, most of all, perseverance. Overall, this was a thoroughly enjoyable and valuable way to spend my summer and absolutely encourages me to pursue a future career in research.

Value of project to supervisor Biochemical Society summer studentships, as well as providing the participating student with an insight into how a genuine research project is undertaken, provide the project supervisor a valuable conduit for engagement with the undergraduate molecular biosciences community. In the case of this particular project, the work performed by the student will form the basis of future studies into the structure and function of two membrane proteins that play a role either in disease processes or in resistance to antibiotic therapy.

References 1Samuel Wagner1, Mirjam Lerch Bader, David Drew and Jan-Willem de Gier. (2010). Rationalizing membrane protein overexpression. TRENDS in Biotechnology. 24 (issue 8), 364-371. 2Robert Novy, Don Drott, Keith Yaeger and Robert Mierendorf. (2001). Overcoming the codon bias of E. coli for enhanced protein expression. inNOVAtions, Newsletter of Novagen. 12, 1-3. 3Ouyang et al.. (2009). A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi. PNAS. 106 (9), 3449–3454. 4 Kunihiko Nishino and Akihito Yamaguchi. (2001). Analysis of a Complete Library of Putative Drug Transporter Genes in Escherichia coli. Journal of Bacteriology. 183 (20), 5803–5812.

Fig.1. Western blot showing that BmtA was expressed in E. coli Rosetta2 cells in lanes 1 and 2.

Fig.2. Western blot showing that MdtM was expressed in E. coli BL21-AI cells in lanes 1 and 2. Lane 3 shows a 120kDa positive control protein. MdtM appears at ~30kDa instead of ~45kDa due to anomalous migration of this hydrophobic protein.

Student: Ross Coron

Supervisor: Professor Frank Sargent

Institution for placement: University of Dundee


Project Title: Recognition of Tat Signal Peptides and Transmembrane Helices

Background:

The Tat (twin arginine transport) pathway is a protein secretion system found in every kingdom of life. In contrast to the Sec transport system which exports unfolded proteins; the Tat pathway acts to translocate fully folded proteins across energy transuding membrane lipid bilayers.

Targeting in the Tat pathway to a membrane-embedded Tat translocase is by a conserved, N-terminal twin arginine (SRRXFLK) amino acid motif which is cleaved following the transportation event.

As the majority of Tat substrates are metal cofactor binding redox enzymes, premature export must be prevented. Proofreading chaperones perform this function by binding the signal peptides of immature proteins only releasing mature protein once fully folded to interact with the translocase.

A key molybdoenzyme under investigation in the studentship was the E. coli Tat-targeted trimethylamine N-oxide (TMAO) reductase (TorA), the signal peptide of which is bound the archetypical Tat proofreading chaperone, TorD.

Part I: Investigation of the TorD Chaperone Protein Release Mechanism

The first part of the summer project was concerned primarily with the isolation of various mutant TorD chaperone proteins irreversibly locked onto a TorA signal peptide.

To identify mutated TorD chaperones, a facile genetic screen was set up in which a TorD mutant library was co-expressed with a fusion protein consisting of the enzyme chloramphenicol acetyltransferase (CAT) and a TorA signal peptide ~ 30kDa.

If cytoplasmically located, CAT acts to provide chloramphenicol resistance. However, due to its fusion to the TorA signal peptide in the construct, CAT will be transported through the Tat system to the periplasm, negating resistance. In this experiment, the fusion protein was co-expressed with a mutant TorD library. If TorD were mutated in such a way that it would irreversibly bind to the TorA signal peptide, transport would be prevented, CAT would not be exported and growth of cells in the presence of chloramphenicol would be possible.

Before this screen, it was first established the level of chloramphenicol the wild type MG1655 E.coli cells could grow at through plating on LB + Cm0 + Cm25 + Cm50 + Cm100 + Cm200. It was determined that cells could grow media containing concentrations of up to Cm50, meaning any growth above this in the screening experiment was due to chloramphenicol resistance conferred by a mutated TorD chaperone.

Growing on plates of Cm100, 8 x Chloramphenicol resistant colonies were selected, miniprepped and sequenced to identify TorD mutations.

Sequence analysis showed an even distribution of mutations with an area of high conservation particularly at the N-terminus. One interesting observation was the apparently ability of several TorD chaperones to bind the TorA signal peptide despite a large truncation in their amino acid sequences.

Using a TorD model created by the PHYRE automatic fold recognition server, mutations were mapped to the structure to allow for visualisation of changes. Again, mutations were shown to be evenly distributed with no discernible areas of concentrated mutations / conservations other than an apparent preference of alpha-helical mutations over disordered loops.

Part II: Blocking the Tat System

In E. coli, five Tat substrates have hydrophobic transmembrane helices (TMs) as part of their structure. As such, the Tat translocase is capable of recognising, handling and inserting TMs into the membrane.

In this part of the studentship, a fusion protein comprising of a TorA signal peptide, fused to CAT, fused to various the TMs of the Tat translocase (derived from TatA, TatB and TatC), fused to Maltose Binding Protein followed by a His tag was utilised. It was hypothesised that by utilising TMs of the Tat translocase; the Tat translocase itself could be blocked.

An interesting phenotype of E. coli blocked for Tat transport is their inability to grow on media containing 1-2% (w/v) SDS. This quality was exploited to determine if the Tat system was being blocked. Disappointingly, through the presence of growth (albeit at a reduced level), it was suggested that the Tat system was not being blocked by the fusion protein and was continuing unaffected.

To determine why the Tat system continued unimpeded, Western blot analysis was utilised. Blotting for the fusion protein showed that although some of the protein existed in its full form, much of it was being degraded within the cell thus negating its effect. As such, although some of the Tat system may have been blocked, enough existed unobstructed so as to continue its normal phenotype.

Further to this, the cells were broken by French press and the membranes and soluble fractions obtained through ultracentrifugation to localise the fusion protein which ultimately appeared dispersed throughout the fractions, again in its full and degraded forms.

Possible Future Experiments:

From just 8 TorD sequences, it is difficult to identify definite areas of conservation / mutation involved in irreversible chaperone binding. Further sequencing of more TorD mutants would allow greater confidence in this matter. Following this, mutations proposed to be responsible for this phenotype could be explored individually. As mentioned, the PHYRE server was used to create a model of TorD – a true x-ray diffraction model of this protein would obviously allow for more accurate mapping of mutations.

In the case of the second fusion protein, the inability of the construct to block the Tat system prevents the pathways biochemical dissection. As such, the fusion protein must be reconstructed.

Departures from the Original Proposal:

None to report.

Value of Studentship:

Not only did the placement provide me with an understanding of a number of laboratory techniques but more importantly, it provided me with a confidence regarding those I am yet to master. Thanks to the friendliness, help and patience of the entire Sargent / Palmer laboratory, I am entering my final year prepared, focussed and eager for the upcoming challenges.

My thanks are also extended to the Biochemical Society and the University of Dundee - Molecular Microbiology Department.

Student: Sam Darby Supervisor: Dr. Nic Harmer Institution for placement: University of Exeter Crystallisation of chaperones from Burkholderia pseudomallei

Background Burkholderia pseudomallei is a Gram-negative bacteria endemic to Thailand and Northern Australia. It is the causative agent of melioidosis. This disease has several clinical forms depending on the route of infection. Inhalation through the respiratory tract can cause pneumonia and pulmonary abscesses. Subcutaneous entry can cause ulceration. Immunocompromised individuals can develop septicemia. This maybe overpowering, with a 90% fatality rate if untreated and death occurring within 24-48 hours. In Northeast Thailand, there are 4.4 cases of melioidosis per 100,000 per year. 1Gene sequencing in B. pseudomallei has identified two genes L1418 and L0659. Both proteins are found in the periplasm and have roles in outer membrane biogenesis. L0659 is a homologue of SurA, a peptidyl prolyl isomerase (PPIase) that assists in the folding of β-barrel outer membrane porins (OMPs).2 Porins exchange materials with the periplasm and the outer membrane. L1418 is a putative exported isomerase that may help fold proteins that make up the outer membrane. Both proteins are putative chaperones. These proteins help fold and protect unfolded proteins from aggregation as they pass through the periplasm to the outer membrane. The crystal structures of both L1418 and L0659 may present new targets for antimicrobials. Aims of the Experiment

