POSTER 1

A CELL-BASED ASSAY FOR THE FUNCTION OF GRP94 Olga Ostrovsky1, Cat Makarewich1, Tali Gidalevitz2, Chhanda Biswas1, Erik L. Snapp3, and Yair Argon1. 1Division of Cell Pathology, Children’s Hospital of Philadelphia, and The University of Pennsylvania, Philadelphia, PA, 2Department of Pathology, The University of Chicago, Chicago, IL, and 3Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY GRP94 is an essential chaperone in the endoplasmic reticulum (ER) whose function remains rather enigmatic. GRP94 has only a few known client proteins, no known co-chaperones, and its mode of action and regulation are obscure. To understand the functions of GRP94 in vivo, we developed a mammalian cell-based assay, taking advantage of our recent finding that GRP94 controls the production of insulin-like growth factor (IGF)-II. Grp94-/- cells are hypersensitive to serum withdrawal and die by apoptosis. Transfected GRP94 confers resistance to serum withdrawal on grp94-/- cells, as does exogenous recombinant IGF. Because it is essential in transient transfections to focus on the transfected cells, we employed a fusion protein of GRP94 with GFP at its C-terminus. This fusion protein is targeted correctly to the ER and is functional, conferring resistance on grp94-/- cells, prolonging their viability in serum-free medium. Thus, expression of this protein allows assessment of the viability of single cells. Using this assay, we tested the effects of specific amino acid substitutions. A mutation that inhibits peptide binding in vitro does not inhibit the ability of the protein to complement cell survival. On the other hand, substitutions of residues that are necessary for nucleotide binding abolish the ability of the protein to promote survival in serum-free medium. Substitution of the catalytic E82, which is required for ATP hydrolysis, also abolishes chaperone function. On the other hand, mutagenesis of the putative catalytic loop in the middle domain, which in HSP90 is involved in ATP hydrolysis, did not dramatically affect chaperone function as measured by this assay. Thus, nucleotide binding and hydrolysis are critical for the function of GRP94 in vivo but its mode of action seems different from that of its cytosolic paralogue HSP90.

POSTER 2

GRP94 IS CRITICAL FOR MUSCLE DIFFERENTIATION BECAUSE IT REGULATES SECRETION OF IGF-II

Olga Ostrovsky1, Sherry Wanderling2, Brigitte Simen2, and Yair Argon1

1 The Children’s Hospital of Philadelphia and The University of Pennsylvania, Philadelphia, PA, 2 Department of Pathology, The University of Chicago , Chicago, IL The physiological role of endoplasmic reticulum chaperone Glucose regulated Protein 94 (GRP94) is poorly understood, in part because only few of its client proteins are known. It is essential, because grp94-/- mice are embryonic lethal. Embryonic Stem (ES) cells derived from grp94-/- mice do not differentiate into muscle lineages. These cells fail to produce insulin-like growth factor (IGF)-II, critical in many differentiation processes, and the muscle differentiation defect can be partially complemented by addition of exogenous IGF-II. These observations suggest that IGF-II is a client of GRP94. To test the hypothesis, we used the well-established myogenic cell line C2C12, which can be induced to differentiate in culture. Inhibition of GRP94 expression by RNAi affects the levels of the early muscle transcription factors MyoD and Myogenin. GRP94-silenced C2C12 myoblasts also failed to fuse into myotube-like cyncitia and to express structural muscle proteins. In addition, IGF-II secretion by these cells was strongly inhibited. Immunoprecipitation analysis showed direct association of GRP94 with the pro-IGF-II isoforms, but not with the mature hormone. Addition of exogenous IGF-II to myoblats with non-functional GRP94 rescues myotube formation and expression of myosin. Thus, we suggest that the muscle differentiation defect of grp94 -/- cells is due to the extreme dependence of IGF-II processing and on the activity of GRP94. These results provide a new paradigm for understanding the molecular pathology of diseases involving IGF-II, such as cancer and muscular dystrophies.

POSTER 3

SMALL MOLECULE SCREENS FOR ACTIVATORS OF THE HEAT SHOCK RESPONSE AND SUPPRESSORS OF PROTEIN AGGREGATION

Anna-Karin E. Svensson, Barbara Calamini, Susan G. Fox and Richard I. Morimoto Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s and Huntington’s diseases, are characterized by aberrant neuronal accumulation of misfolded and/or aggregated proteins that disrupt cellular homeostasis and function. The heat shock response (HSR) is a highly conserved mechanism through which the cell responds to the chronic buildup of aggregation-prone proteins by up-regulating the expression of heat-shock proteins (HSPs), which maintain protein homeostasis by preventing inappropriate protein-protein interactions and promoting the refolding of misfolded proteins. Because of their critical roles in maintaining the correct protein-folding environment, HSP up-regulation through small molecule activators of the HSR is a possible therapeutic strategy for effective treatment of neurodegenerative diseases. In addition to HSR-activators, recent findings have also suggested a role for small molecules in obliterating or suppressing protein aggregation in, for example, transthyretin amyloid diseases and ALS, by directly stabilizing the native protein fold, thus serving as chemical folding chaperones.

In collaboration with the Southern Research Institute, a high-throughput screening methodology to measure the activation of the human hsp70 promoter was developed. Using this methodology, several HSR-activators were identified and these hits are currently being validated here at Northwestern University and assessed for cytotoxicity. Future studies will focus on the characterization of the mechanism-of-action of the compounds and on the evaluation of their potential to reduce protein aggregation in neuronal models of neurodegenerative diseases. Another on-going collaboration is with Cambria Biosciences, in which chemical chaperones for prevention of aggregation associated with ALS are tested. Specifically, candidate compounds are tested for suppression of aggregation in mammalian neuronal cell lines expressing the familial-ALS variant G93A fused to green fluorescent protein. In addition, the same compounds are assessed for their cytotoxicity and HSR activation, using the high-throughput screening facility at Northwestern University.

POSTER 4

THE INTRACELLULAR COMPOSITION AND INFECTIVITY OF [PSI+] PRION AGGREGATES

Sviatoslav Bagriantsev, Elena Gracheva, Janet Richmond, and Susan Liebman Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL [PSI+] is the prion form of the Sup35 protein from the yeast Saccharomyces cerevisiae. Sup35 exists in the cytosol of [PSI+] cells in the form of protein aggregates with a complex two-level structural organization. It was shown that the aggregates contain SDS-stable polymers of Sup35, and possibly other proteins. These additional components and the requirement for the integrity of the aggregates for [PSI+] transmission remained unknown. We analyzed the protein composition of [PSI+] aggregates isolated from yeast and found that their major components are Sup35 polymers and Ssa, a member of the yeast Hsp70 family. We show that in [PSI+] cells, Ssa binds to the N-terminal domain of the Sup35, and exhibits similar binding efficiency to different variants of the prion. However, Ssa interacts poorly with the non-prion Sup35 from [psi-] cells. We found that the minor components of the aggregates are Sis1, Sse1, and Hsp104. We demonstrate that disassembly of the aggregates into Sup35 polymers and Ssa increases their infectivity while retaining their variant-specificity. Our findings elucidate the molecular composition of [PSI+] aggregates and suggest that the structural integrity of the aggregates is not required for the variant-specific transmission of [PSI+] in vivo, which is carried out by Sup35 polymers.

POSTER 5

IDENTIFICATION AND CHARACTERIZATION OF NOVEL ANTIBIOTIC TARGETS Julia E. Bandow1, Michael Hecker2, Heike Brötz-Oesterhelt3, Harald Labischinski4 1 Author is in between jobs. The work presented here was performed at the Department of Microbiology, University of Greifswald, Greifswald, Germany 2 Department of Microbiology, University of Greifswald, Greifswald, Germany 3 AiCuris GmbH & Co. KG, Germany 4 Combinature Biopharm AG, Berlin, Germany Bacteria are able to rapidly adapt to environmental challenges. We are witnessing an impressive example of bacterial adaptability in the area of antibiotic resistance. Several pathogens have acquired multiple resistance and we are running out of treatment options for patients infected with multidrug-resistant tuberculosis or Pseudomonas aeruginosa. In order to develop new effective antibiotics it is crucial to select drug targets which are not negated by pre-existing cross-resistance. We will present how proteomic profiling assisted in the identification of the target of a new compound class. ClpP was identified as target of acyldepsipeptide. The unusual feature of the mechanism of action of acyldepsipeptide is that it does not act as an inhibitor of ClpP, but rather decouples it from the stringent regulation of the ATPase, ClpX, leading to uncontrolled protein degradation.