1. Express recombinant proteins from E.coli. 2. Purify the recombinant proteins for crystallisation and other experiments. 3. Determine the structure of the proteins using X-ray crystallography and other

experiments. Synopsis of work done E.coli cells were used to express either the L1418 or the L0659 gene. Nickel affinity and size exclusion chromatography was used to purify L0659. L1418 underwent hydrophobic interaction chromatography (HIC) before the size exclusion chromatography. A qPCR machine was used to monitor the thermal stability of L1418 under a range of conditions. SYPRO orange was used to monitor protein unfolding in respect to temperature. JCSG+ screen and PACT screen were used to determine favourable conditions for crystallisation. Results from this, allowed construction of screens which should have produced better crystals. Limited proteolysis using several different proteases was used to determine regions of L0659. Discussion SDS PAGE electrophoresis of L1418 showed a protein at 29 kDa, which was in agreement with the predicted value from the sequence. L0659 similarly showed an apparent molecular weight in agreement with the prediction. Nickel affinity chromatography and size exclusion chromatography failed to produce L1418 pure enough for crystallisation, which can be seen from figure 1. I therefore decided to use HIC to improve the purification. The protein bound very strongly to a phenyl-sepharose column, and so I had to use 30% isopropanol to remove the protein from the column. This produced a sample pure enough for crystallisation. SDS-PAGE revealed that the sample was resilient to aggregation in the 30% isopropanol, as no protein was apparently aggregating over 2 hours. One possible reason for this is that L1418 might be a heat shock protein: the chaperone might be produced at higher levels to

Figure 1 SDS PAGE gel. Lane 1 represents the molecular markers. Lane 2 represents the sample with the cells. Lane 3 represents the sample with no cells entered into the nickel column. Lanes 4,5 and 6 are fractions eluted by buffers containing increasing imidazole. Lane 7 is the sample entered into the size exclusion column. Lanes 8, 9, 10 and 11 are fractions from size exclusion chromatography. The band at 29 kDa is believed to be L1418. Contaminates of

stop aggregation of proteins involved in outer membrane biogenesis, and so be naturally resistant to harsh environments. Evolution may have produced a chaperone resistant to a range of denaturing agents so other proteins are not denatured and folded properly. Although L0659 was not purified using HIC and the harsh solvent, it might have the same resistance to aggregation.3 Bioinformatically, I predicted that L0659 will have 3 domains. These are a SurA N-terminal domain; a first, large rotamase domain has, and a second, smaller rotamase domain. Since these domains all share homology with the SurA protein found in E.coli, their functions can be hypothesized as being similar. SurA N-terminal domain may recognise a specific residue pattern on β-barrel porins and may provide most of the chaperone activity, moving the porins from the periplasm to the outer-membrane.4 One of the rotamase domains recognises specific aromatic residues of β-barrel porins.5 Both of these domains act as a highly specific recognition substrate system. This means unfolded proteins which are not destined for the outer membrane will not be incorrectly inserted. The other rotamase domain catalyses cis-trans isomerization of proline imidic peptide bonds in both native and non-native β-barrel porins.6 Limited proteolysis using prothrombin after 30 minutes showed a fragment of the protein at 31 kDa. From the SurA structure (figure 2) it could be assumed the prothrombin cleaved the linker region between the two rotamase domains .7 The rest of the structure without the second rotamase domain would be approximately 31 kDa. Thermofluor analysis of L1418 showed it to be most stable in Sodium acetate pH 4.6. The background fluorescence at the start of the experiment could indicate the protein was already partially unfolded or the protein has high hydrophobicity. Ericsson found samples, which exhibited high initial fluorescence intensity, did not crystallise properly.8 This may offer an explanation as to why L1418 failed to crystallise. L0659 did crystallise. 0.5 µL of L0659 was found to produce the best crystals under 11 % PEG 3350, 0.1 M NaBr, 0.05 M borate pH 8.5 which produced the crystal seen in figure 3. Future ambitions for Project L1418 failed to crystallise under a range of conditions. Using PEG at different MWs may produce precipitation.L0659 crystallised, but the widths of the crystals were not wide enough for diffraction to yield good results. Alterations in pH may slow the rate of supersaturation, decreasing the number of crystals and produce a large enough crystal for X-ray diffraction. Alternatively, L0659 crystals could be used to macroseed crystals. Outcome of the Studentship I found this internship to be valuable for improving both my technical skills and overall competence within the lab. I feel much better qualified to work within in the lab and I am very grateful for what Dr Nic Harmer has taught me over the past 8 weeks. References 1 White NJ (2003). Melioidosis. Lancet. 361, 1715–22 2 Xu, X., Wang, S., Hu, Y.X., McKay, D.B., (2007). The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues. J. Mol. Biol. 373, 367–381. 3 Bergeron, L.M. , Lee, C. , Tokatlian, T. , Hollrigl V. , Clark., D.S. , (2008) .Chaperone function in organic co-solvents: experimental characterization and modeling of a hyperthermophilic chaperone subunit from Methanocaldococcus jannaschii, Biochim. Biophys. Acta 1784 368–378

Figure 2 Ribbon drawing of SurA showing the structural arrangement of the N-domain (blue), C-domain (red), and P1 (green) and P2 (yellow). Bitto, E, and McKay, D. B. (2002). Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins. Structure. 10, 1489-1498 reproduced in Behrens, S., (2002). Periplasmic Chaperones—NewStructural and Functional Insights. Structure. 10, 1469-1471

Figure 3 Crystal of L0659. The conditions for crystallisation were 11 % PEG 3350, 0.1 M NaBr, 0.05 M borate pH 8.5

4,5,6, Xu, X., Wang, S., Hu, Y.X., McKay, D.B., (2007). The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues. J. Mol. Biol. 373, 367–381. 7 Behrens, S., (2002). Periplasmic Chaperones—New Structural and Functional Insights. Structure. 10, 1469-1471 8Erricson, U.B., Halberg, M. B., Detitta, T.G., Depper, Niek., (2006). Thermoflour-based high throughput stability of optimization of proteins for structural studies Anal. Biochem. 357, 289-298

Biochemical Society summer studentship report 2010 Student: Shintaro Aibara Supervisor: Dr. Paul Race

Structure and mechanism of a polyketide synthase trans-acting enoyl reductase

Background Modular polyketide synthases (PKSs) are a family of giant multifunctional enzymes responsible for the biosynthesis of numerous clinically important bioactive natural products, including the broad spectrum antibiotic erythromycin and the anticancer immunosupressant rapamycin. Recently, a novel family of modular synthases termed trans-acyltransferase (trans-AT) PKSs have been identified, whose biosynthetic frameworks differ significantly from those of the well studies canonical cis-AT systems. Trans-AT PKSs are an attractive target for study, as manipulation of this pathway may be used to generate derivative polyketide products with improved pharmacological properties. The aim of this project has been to structurally and functionally characterise the trans-acting enoyl-reductase cPksE from the prototypical trans-AT system bacillaene synthase. Our initial goal was to recombinantly express and purify cPksE to homogeneity and subject this material to crystallisation screening, biochemical, biophysical and kinetic analysis. Description of Work cPksE, a 53 kDa protein, was recombinantly expressed in E. coli BL21 (DE3) cells pre-transformed with a cpksE::pOPIN-F expression vector. In this system the cpksE gene with is under the control of lacZ, allowing IPTG induced expression. The bacteria were harvested by centrifugation, sonicated, and cPksE subsequently purified from the cell lysate using a combination Ni2+ affinity and size exclusion chromatography. Crystallisation plates were set and steady-state kinetic analyses of cPksE were performed. Spectroscopic methods were also used to characterise the proteins identified flavin cofactor. Results Expression and purification. Purification of the His-tagged cPksE by affinity and size exclusion chromatography was performed successfully, with a band corresponding to cPksE identifiable by SDS-PAGE (Figure 1a, 1b). Analysis of the fractions eluted from the final SEC step suggested to the protein to be of >90 % purity and suitable for crystallisation screening and further characterisation (Figure 1). Interestingly cPksE was found to be yellow in colour suggesting a bound flavin cofactor.

a)

b)

Figure 1: Chromatograms and SDS-PAGE analysis of cPksE fractions eluted from (a) Ni2+ affinity column and (b) size exclusion chromatography S200 column. Crystallisation. Purified recombinant cPksE was subjected to crystallisation screening using the sitting drop vapour diffusion method. Nucleation was observed in a single condition (Figure 2), though this was not accompanied by crystal growth.

Figure 2: Nucleation of tagged cPksE observed in 0.1 M Hepes, 10 % PEG 6000, pH 7.0. More crystallisation trays were set up using cPksE from a fresh preparation. To aid crystallisation endoproteinase Glu-C was added to the protein (1:1000 ratio). Such in-drop proteolysis has been shown to improve crystallisability through the generation of truncated fragments of proteins. This protease was

identified as the most suitable by conducting test digests with 6 different proteases (Figure 3). From these experiments a single crystal was identified and is awaiting X-ray diffraction analysis. (Figure 4). 1 2 3 4 5 6 7 8

Figure 3: 15 % SDS-PAGE analysis of protease digestsof cPksE. Lane (1) Mw Marker, (2) cPksE alone, (3) α-chymotrypsin, (4) Trypsin, (5) Elastase, (6) Papain, (7)Subtilisin, (8) Endoproteinase Glu-C.