POSTER 6

THE COLLAPSE OF PROTEIN HOMEOSTASIS IS AN EARLY DETERMINANT OF AGEING IN C. ELEGANS

Anat Ben-Zvi and Richard I. Morimoto

Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL

Aging is associated with the time-dependent accumulation of damaged macromolecules and the concomitant decline in multiple aspects of cellular machinery. The age-dependent accumulation of damaged proteins has been well established as a marker of ageing. In this study, we examine the capacity of the protein homeostasis machinery during ageing to suppress misfolding of diverse metastable proteins with destabilizing temperature-sensitive mutations in Caenorhabditis elegans. We show that proteins with mild folding defects rapidly lose function in multiple tissues very early in adult ageing, shortly post-development. This decline is under the genetic control of the stress responsive transcriptional activators hsf-1 and daf-16, which confer robust capacity to suppress protein damage. The collapse of protein homeostasis represents a new molecular biomarker that can help to explain the plethora of subsequent age-dependent changes in the cell.

POSTER 7

THE ALLOSTERIC STATES OF HSP70 CHAPERONES VISUALIZED BY NMR

Eric B. Bertelsen and Erik R.P. Zuiderweg Department of Biological Chemistry, The University of Michigan, Ann Arbor, MI

DnaK is the canonical Hsp70 molecular chaperone protein from Escherichia coli. The Hsp70 chaperones are central to protein folding, refolding, and trafficking in organisms ranging from Archae to Homo Sapiens, both at normal and at stressed cellular conditions. Recently, Hsp70s have been linked to breast and colon cancer, and to protein folding diseases such as Alzheimer’s, Parkinson’s and Creutzfeldt-Jacob’s. Like other Hsp70s, DnaK is comprised of three structurally independent domains: A 44 kDa N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, a 17 kDa substrate-binding domain (SBD), and a 10 kDa alpha-helical “lid” (LID) domain that modulates the kinetics of substrate binding. Allosteric communication between the domains is required for nucleotide-dependent regulation of substrate protein.

We are studying full-length DnaK, a 70 kDa protein, by solution NMR. Specifically, we are determining the relative domain orientations and dynamics of this protein in different allosteric states, using residual dipolar couplings and paramagnetic restraints. The combined structural and dynamical data reveal that the Hsp70 nucleotide cycle is accompanied by a switch in inter-domain docking. Specifically, in the physiologically relevant ADP- and peptide-bound state, the SBD and LID domains are stably docked together, but tumble largely independently from the NBD. Upon exchange of the nucleotide to ATP, the interaction switches: the NBD and SBD dock, while the LID dissociates from the SBD and tumbles independently. These results form the basis for a general model of Hsp70 conformational change during its reaction cycle.

POSTER 8

NITROSATIVE STRESS TREATMENT OF E. COLI TARGETS DISTINCT SET OF THIOL-CONTAINING PROTEINS

Nicolas Brandes, Andrea Rinck, Lars Ingo Leichert and Ursula Jakob Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI Reactive nitrogen species (RNS) function as powerful antimicrobials in host defense, but so far little is known about their bacterial targets. In this study, we set out to identify Escherichia coli proteins with RNS sensitive cysteines. We found that only a very select set of proteins contain cysteines that undergo reversible thiol modifications upon nitric oxide (NO) treatment in vivo. Of the 10 proteins that we identified, six (AtpA, AceF, FabB, GapA, IlvC, TufA) have been shown to harbor functionally important thiol groups and are encoded by genes that are considered essential under our growth conditions. AceF, the E2 component of pyruvate dehydrogenase (PDH), gets most likely modified by RNS at the two vicinal thiol groups in its lipoyl domain, which have been previously suggested to be the target of environmental toxins such as arsenic. IlvC plays a central role in the biosynthesis of isoleucine and valine. Escherichia coli cells cultivated in the absence of these branched-chain amino acids were significantly more sensitive to micromolar concentrations of the NO-donor diethylamine NONOate (DEANO) than E. coli cells grown in their presence. Targets of oxidative thiol modification in IlvC could be either a cysteine pair (Cys145/Cys156) located within a highly conserved region of the sequence and/or a very highly conserved single Cys226. Interestingly, the majority of RNS-sensitive E. coli proteins differ from E. coli proteins that harbor H2O2-sensitive thiol groups, implying that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. We confirmed this specificity by analyzing the activity of one of our target proteins, the small subunit of glutamate synthase. In vivo and in vitro activity studies confirmed that glutamate synthase rapidly inactivates upon NO treatment but is resistant towards other oxidative stressors. It will be our future challenge to determine what makes these small set of proteins particularly sensitive to oxidative and nitrosative stressors and distinguishes them from the hundreds of cysteine-containing E. coli proteins, whose thiol groups are not affected by ROS and RNS. These proteins then might be used for diagnostic purposes by determining marker proteins that are specifically sensitive to distinct reactive oxygen and nitrogen species. Identification of such marker proteins in cells and tissues can thus be used as direct read-out for the presence of specific reactive oxygen or nitrogen species.

POSTER 9

A MICROFLUIDIC PLATFORM FOR LOCAL THERMAL AND CHEMICAL STIMULATION OF C. ELEGANS

Meghan E. Bush1, Benjamin Umans1, Daniel Choi1, Cindy Voisine2, Daniel Czyz2, Richard I. Morimoto, 2 and Rustem F. Ismagilov1

1Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 2 Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL We develop microfluidic technology for controlling and understanding the dynamics of biological systems in space and time. To characterize the effects of a local stimulus on an individual C. elegans over time, we have created a microfluidic platform that enables immobilization and non-invasive, local perturbations of live, individual C. elegans using a temperature step. This 3-layer microfluidic device is compatible with live-imaging and manipulation of individual C. elegans, eliminating the need for gluing or paralyzing the organism. In addition, the organisms can be removed easily from the microfluidic device for subsequent monitoring or staining, allowing behavioral assays as well as advanced microscopic investigations. Three dimensional numerical simulations and experimental characterization with thermochromic liquid crystals confirms a temperature step within the microfluidic channel. Experiments using transgenic C. elegans with a HSP70::GFP reporter verify that local stress response was induced by the temperature step. This device can be used to stress C. elegans with chemical, thermal, or UV stimuli over any part of their body, and may be useful for experiments with transgenic C. elegans disease models already in use.

POSTER 10

HEAT SHOCK RESPONSE RELIEVES ER STRESS VIA MULTIPLE PATHWAYS Yu Liu and Amy Chang Department of Molecular, Cellular & Developmental Biology, University of Michigan, Ann Arbor, MI Accumulation of misfolded protein in the endoplasmic reticulum (ER) causes stress, and perturbation of normal ER function. To relieve stress and restore homeostasis in the ER, a transcriptional induction pathway called the unfolded protein response (UPR) is activated. Although UPR is not essential for cell viability, cells deficient in UPR are more sensitive to ER stress; UPR-defective ire1Δ cells cannot grow when challenged with tunicamycin to induce protein misfolding or by overexpression of a misfolded luminal protein, CPY*. In these cells, multiple functions are defective such as ER translocation, ER-associated degradation (ERAD), and ER-to-Golgi transport. In this study, we have tested whether heat shock response (HSR), a transcriptional activation program that is induced by heat and other stress conditions, plays a role in relieving ER stress. Using a constitutively active Hsf1 transcription factor to induce HSR in the absence of temperature shift, we find that HSR rescues growth of UPR-defective ire1Δ cells challenged by protein misfolding, partially relieves defects in translocation and ERAD, and selectively relieves impaired ER-to-Golgi transport. Cargo-specific effects of constitutively active Hsf1 on ER-to-Golgi transport, i.e., enhancement of Gas1 and CPY* but not PrA and ALP transport, are correlated with enhanced protein levels of the respective cargo receptors. Finally, HSR is activated in vivo by ER stress caused by loss of UPR and overexpression of CPY* or tunicamycin treatment, albeit to a significantly lower level than that caused by heat. We propose that HSR can relieve ER stress in UPR-deficient cells by facilitating translocation, enhancing ERAD and promoting vesicle transport from the ER. Analysis of heat shock-induced genes reveals many encoding products localized to the ER and/or with functions in the secretory pathway, including vesicular transport and chaperone activity. Therefore, HSR appears to work together with UPR in protein quality control in the secretory pathway.

POSTER 11

USING HIGH THROUGHPUT ASSAYS TO SCREEN FOR INHIBITORS OF HSP70s IN NATURAL PRODUCT LIBRARIES

Lyra Chang and Jason E. Gestwicki Life Sciences Institute, University of Michigan, Ann Arbor, MI

Heat shock protein 70s (Hsp70s) are multifunctional enzymes that participate in many aspects of protein metabolism – folding, transportation, disaggregation, and degradation. The activities of Hsp70s are also related to the prognosis of microorganism infection, cancer, and protein misfolding diseases. Therefore, small molecule modulators for Hsp70s might provide both good chemical genetic tools for studying chaperone biology and lead compounds against various diseases. Despite the obvious importance of Hsp70s, there are only handfuls of compounds reported to interact with Hsp70s due to the lack of good screening methods. In this study, we developed a robust, efficient, and low-cost high throughput assay which can be adapted to 384-well format. Using this method, we are able to screen spice and Chinese medicine extract libraries for active substance that can modulate Hsp70’s ATPase activities.

POSTER 12

PYRIMIDINONE-PEPTOID HYBRID MOLECULES WITH DISTINCT EFFECTS ON MOLECULAR CHAPERONE FUNCTION AND CANCER CELL PROLIFERATION

Raj J. Chovatiya a, Christine M. Wright a, Nora E. Jameson a, David M. Turner b, Guangyu Zhu c, Stefan Werner b, Donna M. Huryn b,d, James M. Pipas a, Billy W. Day c,d, Peter Wipf b,c,d and Jeffrey L. Brodsky a,* a Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, b Center for Chemical Methodologies and Library Development, University of Pittsburgh, Pittsburgh, PA, , c Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, d Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA

The Hsp70 molecular chaperone is a conserved, ubiquitous protein that catalyzes polypeptide folding, degradation, transport, and degradation in the cell. Hsp70s are also required for cell survival and prevent programmed cell death by inhibiting several nodes in the apoptotic pathway. In certain cancers (e.g., breast, endometrial, and colorectal), Hsp70 over-expression correlates strongly with poor prognosis. However, a reduction of Hsp70 levels by treatment with antisense RNA has been shown to selectively kill specific cancer cell types in culture. Based on these collective data, we postulated that a pharmacological approach to modulate Hsp70 activity would represent a more viable attack to kill cancer cells. To this end, a library of compounds based upon one of our previously identified Hsp70 modulators (MAL3-101) was synthesized by the Combinatorial Chemistry Center at the University of Pittsburgh. MAL3-101 induces SKBR-3 breast cancer cell apoptosis in the low micromolar range, and 16 of the newer compounds inhibit SKBR-3 breast cancer cell proliferation with a GI50 of 5-50 mM. The MAL3-101 structural analogues were then examined for their ability to modulate both endogenous and J-domain-stimulated Hsp70 ATPase activity using single-turnover assays. Our data indicate that the newer compounds can be grouped into distinct families based on their activities, and in many cases correlations were noted between their abilities to inhibit chaperone activity and to thwart breast cancer cell growth. The most potent of these compounds were also found to inhibit the growth of MCF-7 breast cancer cells and HT-29 colorectal cancer cells. Three compounds of interest (DMT003052, DMT003088, and DMT003132) have now been selected from this library in order to further characterize their anti-cancer properties. Specifically, we are investigating whether SKBR-3 breast cancer cells undergo apoptosis upon addition of these compounds by assessing caspase-3 cleavage and PARP activation, as previously observed for MAL3-101. In addition, structural derivatives of these derivatives are being synthesized and will be examined in vitro to build structure-activity relationships and to identify molecules with more potent anti-tumor properties.

POSTER 13

ASSESSING THE HOW AND WHY OF POLYGLUTAMINE AGGREGATION USING COMPUTATION AND EXPERIMENT

Scott L. Crick, Xiaoling Wang, Andreas Vitalis, and Rohit V. Pappu Department of Biomedical Engineering & Center for Computational Biology, Washington University in Saint Louis, St. Louis, MO Nine different neurodegenerative diseases, including Huntington’s disease, are associated with mutations in different proteins that generate in frame polyglutamine expansions. Our focus is on developing a quantitative understanding of the driving forces for polyglutamine aggregation and the mechanism of aggregation. Monomeric polyglutamine prefers collapsed structures in water: In previous work, computer simulations were used to show that monomeric polyglutamine forms collapsed structures in aqueous solutions (X. Wang et al. Proteins: Struc., Func. Bioinform. 63, 297-311, 2006; A. Vitalis et al. Biophys. J. 93, 1923-1937, 2007), suggesting that water is a poor solvent for polyglutamine. We tested this prediction using Fluorescence Correlation Spectroscopy (FCS) to show that the size of monomeric polyglutamine increases monotonically with chain length as predicted for polymers in poor solvents (S.L Crick et al. Proc. Natl. Acad. Sci. USA. 103, 1674-1679, 2006). Monomeric polyglutamine forms disordered globules in solution to minimize the interface with the surrounding solvent and maximize self-interactions within the chain. This conclusion contradicts conventional wisdom for describing conformational equilibria of intrinsically disordered molecules that are rich in polar residues such as glutamine. Stabilities of polyglutamine globules increase non-linearly with chain length: Recent atomistic simulations (X. Wang et al. in preparation) show that the stabilities of collapsed globules increase non-linearly with polyglutamine length. Similarly, the cooperativity of coil-to-globule transitions as a function of temperature also increases non-linearly with chain length. FCS experiments that measure denaturation of monomeric polyglutamine provide direct support for these findings (S.L. Crick et al. in preparation). The midpoints of denaturation curves shift to higher GuHCl concentrations and the transitions become sharper with increasing chain length. There is direct correlation between chain length dependent stabilities of polyglutamine globules and oligomer formation: In a poor solvent, polymers form globules at low concentrations and globules self associate eventually leading to phase separation as concentration increases (R.V. Pappu et al. Arch. Biochem. Biophys. 469, 132-141, 2008). Simulations show that there is a thermodynamic equilibrium between monomers and higher order oligomers, and this equilibrium shifts toward higher-order species as chain lengths increase. Our results suggest the need for rethinking the currently accepted mechanisms for polyglutamine aggregation that are based on the tenets of homogeneous nucleation and ignore the formation of oligomers. This is relevant because one can conceive of designing therapeutic strategies to perturb the equilibrium between monomers and higher order species. This should be feasible because we are able to define intermolecular interfaces with atomistic detail.

POSTER 14

SEQUENCE SYMMETRY DETERMINES FIBRIL TERTIARY STRUCTURE IN POLYGLUTAMINE SYSTEMS

Gregory Darnell1, Robert Tycko2, Stephen C. Meredith1,3

1Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 2Laboratory of Chemical Physics, National Institute of Diabetes and Digestive Kidney Diseases, National Institutes of Health, Bethesda, MD, 3Pathology, The University of Chicago, Chicago, IL

Within proteins, anti-parallel β−sheets are more frequent than parallel β−sheets, possibly

because the former allow for shorter, more stable hydrogen bonds. Most amyloid fibrils, however, have parallel, in register β-sheets, which allows for maximal side chain interactions among like amino acids, especially hydrophobic residues. For amyloidogenic peptides formed entirely of the polar amino acids, Gln and Asn, however, it is less clear what determines β−sheet orientation.