Figure 4: cPksE crystal grown in the presence ofendoproteinase Glu-C, 0.1 M NaCl, 0.1 M Na HEPES,1.6 M ammonium sulphate, pH 7.5. cPksE cofactor analysis. To identify whether cPksE bound FAD or FMN as a cofactor, cPksE was thermally denatured, the precipitated protein removed by centrifugation and the resulting free cofactor analysed by UV-vis spectroscopy (Figure 5).

Figure 5: Free cofactor from cPksE has an absorbancepeak at 444.5 nm suggesting it binds FMN, not FAD. Steady state kinetics. The specificity of cPksE for NADH or NADPH was determined by measuring the cPksE catalysed conversion of NAD(P)H to NAD(P) at

340 nm, using molecular oxygen as the second substrate (Figure 6).

Figure 6: cPksE appears to favour NADPH turnover compared to NADH turnover when oxygen is the second substrate. Further directions of the project Although we did identify a crystal in our screening experiments this has yet to be tested for diffraction quality with this being the next priority. Given this came from one of the proteolysis experiments, in the future it would certainly be worth generating some truncated constructs of cPksE for further crystallsation screening. Cleaving off the His-tag from cPksE would also be a logical step. The kinetic data generated all use molecular oxygen as a second substrate. To simulate a more biologically relevant condition, the reactions should be carried out in anaerobic conditions with the natural substrate of the PKS pathway. Value of studentship Student. Working in the lab has given me an opportunity to understand what it is to be a research scientist. I have gained valuable laboratory skills that are not provided as part of my degree, such as techniques involved in protein crystallisation. From this 8 week project I have confirmed my passion for scientific research in the field of crystallography, and will use the experience to help myself excel in my upcoming career. Supervisor. A very worthwhile exercise allowing us to work with a gifted student on one of our priority projects.

Student: Sinéad O’Hara Supervisor: Dr Mark Dillingham, DNA-protein interactions unit, School of Biochemistry, University of Bristol

Mapping the site of interaction between PcrA helicase and RNA Polymerase

Introduction UvrD-like helicases are members of Helicase Superfamily 1 (SF1) and are involved in many cellular DNA transactions including DNA replication, recombination and repair. Recent work suggests that the cellular function of these helicases is often programmed by interactions with partner proteins. For example, an interaction between E. coli Rep helicase and DnaB targets the enzyme to the replisome where it helps the progression of the replication fork through regions of protein-bound DNA (Guy et al. 2009). Using a pulldown approach, the Dillingham laboratory has recently detected a physical interaction between E. coli UvrD and RNA polymerase (RNAP) that is also conserved in the Gram-positive model bacterium B. subtilis since PcrA (a UvrD homologue) also interacts with RNAP. This project aimed to map the site of this interaction by testing individual PcrA domains for their ability to interact with RNAP (Figure 1). The accessory domains 1B and 2B, as well as the C-terminal extension (Ct) of PcrA were considered good candidates because they are the most variable between the UvrD-like family whereas the core domains are involved in the catalytic activity of the protein. Furthermore, this project aimed to investigate the possibility that the PcrA-RNAP interaction was DNA-dependent (and a possible artefact) by testing a mutant PcrA protein that was defective in DNA binding for its ability to interact with RNAP (Dillingham et al., 1999).

Figure 1 –UvrD-like helicases consist of 2 core domains (1A and 2A, red and blue) that contain the catalytic machinery for ATP-dependent translocation and the two accessory domains (1B and 2B, yellow and green). PcrA and UvrD also share a conserved C-terminal extension that is not resolved in the structures and is probably natively disordered.

Aims 1. Express and purify biotin-tagged PcrA constructs (bioPcrA-1B and bioPcrAW259A). 2. Assay for interaction between RNAP and PcrA constructs (bioPcrA , bioPcrAW259A, bioPcrA-1B, bioPcrA-2B, bioPcrA-Ct)

Work carried out Expression and purification involved growing E.coli BL21 DE3 cells containing plasmids pET22b (for helicase expression) and pBirACm (encodes for biotin ligase, Avidity) at 37°C. Biotin tags were incorporated in vivo by covalent modification of an N-terminal 15 amino acid tag contained within the biotinylated helicase constructs. Cells were induced with IPTG, grown for 3 hours, harvested and then lysed by sonication. To remove cell debris the cells underwent centrifugation and the soluble supernatant containing bioPcrAW259A was precipitated by ammonium sulphate before being loaded onto a monomeric avidin column. Free biotin was used to elute the protein which was then applied to a monoQ column to remove contaminating biotin. bioPcrAW259A was concentrated by dialysis and stored in aliquots at -80°C. Pulldown experiments and prey protein identification involved applying the biotinylated helicase constructs to streptavidin-coated magnetic beads. Any unbound proteins were washed off before B.subtilis cell lysate was applied to the beads. The proteins in the cell lysate were left to interact with the biotin-tagged proteins and any unbound proteins from the cell lysate were removed in two wash steps. The samples were boiled in SDS-PAGE loading buffer to remove the biotin-tagged proteins from the magnetic beads. These samples were then analysed by SDS-PAGE. As a negative control ‘mock’ pulldowns were carried out in which the streptavidin beads were un-baited, but all other conditions were the same. As a positive control the established interaction between full length bioPcrA and RNAP was re-capitulated. Results and outcome Expression and purification of the mutant bioPcrAW259A was successful (Figure 2) and this protein was used for subsequent pulldown experiments. The bioPcrA-1B sub-domain was well expressed but found to be insoluble and expression at 27°C did not alter this (data not shown). Therefore we were unable to probe for an interaction between this sub-domain of PcrA and RNAP. As a positive control in pulldown experiments, we confirmed that PcrA interacted with RNAP as had been demonstrated previously (Lane 3, Figure 3A). The β and β’ subunits of RNAP are easily identified on SDS-PAGE gels as a characteristic doublet band running at approximately 150kDa (See red arrow on Figure 3A). The negative control ‘mock’ pulldown experiments showed no interaction with RNAP as expected. The mutant protein bioPcrAW259A showed that it interacts with RNAP in a manner indistinguishable from the wild type (Figure 3A). Together with previous observations that the pulldown still occurs in nucleic acid-depleted cell extracts, this suggests that the interaction is DNA-independent. The subdomain bioPcrA-2B was then tested and did not interact with RNAP (Figure 3C). This either means that there is no site of interaction with RNAP in domain 2B or that

the interaction is too weak to detect in this assay. The final pulldown assay showed that the biotinylated C-terminus of PcrA interacts with RNAP (Figure 3B). From the intensity of the RNAP doublet band, the quantity of RNAP bound to the construct was comparable to the equivalent experiment with full-length PcrA. This result suggests that the C-terminal 80 amino acids of PcrA constitutes the major site of interaction with RNAP.

Fractions off Avidin column

Fractions off monoQ column

M CL L FT E B1 C1 C2 C3 C4

Figure 2 – Stages in bioPcrAW259A purification. M=marker, CL=cell lysate after sonication, L=sample loaded onto column, FT=flow through, E=eluate. The green arrow indicates the position of bioPcrAW259A seen in E.

Figure 3 – Pulldown experiments. The position of the bait and RNAP prey (large subunits) are marked with black and red arrows respectively. (A) Pulldown with bioPcrAW259A. (B) Pulldown with bioPcrA-Ct. (bait too small to resolve on gel) (C) Pulldown with bioPcrA-2B.

Departures from original proposal We were unable to test for interactions between the PcrA-1B subdomain and RNAP as planned due to insolubility problems. In addition to the experiments suggested in the original proposal, we also tested for the ability of a DNA binding defective mutant of PcrA to interact with RNAP. Future direction These experiments have suggested that the C-terminus of PcrA is the major site of interaction with RNAP. To test for a complete loss of this interaction a PcrA construct without the C-terminus should be produced. Multiple sequence alignments of the PcrA/UvrD C-terminal extension suggest several conserved residues as targets for point mutations that might eliminate interaction with PcrA and further refine the interaction site. In vivo analysis of the phenotypes associated with the mutant protein might help elucidate the role and significance of the PcrA:RNAP interaction. To see if this interaction is conserved across bacterial species testing for an interaction between the C-terminus of UvrD and E.coli RNAP should be investigated. However, since the interaction between PcrA and RNAP might not be limited to the C-terminus, further experiments to express the PcrA-1B sub-domain using different approaches should be carried out. These could include attempts to purify the 1B domain from inclusion bodies or a re-design of the expression construct for the 1B domain. Value of internship to lab Sinead’s work has made a key contribution to my lab by localising the major site of interaction between PcrA and RNAP to the C-terminal extension. This result has interesting implications for the structural basis by which helicases interact with partner proteins because we have shown previously that the equivalent region of Rep helicase is involved in interaction with DnaB. Moreover, this work will now rapidly enable us to home in on a UvrD/PcrA mutant protein that is defective for this interaction, but otherwise active as a helicase, enabling us to examine the importance of the interaction in vivo. My experience As early as the first week of my studentship I could see how the work I carried out would help me in my final year project. It has greatly enhanced my existing lab skills making me feel much more confident in approaching the project next year. It was fascinating to see techniques, such as column chromatography and growing and harvesting cells that had been described in lectures, come to life in my work. I really enjoyed learning and executing fundamental techniques as well as new, more complex tasks such as streptavidin pulldown assays. As well as learning new laboratory skills I have developed my ability to work independently, plan experiments and present my findings, all which will be useful in other walks of life. Overall I have gained a valuable insight into working in a laboratory environment that has confirmed my desire to pursue a career in research science. References Dillingham et al. Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation. Nucleic Acids Res. 1999 Aug 15;27(16):3310-7.