We hypothesize that for polyglutamine peptides, β−sheet orientation is governed by the number of side chain hydrogen bonds that can be formed in either orientation. To test this hypothesis, we developed polyglutamine/polyalanine/polyglycine peptides with either a symmetrical or asymmetrical distribution of Gln residues, (R2(QA)9QR2, R2(QA)5QR2, R2(QG)10QR2). We predict that for asymmetric sequences, parallel β−sheets will form more hydrogen bonds between Gln side chains than in the antiparallel orientation. Alternatively, for sequences with symmetrically distributed glutamines, either orientation will allow for the same number of hydrogen bonds, and therefore the anti-parallel arrangement, with shorter hydrogen bonds, will be favored. Electron microscopy show that all the peptides form fibrils after prolonged incubation. Solution and solid-state circular dichroism studies confirm that these peptides adopt β-sheet conformations in solution and upon aggregation in the solid state, typical of amyloid fibrils. We will utilize solid-state NMR experiments (REDOR and fp-RFDR-CT) to test mixtures of site-specific labeled peptides (backbone 15N or carbonyl 13C), which can unambiguously determine the β-sheet orientation of the fibrils formed by these peptides. Here we provide the first structural investigation into sequence symmetry as a determinant of β-sheet orientation in polyglutamine fibrils.

POSTER 15

ENHANCING CAPTURE EFFICIENCIES OF GROEL USING OSMOLYTE MIXTURES Nicholas Degnera, Hiroo Katayamaa, John Seedb and Mark T. Fishera

aDepartment of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, b EdgeBiosystems, Gaithersburg MD. The capture and protein refolding efficiency of GroEL is dictated by the lifetime of the folding intermediate. In the presence of urea/glycerol mixtures, mitochondrial malate dehydrogenase (mMDH) remains in a metastable state that can be refolded by GroEL even after GroEL addition is delayed for extended periods of time (>5 min at 25oC). GroEL remains a stable tetradecamer even at 3-7 M urea/4 M glycerol which allows this chaperonin to capture and refold metastable mMDH monomers over a wide concentration range. This enhanced capture procedure will be extremely useful for column and kit based refolding protocols that employ immobilized GroEL as the folding platform.

POSTER 16

HSP90 SUPPORTS TELOMERASE DNA BINDING AND EXTENSION ACTIVITY D. DeZwaan, Tunji Toogun and Brian Freeman Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, Urbana, IL Telomerase lengthens chromosome termini by specifically associating with single-stranded telomeric DNA and appending nucleotides through a reverse transcription reaction. Telomerase activity is controlled by a number of auxiliary factors including the Hsp90 molecular chaperone. Although Hsp90 was the first cofactor demonstrated to have an effect in vitro, the mechanistic contribution made by this chaperone was poorly understood. Recently it was shown that Hsp90 might assist telomerase in DNA primer loading. Here we show that the yeast Hsp90 molecular chaperone promotes both telomerase DNA binding and nucleotide processivity properties. By isolating telomerase from different Hsp90 allelic backgrounds we observed distinct defects. For example, in an hsp82 G170D background telomerase displayed a loss in both DNA binding and extension activities whereas only nucleotide processivity was decreased in a T101I strain. The decline in both activities correlated with a loss in Hsp90-association, as both nucleotide processivity and DNA binding could be rescued in the mutant backgrounds by addition of recombinant purified Hsp82p. Inherent Hsp82p activity (e.g., in vitro chaperone and ATPase assays) provides no correlative reason for loss of telomerase DNA binding in a G170D background. However, analysis of DNA bound telomerase complex showed loss of Hsp82p binding in a G170D background. Taken together, our results indicate that an Hsp90 protein serves a dual role both to promote telomerase DNA association and to facilitate extension of the DNA substrate once bound by telomerase and that the continual presence of Hsp82p may be necessary.

POSTER 17

OPTIMIZATION OF PROTEIN FOLDING IN VIVO

Linda Foit, Maximillian Kern, Lenz Steimer, Anne-Kathrin von Hacht, and James Bardwell Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI

The production of large amounts of recombinant protein is required for many structural, biotechnological and pharmaceutical purposes. It is, however, often limited by the inability of bacteria to efficiently express high levels of stable and functional polypeptide. We have developed an effective selection system which links the expression level of recombinant proteins to an easily selectable phenotype – the resistance to beta-lactam antibiotics. Our method is based on the insertion of a recombinant protein into TEM1-beta-lactamase, resulting in a tripartite fusion protein. It has been shown that the introduction of small peptides into one particular region of beta-lactamase has little effect on the enzyme’s ability to confer antibiotic resistance. We therefore reasoned that the insertion of larger proteins could be tolerated, as long as the inserted protein is well folded. Poor folding or instability of the inserted polypeptide on the other hand would result in an increased sensitivity of the fusion protein to cellular proteases and aggregation. This would lead to a decrease in antibiotic resistance of cells expressing such a fusion construct.

To test if such tripartite fusion proteins can be used to monitor protein folding in vivo, we chose three different proteins for insertion, Im7, a bacterial protein that has no cofactors or disulfides, Bovine pancreatic Trypsin inhibitor (BPTI), containing three disulfides and cytochrome B562, which contains a heme cofactor. Each of these three proteins has a well studied folding pathway and destabilizing mutations are known. We were able to show for each protein that the introduction of destabilizing mutations lead to an increased sensitivity to penicillin V. For Im7, we furthermore found a direct relationship between antibiotic resistance caused by the beta-lactamase fusion protein and the ΔG of folding for the inserted protein. This suggests that one can easily select for variants of a recombinant protein with increased stability by simply increasing the concentration of penicillin V used in the selection. By using random mutagenesis, we also found mutants of Im7 that lead to higher levels of antibiotic resistance than the WT, with one of them already shown to be more stable than WT in vitro.

So far we have used this system to increase the expression of recombinant proteins by mutating the protein of interest to yield more stable variants. It is possible, however, that folding of a target protein could be enhanced by mutating bacterial folding factors, chaperones, or the host chromosome itself.

POSTER 18

THE PHYSIOLOGICAL ROLE AND MECHANISM OF PROTEIN DISULFIDE ISOMERASE’S (PDI) PROTEIN-UNFOLDING ACTIVITY

Michele L. Forster,1 James J. Mahn,2 and Billy Tsai1 1Department of Cell and Developmental Biology, 2Molecular, Cellular and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI Protein disulfide isomerase (PDI) is an abundant protein within the endoplasmic reticulum (ER) whose ability to form and isomerize disulfide bonds and fold non-native proteins is well known. Less understood is the physiological role and mechanism of PDI’s unfolding activity. Previous in vitro proteolytic analysis revealed that PDI can unfold cholera toxin (CT), an agent that requires ER-cytosol transport, i.e., retrotranslocation, during intoxication. We developed a semi-permeabilized cell system to study CT retrotranslocation and found that PDI facilitates the toxin’s ER-cytosol transport. These data suggest that PDI unfolds CT in preparation for the toxin’s retrotranslocation. We are currently investigating the mechanism of PDI’s unfolding activity. PDI comprises two redox-active thioredoxin domains, a and a’, two redox-inactive thioredoxin domains, b and b’, and an acidic C-terminal tail in the order of a-b-b’-a’-c. To begin to elucidate the mechanism of PDI’s unfolding activity, we determined the contribution of each domain to this activity. Hence, we compared the ability of full-length and truncated PDI variants to render CT sensitive to tryptic digestion. Deletion of the C-terminal tail did not affect PDI’s unfolding activity. While removal of either the a or a’ domain decreased PDI’s ability to unfold CT, removal of the a’ domain had a greater effect on PDI activity. The b’ domain was not required to unfold CT contrary to its essential role in binding other PDI substrates. Although preliminary, these results suggest that PDI employs different mechanisms to fold and unfold proteins.