Student: Sinéad O’Hara Supervisor: Dr Mark Dillingham, DNA-protein interactions unit, School of Biochemistry, University of Bristol Guy et al. Rep provides a second motor at the replisome to promote duplication of protein-bound DNA. Mol. Cell 2009 Nov 25;36(4):654-66

Analysis of ipid raft clustering and assembly of NADPH oxidase in oxidatively stressed

neutrophil-like dHL-60 cells.

Student: Tehreen Dharamshi Supervisor: Dr. H.K.Irundika Dias, Aston University, Birmingham

Background Neutrophils represent the first line of defence against invading microorganisms. An important part of this defence mechanism is the generation of superoxide radicals and its reactive derivatives. Mass production of superoxide radicals termed as respiratory burst is mediated by the activation of NADPH oxidase enzyme complex. Upon stimulation, NADPH oxidase subunits that reside in the cytoplasm; p40phox, p47phox and p67phox are brought into the vicinity of the plasma membrane and then conjugated with its membrane subunits; gp91phox and p22phox. In chronic inflammatory diseases circulating neutrophils show enhanced respiratory burst resulting tissue damage. . Lipid rafts (LRs); cholesterol-rich, detergent-insoluble membrane compartments help the assembly of NADPH oxidase subunits by acting as platforms. LRs are composed of glycosylphosphatidylinositol linked proteins, glycosphingolipids, and cholesterol that are associated with signalling molecules, eg; receptors such as G-proteins. Previous studies have shown that increased LRs formation could enhance the activity of NADPH oxidase complex.

Aims Differentiate HL-60 cells into neutrophil-like cells

Induce oxidative stress and measurement of respiratory burst in differentiated cells

Isolate lipid rafts and investigate assembly of NADPH oxidase subunits, p47phox and gp91phox

Description and results of work carried out………………………………………………………………………………………………………….. HL-60 cell line is a cell line that exhibits an aggressive, malignant and proliferative potential in vivo but can be induced to differentiate terminally when challenged in vitro. HL-60 cells can differentiate into neutrophil-like cells when challenged with 1% DMSO for five days (dHL6 cells). dHL-60 cells can be characterised by the expression of CD11b and the production of respiratory burst stimulated by a pharbol myristic acid (PMA). Initially, I observed HL-60 cells (Figure 1A) increases granularity, a typical phenotypic characteristic of primary neutrophils (Figure 1B) and increased cell surface CD11b expression (Figure2) as measured by flow cytometry.

Figure 2: Expression of CD11b. Both undifferentiated and differentiated HL-60 cells were labelled with RPE anti-CD11b antibody

Figure 1: HL 60 cell size and granularity. Undifferentiated cells are smaller in size and less granular with a lower value of side-scatter (SS) and forward-scatter (FS) values

Undifferentiated HL-60 cells


Differentiated HL-60 cells

Differentiated HL-60 cells


CD11b

Furthermore, the ability of dHL-60 cells to produce an extracellular respiratory burst upon a stimulus was measured by lucigenin-dependent chemiluminescence assay. An increased respiratory burst stimulated by PMA was observed in differentiated dHL-60 cells (Figure 3). dHL-60 cells were treated with Buthionine sulfoximine (BSO) for 24 hours, to deplete cellular GSH level and to induce intracellular oxidative stress. Cells exposed to BSO had five fold less GSH pool compared to none treated dHL60 cells (Figure 4) and showed higher respiratory burst upon PMA stimulation.

Up regulation of respiratory burst in response to stimulus in oxidatively stressed dHL-60 cells and the reduction in the ratio of GSH/protein in cells subjected to BSO suggests that this environment favours NADPH oxidase activity. Therefore, I analysed the distribution of NADPH oxidase subunits into lipid rafts using sucrose gradient ultracentrifugation followed by SDS-PAGE and western blot techniques.

Figure 5: The raft marker flotillin associated within dHL60 cells confirms the presence of lipid rafts and Nadp oxidase subunit gp91phox co-localised with the same fractions in PMA stimulated cells

Departure from the application: The original plan remained unchanged. However, final lipid raft experiments had to cut short due to time constrains.

Value of the studentship to the student: My first experience working in a research laboratory led to the laying of a springboard into the future of pursuing my career as a PhD researcher and carrying out dissertation in my final year. I managed to master the standard molecular biochemical techniques such as tissue culture, PAGE and western blotting and operate the flow cytometry, luminometer, colorimenter and carry out western blots. Moreover, the placement polished my organisational and communication skills and managed to learn the procedures of carrying out a research, including health & safety regulations, laboratory procedures and note-making skills.

Value of the studentship to the laboratory: Tehreen had been an excellent student during her time in lab and research. She very easily integrated herself into lab team and involved in our weekly journal club presentations. Tehreen generated very important data that could be help us in a future publication. This studentship has given me a great opportunity to experience research student supervision as an early career researcher.

Figure 3: Differentiated cells show high respiratory burst.when stimulated with PMA

Figure 4: BSO deplete intracellular GSH pool in dHL60 cells

Student; Therese W. Herling Supervisors; Maksim Tsytlonok and Dr. Laura Itzhaki Institution for placement: MRC Cancer Cell Unit, Cambridge

Page 1 of 3

Report for the Biochemical Society Introduction PR65/A is the scaffolding subunit of the heterotrimeric phosphatase 2A, PP2A, where it binds a catalytic- and a regulatory subunit. PP2A is involved in the regulatory pathways of many important events in the cell cycle such as; DNA replication, RNA splicing and translation, and cytoskeleton rearrangement. Mutations in the subunits of PP2A are associated with a number of cancers. PR65/A is a 65 kDa linear repeat protein consisting of a tandem array of 15 HEAT repeats, each of 39 amino acids with α-helical secondary structure. As opposed to globular proteins linear repeat proteins such as PR65/A lack long range interactions within the primary structure, which makes them good systems for folding studies. Aims The Itzhaki lab is interested in understanding the folding, stability and function of linear repeat proteins in order to use them as relatively simple models for studies of biological folding and unfolding processes. The aims of this project were to obtain PR65/A mutants for single-molecule, sm, experiments; engineering cysteine-to-serine mutations for sm fluorescence resonance energy transfer, smFRET, experiments, and truncated variants for atomic force microscopy, AFM. Once a non-labelling phenotype without any exposed cysteine residues was obtained, pairs of cysteines could be (re)introduced for fluorophore labelling and FRET measurements. If the repeat protein folds and unfolds via a number of pathways, different populations would be detectable via single-molecule, sm, experiments, whereas ensemble studies only show an average population. Materials and Methods • A pET28a vector construct was used. Site-directed mutagenesis, SDM, and round-the-horn polymerase

chain reactions, PCR, were used to replace the cysteines. Parental DNA was digested with Dpn1, and the result verified by EtBr agarose gel electrophoresis.

• E. coli XL-1 cells were used for generation of DNA for minipreps, and C41 cells were used for protein expression. Kanamycin resistance was used to select for transformed cells.

• C41 cells were grown to an OD of 0.6-0.9 at 37˚C, expression induced with 0.25 mM IPTG, and the temperature reduced to 26˚C overnight.

• Cells were collected, lysed using a cell cracker, centrifuged, and PR65/A isolated from the soluble fraction with a Ni-NTA2+ resin, anion exchange chromatography and gel exclusion chromatography. Protein expression and purity were verified and monitored throughout with SDS-PAGE.

• Protein was incubated in solutions conatining 0-8 M urea, Trp fluorescence was measured from 320 nm to 370 nm, and the denaturation curves were then plotted with Trp fluorescence/A.U. against [urea]/M at 340nm. Unfolding of the α-helical structure at increasing urea concentrations was monitored by CD via a decrease in ellipticity at 222 nm.

• The fluorescent dye Alexa-488 was used to test the labelling efficiency of the cysteine mutants; intermolecular disulfide bonds were first removed with an excess of DTT, the DTT was then removed by buffer exchange, using a PD10 column, before the dye was added at a 1:1 protein:dye ratio. Unbound dye was removed after 1h incubation, and the labelling efficiency found by calculating the dye:protein ratio from absorbance measurements at 280nm and 488nm. In order to avoid photobleaching of the dye, the experiment was performed in the dark as far as possible.

An assessment of the results and the outcomes of the studentship PR65/A has 14 cysteine residues in total, six of which had been identified as solvent exposed from the crystal structure of PR65 (fig. 1), four of which had already been replaced before I began (C86, C315, C375 and C390).


Page 2 of 3

The stability of PR65/A with C294 and C327 removed was tested by Trp fluorescence, (fig. 2). PR65/A unfolds via a hyperfluorescent intermediate. The stability of the tertiary structure did not appear to be altered drastically by the cysteine mutations, with only slight shifts in the unfolding transitions, when monitored with Trp fluorescence. However, once obtained, the hexa-mutant was found to still bind the fluorescent dye, Alexa-488, with a labelling efficiency around 30%. CD was used to investigate whether the mutations introduced had destabilised the α-helical secondary structure. Here the first transition, from the folded state to the intermediate state, had been shifted from 2 M to 1.5 M urea, indicating a loss of stability. Although the cysteine residues appear inaccessible for labelling in a space-filling model of the crystal structure, the dynamics of the protein in solution may have transiently exposed some of the cysteines for labelling.

At this point the studentship was coming to an end. So, although I removed a further three cysteines, C146, C151, C510, I did not have time to purify this variant or characterise its stability and labelling efficiency. I also made several truncated variants for AFM studies. Some gave no expression (repeats 3-9.5, 6-15 and Δ2-3), but 1-9.5 and 1-14 were purified. Future directions in which the project can be taken That PR65/A accommodates the multiple cysteine to serine mutations, without great losses of stability, is encouraging with respect to further replacement of cysteines. Additionally studies of the dynamics of the cysteine mutants would be interesting to see, whether increased dynamics could have exposed buried residues

for labelling. Introduction of cysteine pairs in different repeats would enable folding analysis by FRET. Any departures from the original proposal The original proposal was to look at degradation of repeat proteins by the Clp proteins, but the lab had just started to do sm experiments on various repeat proteins with exciting results. Therefore it was decided that I would make labelled repeat proteins for sm experiments of Clp-mediated degradation. However, the molecular biology to create a non-labelling phenotype of PR65/A took longer than expected which delayed the project and therefore there was not enough time to perform degradation assays on the repeat proteins. Value of the studentship During the studentship I have undertaken some of the ongoing projects in the lab and made progress in purifying truncations and removing cysteines from PR65/A. The work has contributed to the data on PR65/A and will be published. To me the studentship has been very valuable in that I have become familiar with many of the procedures in protein expression and purification, techniques for the molecular biology to create

Figure 1. The structure of PR65/A (PDB ID; 1B3U) with the exposed cysteine residues shown in red, and those deemed to be buried in green.

Figure 2. Trp fluorescence curve at 340 nm of wild-type (WT), quadruple mutant (QM) and hexa-mutant (HM). The data are normalised to the highest fluorescence intensity.


Page 3 of 3

desired mutants, and labelling with fluorescent dyes. I will be able to apply many of these skills in future lab projects. Furthermore the experience has made me certain in my wishes to pursue a research based PhD.

Student: Tom Lund, Supervisor: Alan Berry, Institution for placement: The University of Leeds

Figure 1 The chemical mechanisms of both Class I ( A) and Class II (B) aldolases. (A) The chemical mechanism of Class I aldolases such as NAL, which form a Schiff base using a catalytic lysine residue in their active site, resulting in an enamine intermediate. (B) The chemical mechanism of Class II aldolases such Fructose-bisphosphate aldolase forms an enediolate intermediate using a bound Zn2+ ion within their active site. Bolt, A., A. Berry, and A. Nelson, Directed evolution of aldolases fo rexploitation in synthetic organic chemistry. Archives of Biochemistry and Biophysics, 2008. 474(2): p. 318-330.

THE BIOCHEMISTRY OF ALDOLASES – THE DIRECTED EVOLUTION, REFOLDING CONDITIONS

AND THE EFFECTS OF LYOPHILISATION OF N-ACETYLNEURAMINIC ACID LYASE.The aldolase enzymes catalyse a reversible aldol condensation reaction, forming a new carbon-carbon bond, thus establishing

them as important components in synthetic organic chemistry. The aldolase family of enzymes fall into to two classes; Class I uses a catalytically active lysine reside within the active site to form a Schiff base with the substrate, subsequently forming an enamine intermediate, and Class II enzymes are metalloenzymes with a bound Zn2+ ion within the active site, which helps in the formation of an

enediolate intermediate. Both Class I and Class II aldolases form a new carbon-carbon bond and new chiral centres within the formed product, the mechanism of which are shown in figure 1.

N-acetylneuraminic acid lyase (NAL) is a class I aldolase which catalyses the reaction between N-acetyl-D-mannosamine (ManNAC) with pyruvate to form the sialic acid, N-acetylneuraminic acid. Sialic acids are common components of complex carbohydrates found on the surface of many mammalian cells and play a central role in intracellular adhesion, cell- cell and cell-small molecule recognition, and cell signalling [1]. However, two features of NAL limit the application of its synthetic abilities. Firstly, 2-3 carbon aldehydes are very poor substrates, and secondly it shows poor facial stereoselectivity. Thus studies into the directed evolution and alkylation of NAL to incorporate these truncated substrates would help to greatly increase the possible applications of NAL in synthetic chemistry.

Chemical modification

Alkylation reagents causes a modification of cysteine residues by the addition of alkyl groups onto the thiol. Alkylation of a key catalytic residue (165), to produce a lysine analogue with longer reach into the active site pocket is difficult due to the buried nature of this residue and requires a K165C mutation to react with the alkylating reagent. Thus the enzyme needs to be unfolded to effectively allow the alkylation procedure to take place. NAL has four cysteine residues. For the alkylation to be successful however, the protein must be unfolded, and consequently four cysteine residues have to be mutated. Here we mutate to either serine or alanine, creating the mutants C82S/C119S/k165C/C239S/C270S and C82A/C119A/K165C/C239A/C270A respectively.

Semi-rational redesign

The problem with many wild-type enzymes is that they are highly specific for both their substrate and its stereochemistry. We use semi-rational redesign to create a saturation DNA library at a specific amino acid site, Y43X. This combined method allows a non-specific single point mutation of the targeted residue Y43. Many new exciting developments have been made with incorporating fluorine into pre-established compounds, such as Cerivastatin, a new second generation HMG CoA reductase inhibitor[2]. This is a fluorinated analogue of first generation drugs Fluvastatin and Lovastatin, and has been shown to be much more efficient then its predecessors[2]. However, incorporation of fluorine into any compound can be difficult, but by evolving NAL to utilises a fluorinated substrate we could create a highly useful tool for organic synthetic chemistry.

Project Aims

To address the evolution of NAL to accept fluorinated substrates, the initial aim was to produce a saturation library for screening. Second, to further research into the possible alkylation of NAL, the necessary refolding conditions needed for NAL to refold back into its active conformation after alkylation were researched. Finally, for long term storage of the enzyme, the effects of freeze-drying the enzyme were studied.

Results and Discussion

Y43X Saturation Library

Tyrosine 43 was identified by the Berry Lab as a possible key residue in the activity of NAL because of its close proximity to the C3 of pyruvate and fluorinated substrates such as fluoropyruvate, in its Schiff base with K165. We used semi-rational design to create a saturation DNA library Y43 (Y43X). This combined method allows a non-specific single point mutation of Y43 and provides us with a profile of that particular position; what factors (hydrophobicity, size, reach in the active site etc.) contribute to the activity for both native and


fluorinated substrate and how these are changed by mutation at this site. To achieve this the Y43X FOR and Y43X REV primers kindly donated by the Berry lab have been designed to incorporate codons corresponding to residue 43, that contains an NNK codon to create mutants with all 20 possible amino acids at position 43. The addition of K (either T or G) helps to remove elements of codon bias, as well as eliminating 2 possible stop codons. After isolation and purification of each of the 20 possible different mutations (i.e. incorporation of each of the remaining 19 standard amino acids and 1 remaining stop codon), the effects on KM and kcat for both fluorinated compounds and for the native substrate pyruvate, can then be elucidated by further screening of these libraries.