POSTER 19

EXPLORING TORSINA DEGRADATION: POTENTIAL ROLE IN DYT1 DYSTONIA PATHOGENESIS AND THERAPEUTICS

Gordon, KL1; Bode, N2; Paulson, HL4; Glenn, K3,5; and Gonzalez-Alegre, P1,2. Graduate Programs in Neuroscience1 , Departments of Neurology2 and Internal Medicine3, University of Iowa Carver College of Medicine, Iowa City, IA, Department of Neurology4, University of Michigan, Ann Arbor, MI and Veterans Hospital5 Iowa City, IA

DYT1, the most common form of inherited dystonia, is a dominantly inherited neurological disease caused by a single amino acid deletion in the protein torsinA (torA). Whereas wild type torA (torA(wt)) is an endoplasmic reticulum (ER)-resident glycoprotein, the mutant form (torA(mut)) redistributes to the nuclear envelope (NE). The different subcellular localization might influence the accessibility of the proteolytic machinery to torA(wt) and torA(mut). ER proteins are normally degraded by the proteasome through ERAD or autophagy. Little is known, however, on how cells handle NE proteins.

Because variations in the torA(wt):torA(mut) expression ratio are predicted to modulate DYT1 pathogenesis, we aimed to identify differences in the degradation routes for both forms of torA that could be exploited for therapeutic design through the pharmacological manipulation of catabolic pathways. Pharmacological inhibition of proteasomal and macroautophagic pathways in an inducible cell model overexpressing torA reduced clearance of both forms of torA, demonstrating they follow both proteolytic routes, perhaps the mutant form depending more on autophagy. When the same experiment was performed in cells expressing torA(wt) at endogenous levels, autophagy was the preferential degradation pathway.

We next investigated the role of a candidate family of ubiquitin ligases on torA degradation and, consequently, as potential modulators of DYT1 pathogenesis. The FBG family of ubiquitin ligases targets glycoproteins for proteasomal degradation through ERAD. We hypothesized that, as an ER glycoprotein, torA(wt) is a substrate for FBG ligases. Initial co-expression experiments confirmed that FBG proteins reduce steady-state levels of torA(wt), showing a less prominent effect over torA(mut). Dominant negative forms of FBG proteins did not lower torA levels, excluding a potential artifact of overexpression. Furthermore, silencing an FBG protein through RNAi resulted in increased levels of endogenous torA. Finally, co-immunoprecipitation experiments demonstrated the presence of torA-FBG interactions. Surprisingly, the effect on FBGs on torA was not rescued after protesomal inhibition, but was partially restored when macroautophagy was blocked.

In summary, torA is degraded both by macroautophagy and the proteasome, with some differences for torA(wt) and torA(mut) likely due to their different subcellular localization. As a result, pharmacological induction of macroautophagy could perhaps represent a therapeutic strategy in DYT1. Furthermore, owing to their role on torA degradation, FBG ubiquitin ligases could be modifiers of DYT1 pathogenesis and penetrance.

Supported by: NIH/NINDS K02 NS058450 (PG-A) and R01 NS047535 (HLP), V. A. Research Career Development Award 2006-12 (KG).

POSTER 20

INFLUENCE OF C-TERMINAL SPLICING ON THE ENZYMATIC ACTIVITY AND AGGREGATION OF ATAXIN-3, A POLYGLUTAMINE DISEASE PROTEIN

Ginny Marie Harris,1,2 Pedro Gonzalez-Alegre3 and Henry Paulson4

1Interdisciplinary Program in Molecular and Cellular Biology, 2Medical Scientist Training Program, 3Department of Neurology, University of Iowa, Iowa City, IA, and 4Department of Neurology, University of Michigan, Ann Arbor, MI Spinocerebellar ataxia, type 3 (SCA3) is one of at least nine polyglutamine (polyQ) neurodegenerative diseases caused by the expansion of polyQ-encoding CAG repeats. Though all polyQ diseases share the hallmarks of protein misfolding, oligomerization, and aggregation, it is becoming increasingly clear that the protein context in which each polyQ expansion occurs influences toxicity. Alternative splicing, a mechanism for achieving functional diversity from a given gene, has rarely been investigated as a contributing factor to protein context in polyQ diseases. Ataxin-3, a deubiquitinating enzyme that is the disease protein in SCA3, is alternatively spliced to produce a protein containing a Josephin protease domain, two ubiquitin interacting motifs (UIMs), and either a hydrophobic stretch or a third UIM at its extreme C-terminus (2UIM and 3UIM ataxin-3 isoforms, respectively). Here we have taken advantage of emerging insights into the biological function of ataxin-3 to examine the significance of this alternative splicing. In brain tissue from YAC transgenic mice expressing either normal (Q15) or expanded (Q84) alleles of the complete human ATXN3 gene, we confirmed the expression of both known alternative C-termini of ataxin-3 at the mRNA level. Both in neonatal and adult brain from mice expressing Q15 or Q84 ataxin-3, the predominant ataxin-3 transgene contains the C-terminus encoding a third UIM. Although the two C-terminal splice variants of ataxin-3 display similar in vitro deubiquitinating activity, the originally characterized 2UIM ataxin-3 splice variant is more prone to aggregate than the 3UIM variant when overexpressed in mammalian cells. Further characterization of the biochemical and cellular behavior of these splice variants will be important to our understanding of ataxin-3 function in vivo and may shed light on the selective neurotoxicity of expanded ataxin-3.

POSTER 21

GLOBAL TRANSCRIPTIONAL ANALYSIS OF THE YEAST RIBOSOME-ASSOCIATED CHAPERONE SSB DELETION STRAIN

Samantha A Herbst1, David Berry2, Audrey Gasch2 and Elizabeth Craig1

1Department of Biochemistry, 2Department of Genetics, University of Wisconsin-Madison, Madison, WI Ribosome-associated chaperones are found in both prokaryotes and eukaryotes. By transiently binding and releasing nascent chains emerging from the ribosome, these chaperones are thought to promote productive folding and prevent protein aggregation. In the yeast Saccharomyces cerevisiae, the primary Hsp70 to carry out this role is Ssb. An Ssb deletion strain is slow-growing, cold sensitive, and cation hypersensitive. To better understand the underlying causes of the deletion phenotype, we carried out transcriptional microarray analysis on wild-type and Ssb deletion strains in order to examine the global transcriptional response to the absence of Ssb. Functional analysis indicates that stress response and carbon metabolism genes are enriched among the up-regulated genes, while protein translation-related genes are among those down-regulated. We are further examining the effects of a time-dependent decrease in the level of Ssb upon the most highly up-regulated genes identified in the microarray.

POSTER 22

ALZHEIMER'S DISEASE IS DUE TO FAULTY ER, POSTTRANSLATIONAL PROCESSING

Jordan L. Holtzman Departments of Pharmacology and Medicine, and Division of Environmental Health Sciences, University of Minnesota, Minneapolis, MN Background: Alzheimer's disease is characterized by dementia and extracellular deposits of a garbage protein, β-amyloid. β-amyloid is produced during the normal ER processing of the amyloid precursor protein (APP); a single transmembrane protein which is cleaved to give sAPP and β-amyloid. Since β-amyloid is derived from the transmembrane portion of APP, it is not water soluble. sAPP is a growth factor which is produced in everyone; raising the question of why β-amyloid is deposited only in the brains of the elderly. Methods: Human CSF and rat hepatic microsomes were immunoblotted with antibodies to β-amyloid and 6 ER chaperones. Results: In human CSF β-amyloid is complexed with ERp57 and calreticulin. The binding to these 2 chaperones suggests that it is N-glycosylated on ASN27. The ERp57 appeared to be covalently bound, since the complex was stable under denaturing conditions. The complex was isolated by immunoprecipitation with either anti-ERp57 or -βamyloid. In aging studies in rat liver several ER chaperones, including ERp57, but not calreticulin, declined with age. Patients with low CSF levels of ERp57 invariably had β-amyloid deposits in their brains at autopsy. Conclusions: Our data suggest that β-amyloid deposits when it is not N-glycosylated and complexed with ER chaperones. And that the dementia is probably not due to β-amyloid toxicity but rather to decreased processing of the membrane proteins which are necessary for memory formation. This decline is associated with decreases in ER chaperones as noted both in rat liver and in the CSF of elderly patients. The decreased processing could relate to the declines in N-glycosylation suggested by the increased levels of dolichol and dolichol phosphate found in aged animals. These data would suggest that there is a decrease with age in the first step of the pathway.