Protein Purification

The SDS-PAGE gel shown in Figure 3 shows the successful purification of NAL. The NAL band seen after each wash is due to the presence of imidazole within the washing buffer. The presence of a small concentration of imidazole within this buffer increases the specificity of the interactions of the Ni bound to the resin and the protein. The Ni will bind to any aromatic group, potentially forming unwanted non-specific interactions with other proteins which have not been hexa-his tagged, and retaining them within the resin. Including a low concentration of

imidazole within the washing buffer out competes these non-specific interactions, however a small amount of NAL is removed (as can be seen by the presence of the NAL band after each wash) but helps to increase the purity of the final protein sample.

From figure 3, the NAL band shown by the green arrow in lane 3 is not present in lane 4 (purple arrow). NAL has successfully bound to the Ni-resin by the hexa-his tag and is therefore not precent in lane 4. Lane 5 - 8 have decreasing intensity of all other surrounding bands around the NAL band. This shows that after each wash, the protein is becoming more purified and more of the other protein is being lost per wash.

Protein refolding

The initial trial of this experiment lead to the inactivation of WT-NAL in 6 M urea by using dialysis and quick dilution methods, and so for practical purposes it was not necessary to

continue using the dialysis method as the quick dilution worked and took much less time and materials. However, this result was not then seen again thought out re-trials. Upon further investigation it was found that the urea concentration was not accurate in the original trial, but had to be corrected for using a volumetric flask. After this correction, both dialysis and quick dilution method could not take place as the WT-NAL would not unfold up to 8M urea, therefore refolding could not be attempted. The activity of WT-NAL could not be removed by urea up to a concentration of 8 M. The CD spectra as shown in figure 4 show that NAL is fully folded from 1 M Urea to 7 M Urea, and is partially unfolded at 8 M Urea.

Freeze drying

As can been seen by the results after freeze drying, in all three cases the KM and kcat for each of the samples were within acceptable experimental variables of that of WT-NAL that has not undergone freeze drying, thus it can be concluded that full kinetic activity has returned.

kcat (Average) / min-1

KM (Average) / mg/mL

kcat/KM

WT-NAL No freeze drying 800 1.9 400

WT-NAL after freeze drying in Ammonium acetate

900 4.3 200

WT-NAL after freeze drying in Ammonium Bicarbonate

500 2.9 200

WT-NAL after freeze drying in H2O 900 4.6 200

1

Figure 3. An SDS-PAGE Gel to show protein purification procedure. Lane 1 – Cell sample (10 x dilution),Lane 2 – Protein Ladder, Lane 3 – Soluble Sample, Lane 4 – Sample after Binding, Lane 5 – Sample after wash 1, Lane 6 - Sample after wash 2, Lane 7- Sample after wash 3, Lane 8 - Sample after wash 4 and Lane 9 – Eluted sample (5 x dilution)

5 6 8 7 3 2 4 9

NAL band

- 5 0

- 4 0

- 3 0

- 2 0

- 10

0

10

2 0

19 0 2 10 2 3 0 2 5 0 2 7 0 2 9 0

Wa v e l e ngt h / nm

WT - NAL

WT - NAL + 1M URE A

WT - NAL + 2 M URE A







0

200

400

600

800

1000

1200

190 210 230 250 270 290

Wavelength (nm(

HT

Volta

ge

Figure 4. The CD spectra of WT-NAL with increasing concentration of Urea. WT-NAL in the absence of urea has been

zeroed with NaPi, and all the values containing urea have been zeroed with a blank containing NaPi and Urea

Table 1. The kcat , KM and kcat/KM values for WT-NAL before it has been freeze-fryed, and after it has been freeze dryed in ammonium acetate, ammoinium bicarbonate buffers and H2O. kcat/KM is a measure of an enzyme’s catalytic effiency


References

1. Severi, E., D.W. Hood, and G.H. Thomas, Sialic acid utilization by bacterial pathogens. Microbiology, 2007. 153(Pt 9): p. 2817-22. 2. Ismail, F.M.D., Important fluorinated drugs in experimental and clinical use. Journal of Fluorine Chemistry, 2002. 118(1-2): p. 27-

33. Acknowledgements

I would like to thank Prof. Alan Berry for giving me the opportunity to undertake this project and for his guidance and support throughout. I would like to thank Nicole Timms and Adam Daniels for their continued support and supervision throughout this project, at the bench and with writing this report, without which any of this wouldn’t have been possible. And finally I would also like to thank the Biochemical Society for funding.

Value of the Summer Studentship to the student

This summer studentship was my first experience in a research lab. It has taught me many new practical skills (protein purification, SDS-PAGE, enzyme kinetic studies, protein refolding studies etc.) and has reinforced my existing biochemical knowledge. It has shown me how a modern biochemical lab functions on a day to day bias. Other important skills I have picked up along the way include time management, the ability to plan experiments from scratch, and how to incorporate the correct controls to validate the experiment. I have thoroughly enjoyed my short time in the Berry lab and it has been a rewarding and challenging experience which has reinforced my position to undertake a PhD after my degree.

Value of the Summer Studentship to the Lab

The Berry lab has benefited from the completion of this project, not only through the new understanding it has brought to light but also through the presence of the student in the lab. Tom always contributed to group lab activities and often presented a novel perspective in pursuit of answering ongoing questions. His help in setting up the freeze drying methodology will be of great value to the lab and his methods have already been followed on recent protein preparations. Furthermore, his documentation of the work done in the lab has been written up to a very high standard, providing excellent methodology resources for the group.

Student: Tom Green Supervisor: Dr Raffael Schaffrath Institution for placement: University of Leicester Kti12, a partner of the disease‐linked Elongator complex

Background It is essential for correct gene expression that tRNAs are able to accurately decode mRNAs; a process that relies on diverse tRNA processing steps, one of which is the modifications of wobble bases in the anticodon stem loop [1]. The importance of such modifications is highlighted when defects in genes that code for tRNA modification enzymes, and mutations in tRNA that prevent such modifications from being generated are associated with developmental and neurological disorders [1, 2]. Further more recent evidence demonstrates that such neurological disorders are associated with defects in Elongator [2], a six-subunit protein complex that is conserved from yeast to man [3], and exchangeable between yeast and plants [4]. Elongator has also been shown to be required for the modification (directly or indirectly) of the wobble uridine bases in tRNA anticodons [5]. It is still unclear as to whether Elongator is constitutively active or subject to dynamic regulation, however the supervisor’s lab have produced convincing evidence that Elongator is conditionally regulatable by reversible phosphorylation of the Elongator subunit 1 (Elp1) by kinase (Hrr25) and phosphatase (Sit4) activities [7, 8]. Kti12, a partner protein of Elongator, has been found to have a positive effect on tRNA modification [9]; with it being required for phosphorylation of Elongator by Hrr25 (directly or indirectly) [8]; however the exact method of Kti12’s action remains elusive. Intriguingly, Kti12 and its mouse homologue have been characterised as overexpression suppressors in a yeast model system for Huntington’s disease (HD). Also mutant huntingtin (the protein underlying HD) interacts physically and genetically with the mammalian homologue of Elp1 (IKAP). This reinforces the neurological relevance for Kti12 and Elongator, suggesting that the roles these proteins play in tRNA anticodon modification function are crucial for the development and generation of neuronal functions in mammals. Aims

• To produce double knockout (KO) strains Elp1ΔKti12Δ and Elp1Δ Sit4Δ. • To KO Elp1 in existing strains containing tagged proteins in the following combinations; Elp2- c-myc

Elp3-HA, Elp3-c-myc Elp5-HA, Elp2-c-myc Kti12-HA and Elp5-HA Kti12-c-myc. • To observe the affects on protein-protein interactions and tRNA modification when introducing amino

acid substitutions in the Elp1 subunit, via plasmids, into the yeast double KO strains and the tagged strains.

Methods The lab already had single KO strains with Elp1∆ for Tryptophan and Kanamycin, Sit4∆ for Histidine and Kanamycin, and Kti12∆ for Kanamycin. To create the double KO strains a PCR was set up to amplify the region of the deleted gene in the single KO strain. This PCR product was then transformed into a second KO strain to give the appropriate combination of KOs using the marker genes for selection. This same process was applied for the creating the tagged KO strains, where by the PCR product from an Elp1Δ for Kanamycin strain was transformed in the four tagged strains. Plasmids (WAP05, 06, 09, 19, 25, 26, 34, 46, 59, 64, 70, 71, 72, 73, 74, 75 and 76) provided by Prof M Stark (University of Dundee) contain the Elp1 subunit with various amino acid substitutions (see Fig.1. and Fig.2.). Plasmids were selected based on their substitutions (glutamate substitutions mimicking phosphorylation were selected for Kti12∆, while alanine substitutions mimicking dephosphorylation were selected for Sit4∆) and transformed into E.coli, grown for 24 hours, and then a plasmid preparation was carried out. The appropriate plasmids where transformed into the new KO strains. The tagged strains where analysed by immune precipitation whilst the double KO strains where assayed by zymocin, caffeine and temperature sensitivity. Departure from Original Proposal The project differed from the original proposal due to developments in the group’s research between the time of the original proposal submission and undertaking the placement. Rather than producing mutations in the Kti12 protein, mutations were introduced into the Elp1 subunit and the affect observed.

Results and Discussion S. cerevisiae becomes sensitive to caffeine with the loss of Elp1 and/or either Sit4 or Kti12. From the caffeine phenotype for the Elp1ΔSit4Δ double mutant (Figure 1) showed that most of the plasmids, with the exception of WAP5, WAP74, WAP75 and WAP76, were not able to compensate for the loss of Sit4 and Elp1 and thus where sensitive to caffeine. The Plasmids that did not show the caffeine sensitivity, WAP5, WAP74, WAP75 and WAP76 grew similar to the wild type. Of these caffeine resistant plasmids, WAP5, WAP74 and WAP75 all had phosphorylation-related substitutions at distinct Elp1 residues. This suggests that Sit4 dephosphorylates these residues, either directly or indirectly. This was also carried out with an Elp1ΔKti12Δ strain, however all of the transformants remained sensitive to caffeine. Due to problems with transforming the zymocin plasmids we were unable to get reliable results with regards to the strains zymocin sensitivity. We were also unable to get reliable results with the temperature sensitive tests.

Using the tagged strains we first established that the Elp1-HA and Elp3-HA subunits were both present when doing the immune precipitations for Elp2-c-myc with the Elp1∆ substitutions on WAP5, WAP 26, WAP72, WAP73, WAP74 and WAP75. From this we can conclude that the core Elongator complex of Elp1, Elp2 and Elp3 was interacting independent of modifications to these particular residues. We were unable to use the results from either of the strains that contained Elp5-HA as it appeared to be binding unspecifically to the agarose on the antibodies and thus was present in the positive control. The immune precipitation for the final tagged strain (Figure 2.) was done with Elp2-c-myc and showed that with all the substitutions in Elp1, Kti12 is still present. It was shown that Kti12 will only associate with the complete Elongator complex [10], and thus as all variations of the Elp1 subunit have Kti12 associated we can assume

that the Elongator complex is both complete and that the substitutions do not prevent the association of Kti12. Furthermore there is noticeably more Kti12 with WAP72 and WAP26, both of which have a common substitution in a phosphorylation-related Elp1 residue. Whilst a repeat of this experiment would be required we could suggest that because Hrr25 mutants are able to increase the quantity of Elongator-Kti12 interactions this shared residue in Elp1 could be phosphorylated by Hrr25 [8]. Thus a mutation in the residue that mimics phosphorylation could increase Kti12 interaction.

Fig.2. Interaction between Kti12 and Elongator Immune precipitates of strain expressing mutant forms of Elp1 were probed with anti-HA antibody to detect HA-tagged proteins and anti c-Myc for Elp2. Their positions are indicated by the arrows. Vector serves as positive control as cell is Elp1 deficient. Note increased Kti12 levels with WAP72 and WAP26. TE: total extract.

Fig. 1. Elp1 substitutions affect on Sit4∆ cells and caffeine sensitivity. ELP1 elp1∆ show WT response. Sit4∆ cells are caffeine sensitive. Only WAP5, WAP74, WAP75, WAP76 are able to compensate for Sit4∆ giving caffeine resistance.

References

[1] Hopper AK, Phizicky EM. (2003) tRNA transfers to the limelight. Genes Dev 17: 162-80. [2] Nguyen L et al. (2009) Elongator - an emerging role in neurological disorders. Trends Mol Med, epub ahead of print. [3] Hawkes NA et al. (2002) Purification and characterization of the human elongator complex. J Biol Chem 277: 3047-3052. [4]Chen Z et al. (2006) Mutations in ABO1/ELO2, a subunit of holo-Elongator, increase abscisic acid sensitivity and drought tolerance in Arabidopsis thaliana. Mol Cell Biol 26: 6902-6912. [5] Huang B et al. (2005) An early step in wobble uridine tRNA modification requires the Elongator complex. RNA 11: 424-436. [6] Esberg A et al. (2006) Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis. Mol Cell 24: 139-148. [7]Jablonowski D et al. (2004) The yeast elongator histone acetylase requires Sit4-dependent dephosphorylation for toxin-target capacity. Mol Biol Cell 15: 1459-1469. [8]Mehlgarten C et al. (2009) Elongator function depends on antagonistic regulation by casein kinase Hrr25 and protein phosphatase Sit4. Mol Microbiol 73: 869-881. [9]Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action. [10]F, Jablonowski D, Fichtner L, Schaffrath R. (2003) Subunit communications crucial for the functional integrity of the yeast RNA polymerase II Elongator (�-toxin target (TOT)) complex. J Biol Chem 278: 956-961.

Student: Tsz Fong Cho

Supervisors: Dr Einat Cinnamon and Dr Alex Gould

IDENTIFYING NEW LIPID METABOLIC GENES IN DROSOPHILA LIVER-LIKE CELL

DESCRIPTION OF PROJECT

Regulation of lipid metabolism by nutrition is important for growth. During starvation, it also helps to maintain the energy supply by increasing serum fatty acids and lipids via lipolysis of triglycerides (TG) stored in adipocytes. Hepatocytes then transform serum fatty acids into an alternative energy source, ketone bodies, by fatty acid oxidation. In addition to this lipid catabolism, hepatocytes can synthesise fatty acids and TG. Imbalance in fatty acid synthesis and breakdown underlie certain human metabolic diseases such as type II diabetes and non–alcoholic fatty liver disease (NAFLD). In Drosophila, the fat body (FB) is analogous to the adipose tissue and hepatocyte-like cells termed oenocytes (OE) have been shown to accumulate lipid droplets (LD) during starvation. To study how the OE regulate lipid metabolism, I knocked down genes specifically in OE using the binary GAL4/UAS system combined with RNA interference (RNAi) (Fig. 1). The metabolic phenotypes in the OE (cell autonomous) and in the FB (cell non-autonomous) were then assessed by staining for neutral lipids using Oil Red O.

AIMS

- To perform a miniscreen of OE gene functions using tissue-specific RNAi and then to examine the cell autonomous and cell non-autonomous effects on lipid metabolism.

- To investigate how the expression of several different OE proteins are regulated by nutrition.

DEPARTURES FROM ORIGINAL PROPOSAL

20 OE-specific genes were screened rather than the proposed 50 since I found 2 hits out of the first 20 and some experiments were repeated 3 times. An additional related project, providing training in a second technique was to characterise affinity-purified antibodies raised in the lab against proteins encoded by 4 OE genes.

MINISCREEN OF OENOCYTE GENES

I conducted a miniscreen of 20 genes that were specifically knocked down in the OE during larval stages using an OE-specific driver and UAS-RNAi lines. The larvae were starved for 18 h or fed and the OE and FB were then stained with Oil Red O or visualised with Nomarski microscopy. 1 of the 20 genes screened, acyl transferase A showed a clear knockdown phenotype; reduced size and

Figure 1. The GAL4/UAS binary system. The driver line (GAL4 specifically expressed in OE) is crossed to the Upstream Activation Sequence (UAS) responder line (UAS-RNAi). The transcriptional activation only occurs when GAL4 binds to its UAS binding sites. This triggers expression of a double-stranded RNA which targets and degrades endogenous mRNA. (From Dahmann, C. 2008, “Drosophila Methods and Protocols”, Humana Press, Chapter 5, pp. 81.)

abundance of LD in the OE (Fig. 2A). In contrast, the FB shows an increase in the LD abundance and cell size (Fig. 2B). Confocal microscopy, using phalloidin to stain the cell cortex, confirms the non cell autonomous effect on the enlargement of FB cells under starvation (Fig. 2C). Knock down of acyl transferase A in fed larvae had no detectable phenotype and resembled the controls (data not shown).

Acyl transferase A is predicted to encode an enzyme which converts diacylglycerol (DG) into TG. The reduced TG levels in the OE of starved larvae where acyl transferase A was knocked down suggest that it is required for TG production in the OE upon starvation. This is consistent with DG being the major form of circulating lipid which could be taken up by OE and converted by acyl transferase A into TG. More LD in the FB suggests the existence of an OE-to-FB lipid mobilising signal that depends on OE TG synthesis, which stimulates lipolysis in the FB in starved conditions. To further investigate the relationship between OE and FB, a FB driver (Cg-GAL4) was used to knockdown acyl transferase A in the FB. Starved larvae showed less LD in the OE (data not shown) suggesting that conversion of DG to TG in the FB is necessary for breakdown of TG and export to OE. Further experiments to support this hypothesis would be: 1) To measure TG abundance in the FB of starved larvae using gas chromatography 2) To test if knockdown larvae are more sensitive to starvation, anticipated if fat mobilisation from the FB is blocked. 3) It would also be of interest to generate acyl transferase A null mutants.