POSTER 23

THE REDOX-SWITCH DOMAIN OF HSP33 FUNCTIONS AS DUAL STRESS SENSOR

Author(s): Ilbert M., Schmitt S., Horst J., Ahrens S., Graf P.C.F and Jakob U.

Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI Hsp33 is a potent holdase chaperone which presents an unusual regulation. It requires extreme conditions, such as peroxide stress and elevated temperatures to be activated as a chaperone. Hsp33 can then protect the vast majority of unfolding proteins against non-specific aggregation. To detect these stresses, Hsp33 presents a highly sophisticated mechanism of regulation composed of two interdependent sensors. Hsp33 contains a redox sensor domain, which consists of four highly conserved cysteines that coordinate one zinc ion. Upon exposure to peroxide stress, the cysteines get oxidized and zinc is released (6). While this redox event is absolutely crucial for the activation of Hsp33, it is not sufficient. Hsp33 harbors an additional “folding” sensor region, which senses unfolding conditions. These two stress-sensing regions, located in the C-terminal redox-switch domain of Hsp33, are interdependent: neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33’s full activation. This atypical regulation nicely reflects the in vivo requirement of Hsp33, which is to compensate for the loss of activity of other E. coli chaperones under these severe oxidative stress conditions. Indeed, ATP-dependent chaperones such as the DnaK system get inactivated by the exact conditions which activate Hsp33. This emphasizes the crucial role of Hsp33 in vivo. To fully understand the complete regulation of Hsp33, the exact modus operandi of Hsp33 inactivation and substrate transfer to the ATP-dependent chaperones upon return to normal conditions need to be further investigated. We show that Hsp33 can be reduced and zinc is reincorporated, creating a stable reduced dimer which is still able to bind substrates. Another crucial step seems necessary for the full return of Hsp33 to an inactive monomeric form. The possibility that the DnaK system could play an active role in the inactivation of Hsp33 is proposed.

POSTER 24

IDENTIFICATION OF THE REQUIREMENTS FOR THE IN VIVO INTERACTION OF THE HSP40 YDJ1 WITH TWO DISTINCT HSP90 CLIENT PROTEINS

Gary Flom, Marta Lemieszek and Jill L. Johnson Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID The Hsp40 Ydj1 is required for the activity of Hsp90 client proteins in yeast, including both heterologous clients such as steroid receptors and the v-src kinase, as well as native client proteins such as the Ste11 kinase and the Hap1 transcription factor. The substrate-binding domain of Ydj1 is required for these functions, but the specific regions within the substrate domain required for Ydj1-client interaction are unknown. We examined the effect of mutations within Ydj1 on the physical and functional interaction of Ydj1 with two distinct clients proteins: Ste11 and the glucocorticoid receptor. We found that mutations within the proposed client-binding site (domain I), did not disrupt interaction with either client protein. Instead, the most dramatic effects on client interaction and activity were observed upon mutation of the zinc-binding domain (domain II) and/or the farnesylation signal, which dictates localization of Ydj1 to the cytoplasmic face of ER and nuclear membranes. Hsp40s are modular proteins, and the zinc-binding domain and farnesylation signal of Ydj1 distinguish it from another cytosolic Hsp40, Sis1. Although Sis1 and Ydj1 share some overlapping functions, Sis1 is unable to support activity of Ste11 or the glucocorticoid receptor in the absence of Ydj1. Our results suggest that the unique domains of Ydj1 are major contributors to its specific functions in maturation of Hsp90 client proteins, and agree with prior results that the unique features of Sis1 dictate its specific functions in yeast prion maintenance.

POSTER 25

CAENORHABDITIS ELEGANS’ PEROXIREDOXIN-2 PLAYS A CRUCIAL ROLE IN THE RESISTANCE AGAINST OXIDATIVE STRESS

Caroline Kumsta, Maike Thamsen and Ursula Jakob

Cellular and Molecular Biology Graduate Program and the Molecular, Cellular and Developmental Biology Department, University of Michigan, Ann Arbor, MI

Aging is a complex physiological process and numerous aging theories have been proposed. One of the leading models is the free radical theory of aging, which suggests that the accumulation of reactive oxygen species (ROS) is causally linked to aging and cell death. Aging cells have been found to accumulate proteins with oxidative modifications, including side chain carbonylation and thiol modifications. It is this oxidative damage to specific cellular proteins that is often proposed to constitute one of the major mechanisms that link oxidative stress to age-associated loss of critical physiological functions.

Our goal is to identify C. elegans proteins that are sensitive to oxidative stress and are targets for aging-induced oxidative modifications. Age-related changes in protein activity can be often mimicked by exogenous exposure of proteins to ROS. We found that short-term incubation of synchronized wild-type L4 larvae in sublethal concentrations of hydrogen peroxide leads to two distinct phenotypes. The movement is highly impaired during the first 12 hours after the stress treatment and ranges from no movement to slow and uncoordinated motility. Within 24 hours, surviving worms undergo a complete recovery and reveal the typical fast, sinusoidal body movement. The second phenotype is a significantly reduced brood-size throughout the complete selffertile reproductive span. We have now started to monitor and visualize the effects of oxidative stress treatment on the proteins of oxidatively stressed C. elegans using 2D gel electrophoresis. This analysis revealed a number of proteins that show substantially increased levels of oxidative protein modifications. One of these proteins is PRDX-2, a 2-Cys Peroxiredoxin, responsible for the detoxification of peroxides. We found that oxidative stress treatment causes the rapid and reversible overoxidation of PRDX-2’s catalytic cysteine. This might turn PRDX-2 into a molecular chaperone, as observed in other organisms. Interestingly, prdx-2 knockout worms are highly susceptible to oxidative stress conditions and reveal a substantial lifespan decrease at 15°C, suggesting that PRDX-2 plays an important cytoprotective role in C. elegans.

POSTER 26

N-METHYL PEPTIDE INHIBITORS OF POLYGLUTAMINE FIBRILLOGENESIS Jennifer Lanning1, Tali Gidalevitz2, Richard Morimoto2 and Stephen Meredith1

1Department of Pathology, University of Chicago, Chicago, IL 2Department of Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL

Polyglutamine (polyQ) expansion in the exon 1 domain of huntingtin leads to aggregation into β-sheet-rich insoluble aggregates associated with Huntington’s Disease (HD). One therapeutic strategy might be to develop inhibitors that bind to the growing β-sheet and hamper fibril formation. To this end, we have developed N-methylated polyglutamine homologs in which hydrogen-bond donor sites on one face of the peptide have been removed. This arrangement would permit binding to, but not propagation of the β-sheet, and would therefore inhibit fibrillogenesis.

We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides. Surprisingly, the most effective inhibitor, 5QMe2 (Anth-K-Q-Q(Me2)-Q-Q(Me2)-Q-CONH2, Anth = anthranilic acid, Q(Me2) = side chain N-methyl Q) includes only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyQ fibrillogenesis. 5QMe2 can completely prevent fibrillogenesis of YAQ12A at ratios of 10:1 (5QMe2:YAQ12A) for at least 110 hrs, and also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance measurements show Kd in the mM range with very fast kon and koff.

We are currently examining the effect of 5QMe2 in a HD model organism: C. elegans expressing toxic levels of GFP-polyQ proteins in either muscle wall or neuronal cells. Preliminary experiments indicate that treatment of the worms with 5QMe2 causes a clear reduction in the cellular load of insoluble aggregates, suggesting that the peptide can enter cells and prevent fibril formation. Future experiments include further quantification of aggregate burden, coupled with behavioral assays of neuro- and cyto-toxicity.