EXPRESSION OF OENOCYTE PROTEINS

The expression of 4 different proteins in fed and starved larvae were tested using affinity-purified rabbit primary antibodies and secondary antibodies conjugated to a fluorophore. By Confocal microscopy, protein expression was seen in various tissues (Fig. 3) with 1 protein showing stronger expression in the OE of starved larvae than fed larvae (data not shown).

VALUE OF STUDENTSHIP OF THE STUDENT

Working in this research laboratory was extremely enjoyable and educational in improving my knowledge and skills such as planning experiments and writing concise accounts of the results. It has also provided me with the opportunity to use various experimental techniques (larval OE and FB dissections, Oil Red O staining, Nomarski and Confocal microscopy). This experience provides me with a strong foundation for my research project next year and an understanding of the challenges and rewards of working in biomedical research which further propels my pursuit to work in biomedical research.

Figure 3. Protein expression in starved larvae. Expression is observed in the (A) muscles, (B) nuclei of epidermal cells and (C) OE using primary antibodies MWIL-5 or MWIL-15.

Figure 2. Acyl transferase A knock down in OE. Reduced amounts of LD are observed in the OE (A) and larger FB cells can be visualised by Nomarski microscopy (B) or stained with phalloidin to outline the cell cortex (C).

A CB

MWIL-5 MWIL-15 MWIL-15

Acyltransferase Aknockdown

Control

A B C

VALUE OF STUDENTSHIP TO THE LABORATORY

This project identified acyl transferase A as a regulator of lipid metabolism in the Drosophila OE and suggests the existence of a lipid mobilisation OE-to-FB signal which requires further investigation. Purified antibodies were confirmed to work well in larvae and will serve as an important tool in testing the regulation of these proteins by nutrition and by other genes.

SSTTUUDDEENNTT:: ZZEEEENNAATTUU MMUUSSTTHHAANN

SSUUPPEERRVVIISSOORR:: MMAANNUUJJAA KKAALLUUAARRAACCHHCCHHII

IINNSSTTIITTUUTTIIOONN FFOORR PPLLAACCEEMMEENNTT:: MMIIDDDDLLEESSEEXX UUNNIIVVEERRSSIITTYY

BBIIOOCCHHEEMMIICCAALL SSOOCCIIEETTYY-- SSUUMMMMEERR SSTTUUDDEENNTTSSHHIIPP 22001100 RREEPPOORRTT

AAFFFFIINNIITTYY CCAAPPTTUURREE MMAALLDDII TTOOFF MMSS FFOORR TTHHEE AANNAALLYYSSIISS OOFF hhCCGG IINN PPRREEGGNNAANNCCYY AANNDD DDIISSEEAASSEE:: TTEESSTTIINNGG hhCCGG AANNTTII-- BBOODDIIEESS

Background. Human chorionic gonadotrophin, hCG is a heterodimer glycoprotein hormone consisting of an α-subunit and a β-subunit that are non-covalently bonded. Glycosylated variants of hCG shown great diagnostic potential in clinical research. Structural changes in the glycosylation of hCG have been associated with abnormal pregnancies and malignancies such as hydatidiform mole, pre-eclampsia and hyperemesis gravidarum. hCG antibodies specifically recognise the glycoprotein hormone and its increased affinity to the beta chains cause bindin. This property is very useful in various analysis of hCC in pregnancy and diseases, hence working towards a possible treatment to these conditions.

Aims of the project.

• Establish the minimum dilution standard for the recombinant hCG that enables detection both on the SDS-PAGE gel and the MALDI-TOF mass spectrometer.

• Investigate the effect of incubating hCG antibodies with the glycoprotein hormone by running an SDS-PAGE.

• Confirming the results of the SDS-PAGE experiment with the MALDI-TOF mass spectra for the same samples used.

Description of the work carried out.

The two main experimental techniques used were namely SDS-PAGE and MALDI TOF MS.

1. SDS-PAGE: Samples of different dilutions of neat rhCGβ (1µg/µl) were run on the SDS PAGE gel. The Blue Stain reagent was used to identify any bands produced. This experiment determined the limit of detection of the glycoprotein hormone- rhCGβ produced conspicuous bands near the 25KDa band of the colour marker that could however not be detected beyond the 1.40 dilution.

Monoclonal mouse anti-hCG antibody MCA1024 (AbD Serotec) was then used and the affinity capture was analysed through another SDS PAGE experiment. Different ratios of rhCGβ and MCA1024 were incubated. The bands produced by the samples on the electrophoresis gel revealed significant reduction in the intensity of the rhCGβ band when the antibody was added. Furthermore, an additional band seen around the 100KDa band of the colour marker could be attributed to the antibody.

The above procedures were repeated using rabbit polyclonal IgG SC-67357 (Santa Cruz). The electrophoresis results showed similar trend to those with the MCA1024 antibody. The affinity capture of the rhCGβ antigen by the antibodies result in a smaller concentration of unbound antigen in the sample that produces the lighter band compared to the ones from the first practical. These findings correlated with the MALDI data generated.

2. MALDI-TOF MS: MALDI-TOF mass spectra were generated for different ratios of rhCGβ incubated with MCA1024 monoclonal mouse antibody using sinapinic acid as the spotting matrix. An overlay of the spectra of the neat rhCGβ without and with the antibody highlighted major decrease in the peak intensity at around 23KDa for the rhCGβ antigen and around 45KDa for the dimeric molecule of the antigen. Moreover, the antibody peak could also be identified at around 140KDa on the spectrum.

(ATTACHED FIGURE 1) Figure 1: Overlap of three MALDI-TOF MS showing the action of the antibody on the hCG peak. Diluting the rhCGB to a 1.5 results in a considerable decrease in the peak intensity observed at for the monoprotonated beta molecule. When the antibody is incubated in the pure samples, binding to the epitopes of the glycoprotein occurs, resulting in a reduction in both the rhCGB+ and the dimeric B/B+ peaks..

Similarly, rhCGB samples incubated with a constant volume of 5µl of the rabbit polyclonal IgG SC-67357 were subjected to MALDI-TOF analysis. This antibody also produced significant decrease in the rhCGβ peak on the MALDI spectra generated. An overlay of the peaks obtained for the different dilutions of rhCGβ used further established that the rhCGβ peak was completely reduced by the antibody at the 1.20 dilution and could not be detected at higher dilutions. These findings correlated closely to the SDS-PAGE results.

Before proceeding to building the biolayers on the gold plate, initial experiments were

carried out to generate peaks for the reagent components to be used for this method namely DTSP, Protein G and DTSP+Protein G by spotting on a normal steel MALDI plate. These were analysed using sinapinic acid, CHCA and THAP matrices, in line with published data.

Assessment of results and outcome of the studentship. The calibration mix spectra generated were used to calibrate the MALDI prior to analysing

the samples. The experiments were initially carried out at varying laser power so as to establish the ideal one for the particular mass range used. The samples were fired evenly throughout so as to get a resulting high voltage which would give better signal on the spectra. The results of both the SDS-PAGE and the MALDI experiments were analysed in parallel and they showed good correlation.

It is essential to generate individual spectra for DTSP and PrG in the steel plate before

proceeding to building the biolayers on the gold plate.

Further directions in which the project can be taken. Further tasks on this project would include method development for the

immobilisation of antibodies on the gold plate. Other antibodies can therefore then be characterised in the same way.

Any departures from the original proposal.

I have not diverged from the original plan. Yet, I feel that there is a lot more work to be done that goes beyond the time frame of this studentship that needs to be carried out in order to achieve a usable affinity capture MALDI-TOF MS assay.

The value of the studentship to me.

This studentship has been a step forward from the student life at Middlesex University; I felt as member of the teaching and research team. This unique opportunity enabled me to work independently in a laboratory and acquaint myself with the different equipment and experimental techniques used in analysis. I have been given the opportunity

to work independently from sample preparation to generation of useful results. It has been a hugely pleasant journey to liaise with various members of staff and students and share practical tips about the protocols used and the ways to carry out the experiments. I now see myself as more motivated and confident in working in a laboratory as I developed the key skills such as time management, abiding by work schedules and health and safety procedures.

Value of the studentship to the lab.

The results generated during this studentship will indeed be of great help to the current PhD students who are probing into various analysis on hCG. The findings would also be a starting for further research projects and further grants applications as further method development would take this study to a newer dimension. Furthermore, the staff and students using the MALDI-TOF have now a better insight of the instrumental settings which generate best spectra for the glycoprotein hormone hCG.

SVS Reports 2010

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Transcript of SVS Reports 2010