POSTER 27

FACTORS INVOLVED IN THE AGGREGATION OF YEAST PRIONS Anita L. Manogaran, and Susan W. Liebman Department of Biological Sciences, Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, IL The presence of cellular aggregates is a hallmark of many neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease and prion diseases. Emerging evidence has suggested that other factors, such as chaperones and ubiquitin, play an integral role in the formation and maintenance of these aggregates. Prions are set apart from other neurodegenerative aggregation diseases in that they are infectious. Cellular aggregates have also been associated with yeast prions, including the self-perpetuating form of the Rnq1 protein, [PIN+]. The Rnq1 protein, when fused to the green fluorescent protein (GFP), is localized into distinct cytoplasmic foci in the [PIN+] prion state and is diffuse in the non-prion, or [pin-], state. A screen was performed to identify non-essential gene deletions that fail to maintain [PIN+] aggregates. Surprisingly, of over 4800 strains screened, only two genes failed to maintain [PIN+] aggregates, hsp104Δ and rnq1Δ. Upon closer inspection of [PIN+] aggregates in library strains, Rnq1:GFP aggregation was considerably altered in strains lacking the CUE2 gene. Distinct [PIN+] foci observed in wild type strains were extended and spider-like in cue2 deletion strains, suggesting that the Cue2 protein, a monoubiquitin binding protein of unknown function, may play a role in [PIN+] aggregation. Biochemical and genetic studies are underway to better understand how CUE2 contributes to prion formation and provide possible clues into how the Cue2 protein functions within the cell.

POSTER 28

THE ROLE OF THE ACTIN CYTOSKELETON IN THE FORMATION OF THE NON-Q/N-RICH PRION [HET-S]Y IN SACCHAROMYCES CEREVISIAE

Vidhu Mathur, Vibha Taneja, and Susan W. Liebman Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL Mammalian prion diseases like Creutzfeldt Jacob in humans, scrapie in sheep and mad cow disease in cattle are infectious, neurodegenerative diseases caused by a misfolded form of the prion protein, PrPSc. In Saccharomyces cerevisiae, the prions [PSI+], [URE3] and [PIN+] are characterized by the aggregation of Sup35p, Ure2p and Rnq1p, respectively. Various cellular factors, including molecular chaperones, govern the inheritance of the known yeast prions. Particularly, the heat shock protein Hsp104p, plays an essential role in prion propagation in yeast. Inhibition of Hsp104p activity in yeast leads to the loss of all known native yeast prions. Members of the Hsp70 and Hsp40 families are also important for prion propagation. [Het-s] is a native prion of the filamentous fungus Podospora anserina. A fusion of the [Het-s] prion domain and GFP has been shown to propagate as a prion in yeast, called [Het-s]y. Although Hsp104p is not absolutely required for [Het-s] propagation in Podospora, in yeast [Het-s]y propagation requires Hsp104p activity. All known native yeast prions have glutamine/asparagine (Q/N) rich prion domains. In contrast, [Het-s]y lacks a Q/N-rich region, making it similar to the non-Q/N rich mammalian prion PrPSc. To elucidate the role of other cellular factors that affect prion formation, we show that components of cortical actin cytoskeleton that are involved in endocytosis play a role in [Het-s]y aggregation. We examined the effect of mutations in actin cytoskeletal proteins like Sla1p, Sla2p, End3p and Act1p, on the induction of [Het-s]y . While mutations in Sla1p and Act1p do not considerably affect [Het-s]y formation, deletion of END3 clearly reduces, and deletion of SLA2 completely inhibits, the formation of [Het-s]y. The rare cells containing [Het-s]y aggregates in these mutants can propagate [Het-s]y and show no growth defect. Sla2p also interacts with [Het-s]y in a co-immunoprecipitation assay. We are currently testing if sla2 mutants infected with [Het-s]y can propagate the aggregates. Analogous to what is seen during de novo appearance of [PSI+], overexpression of Het-s-GFP fusion leads to the formation of large ring or worm-like aggregates which can be peripheral, surrounding the cell membrane, or internal, surrounding the vacuole. Likewise, we have seen that the internal rings of [Het-s]y colocalize with the vacuole. Since, the actin cytoskeleton was also shown to affect the formation of the Q/N-rich yeast prion [PSI+], our findings suggest that the Q/N-rich and the non-Q/N-rich prions require the same cellular machinery for their formation and propagation.

POSTER 29

PKC-α MEDIATES TRANSCRIPTIONAL ACTIVATION OF THE INDUCIBLE HSP70 VIA AP1 SIGNALS

Osman Mirza, Tina Griffin, and Ruben Mestril Department of Physiology and the Cardiovascular Institute, Loyola University Medical Center, Maywood, IL Recent research has demonstrated the involvement of both PKC and HSP families in cardioprotection. Investigations into the interaction between these proteins implicate a PKC-α mediated induction of Hsp70 and subsequent protection against simulated ischemia-reperfusion in neonatal rat ventricular myocytes (NRVM). Interestingly, the induction by PKC-α was found to be both highly specific for Hsp70 and independent of the conventional heat shock transcription factor (HSF-1) activation (1). In an effort to further elucidate this mechanism, we linked a luciferase reporter gene to chimeric constructs of the promoter region of the rat Hsp70. Constructs were transfected into myogenic H9c2 cells followed by adenoviral mediated overexpression of PKC-α . Considering that regulation by PKC-α signaling was found to be independent of HSF-1, the significance of other transcription factors exerting their action on the promoter region of Hsp70 are presently being investigated. Our results demonstrate that induction of the Hsp70 promoter by PKC-α is preserved within the first 175 base pairs from the start of transcription. The sequence of the Hsp70 promoter in this segment contains at least two potential Ap-1 signaling elements. Further mutation of the -175 bp promoter region may disclose the possible role of these Ap-1 sites in the transcriptional activation of Hsp70 by PKC-α . In addition, the insertion of Ap-1 signals as oligomers upstream of a basal promoter-luciferase construct could further confirm their significance. (1) Coaxum S, Griffin TM, Martin JL, Mestril R. (2007) Influence of PKC-alpha over-expression on hsp70 and cardioprotection. Am. J. Physiol. Heart Circ. Physiol. 292:H2220-H2226.

POSTER 30

ACTIVATION OF HEAT SHOCK AND ANTIOXIDANT RESPONSES BY THE NATURAL PRODUCT CELASTROL: TRANSCRIPTIONAL SIGNATURES OF A

THIOL-TARGETED MOLECULE Amy Trott*, James D. West†, Lada Klaić‡, Sandy D. Westerheide†, Richard B. Silverman‡, Richard I. Morimoto† and Kevin A. Morano*

*Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX, †Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL , ‡Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Center for Drug Discovery and Chemical Biology, Northwestern University, Evanston, IL Stress response pathways allow cells to sense and respond to environmental changes, such as those caused by pathophysiological disease states. Pharmacologic modulation of cellular stress pathways has implications in the treatment of a variety of human diseases including neurodegenerative disorders, cardiovascular disease and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous clinically relevant properties and is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates the homologous Hsf1 of Saccharomyces cerevisiae at the same effective concentration seen in mammalian cells. Transcriptional profiling revealed that in addition to heat shock genes, celastrol treatment induces a battery of oxidant defense genes in yeast. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant genes were likewise induced in mammalian cells, demonstrating that celastrol simultaneously activates two major cellular stress-mediating pathways in distantly related eukaryotes. We demonstrate that celastrol’s biological effects, including inhibition of Hsp90-mediated glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism to explain the cellular activity of this therapeutically promising compound.

POSTER 31

STRUCTURAL GENOMICS OF EUKARYOTIC CHAPERONE PROTEINS Jerzy Osipiuk1, Minyi Gu1, Monireh Bargassa1, Rory Mulligan1, Min Zhou1, Natalia Maltseva1, Erika Duggan1, Hui Li1, Catherine Hatzos1, Cindy Voisine2, Chandan Sahi3, Elizabeth A. Craig3, Richard I. Morimoto2 and Andrzej Joachimiak1 1 The Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne, IL 2 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 3 Department of Biochemistry, University of Wisconsin, Madison, WI Molecular chaperones assist protein folding and are essential for cell viability. These proteins have recently become a renewed biomedical focus because of observations that accumulation of misfolded proteins can lead to cellular dysfunction and death. The activities of molecular chaperones suppress aggregation and toxicity allowing cellular functions to be restored. It is believed that more than 100 human diseases including neurodegeneration, ischemic heart disease and diabetes are associated with the expression of misfolded proteins. The Midwest Center for Structural Genomics pilot program of structural genomics applied to chaperone proteins from Caenorhabditis elegans and Saccharomyces cerevisiae started in year 2006. We focused on two major families of chaperones: Hsp40 and Hsp70/Hsc70 proteins. 66 cDNA full-length and 318 partial-length clones were used in first stage of the program. All clones were cloned into pMCSG7 plasmid. The proteins were screened for solubility and purified using His-tag affinity chromatography and TEV protease cleavage. We obtained 8 diffraction-quality crystals of Hsp40 and Hsp70 protein domains. Those were 4 crystals of Hsp40 J-domains, 3 crystals of Hsp70 peptide-binding domains and 1 crystal of Hsp70 ATPase domain. 4 structures were deposited in PDB including first X-ray structure of Hsp40 J-domain at 1.24 Å resolution. Currently, we are expanding our studies by adding more targets and attempting to crystallize protein complexes. This work was supported by National Institutes of Health Grant GM074942 and by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

POSTER 32

A CONSERVED STABLE CORE STRUCTURE IN THE PASSENGER DOMAIN β-HELIX OF AUTOTRANSPORTER VIRULENCE PROTEINS

Jonathan P. Renn and Patricia L. Clark Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN In Gram-negative bacteria, a wide variety of virulence factors are secreted via the autotransporter (AT) pathway. Intriguingly, there is no significant concentration of ATP in the periplasm, nor a proton gradient across the OM, so the energetic origin of efficient secretion of AT proteins is unknown. More than 97% of AT proteins are predicted to contain right-handed parallel β-helical structure, and the three crystal structures available for AT passenger domains each contain a long right-handed parallel β-helix. Previous studies have shown that pertactin, an AT from Bordetella pertussis, exhibits three-state folding and has a C-terminal stable core structure. Here, we show that Pet, an unrelated AT from Escherichia coli, also exhibits three-state unfolding and also has a stable core structure. Deletion mutants, mass spectrometry, and N-terminal sequencing demonstrate that the Pet stable core is also located near the C-terminus of the passenger domain. Moreover, sequence analysis suggests that three-state folding and a C-terminal stable core structure could be important general features of the biogenesis of AT proteins in vivo.

POSTER 33

DJ-1-MEDIATED NEUROPROTECTION AGAINST ALPHA-SYNUCLEIN TOXICITY IN PARKINSON’S DISEASE

Jean-Christophe Rochet, Fang Liu, John Hulleman, and Jagadish Hindupur Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN Parkinson’s disease (PD) is a neurodegenerative disorder that involves a loss of dopaminergic neurons in the substantia nigra. The postmortem brains of PD patients are characterized by abnormally high levels of oxidative damage. Some surviving neurons contain inclusions named Lewy bodies, which consist largely of fibrillar forms of the presynaptic protein alpha-synuclein (αSyn). Three αSyn mutants, A30P, E46K, and A53T, are associated with rare cases of early-onset, familial PD. All three mutants have a greater tendency to self-associate than the wild-type protein, suggesting that αSyn aggregation plays a role in the pathogenesis of PD. Other cases of early-onset, familial PD result from loss-of-function mutations in the gene encoding DJ-1, a homodimeric protein with proposed antioxidant and chaperone functions. Moreover, age-dependent over-oxidation of DJ-1 is thought to contribute to sporadic PD. The neuroprotective function of DJ-1 is modulated by oxidation of cysteine 106, a residue that may act as an oxidative stress ‘sensor’. In this study we examined whether DJ-1 protects against dopaminergic cell death induced by αSyn, and we investigated the underlying mechanisms. Expression of wild-type DJ-1 suppressed αSyn neurotoxicity and aggregation in primary dopaminergic neurons transduced with αSyn-encoding lentivirus. Neurons with reduced levels of endogenous DJ-1 were sensitized to αSyn toxicity, and mutant forms of DJ-1 involved in familial PD exhibited decreased neuroprotective activity. Suppression of αSyn neurotoxicity by wild-type DJ-1 correlated with upregulation of the stress-inducible form of Hsp70 (iHsp70). Using cultures co-transduced with A53T- and iHsp70-encoding lentivirus, we further showed that upregulation of the chaperone was sufficient for DJ-1-mediated inhibition of αSyn toxicity and aggregation. In contrast, siRNA analyses revealed that iHsp70 is not strictly necessary for DJ-1-mediated neuroprotection, although it is essential for the inhibition of αSyn aggregation. In parallel studies, we showed that DJ-1 suppresses αSyn fibril formation in a test-tube model. This DJ-1 chaperone activity was modulated by the oxidation of cysteine 106, and it correlated with the binding of DJ-1 to prefibrillar αSyn oligomers termed ‘protofibrils’. Our findings suggest that DJ-1 interferes with αSyn neurotoxicity and aggregation via two mechanisms: an indirect mechanism, involving upregulation of Hsp70, and a redox-sensitive direct mechanism, involving the binding of potentially toxic αSyn protofibrils. Activation of both mechanisms may be a reasonable strategy to alleviate dopaminergic cell death in PD.

POSTER 34

C. ELEGANS MODELS FOR POLYGLUTAMINE AND MUTANT SOD1 EXPRESSION AS TOOLS TO EXAMINE THE GENETIC BASIS FOR THE TOXIC OLIGOMER

Maria Catarina Silva1, Monica Beam2, Happy Thakkar2, Susan Fox2, Margarida D. Amaral1, and Richard I. Morimoto2 Faculdade de Ciencias da Universidade de Lisboa, Portugal1 Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston IL, USA2 Protein misfolding, aggregation and toxicity are a common feature of many neurodegenerative diseases. However, whether the aggregates, as the end point of a protein sequestration event, are the toxic species or result from a cellular protective mechanism is still controversial. The cellular quality control may promote aggregation as a mechanism to sequester intermediate oligomers that are highly reactive and toxic. In light of the ongoing efforts to better understand the aggregation-induced toxicity, we started by determining if the presence of polyQ and mutant SOD1 protein aggregates and toxicity are directly correlated. We used an unbiased genetic approach to suppress aggregation, followed by analysis of its effect on toxicity, and found that suppression of aggregation by genetic knock-down of individual genes can either increase or decrease toxicity. Furthermore, we explored if there is a direct correlation between protein biophysical properties, as oligomeric state, and toxicity. We verified that no specific oligomeric size can be associated with increased toxicity, contradicting the ‘toxic oligomer’ hypothesis.

POSTER 35

CONFORMATIONAL PROPERTIES OF RIBOSOME-BOUND GFP

Krastyu Ugrinov and Patricia Clark University of Notre Dame, Department of Chemistry and Biochemistry, Notre Dame, IN A growing amount of evidence suggests that the folding of nascent polypeptides may be influenced by the vectorial appearance of the polypeptide at the surface of the ribosome. Yet little is known about the conformational flexibility of nascent, ribosome-bound polypeptides. Our goal is to determine the effects of the ribosome on the conformations and dynamics of newly synthesized polypeptide chains. We are using green fluorescent protein (GFP) as a model for co-translational folding of β-sheet structures. GFP is an excellent model for these studies because the residues that make contacts in the native structure are often far apart in the primary sequence. This high ‘contact order’ renders GFP (and many other β-sheet proteins) prone to aggregation during refolding in vitro, and suggests co-translational nascent chain conformations may be particularly important for productive folding. In this study, we have used fluorescence anisotropy as a powerful tool to provide information on the overall flexibility of polypeptide chains. For this purpose, we designed a series of GFP constructs with an N-terminal CCPGCC motif, which specifically binds the biarsenical fluorescein dye FlAsH. Experiments with ribosome-bound GFP chains have revealed that conformational freedom increases with increasing nascent chain length. Moreover, ribosome-bound full length GFP can fold completely on the ribosomal surface. The complete folding is possible only if the entire GFP sequence is exposed on the ribosome. Conformational flexibility and properties for nascent versus released polypeptide chains was determined.

POSTER 36

DECIPHERING THE CHAPERONE CODE USING C. ELEGANS MODELS Cindy Voisine, Michael Schieber, Kai Orton, Sally McFall, and Richard Morimoto Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston IL Molecular chaperones, key components of the cellular folding machinery, interact with misfolded proteins and prevent aggregation. We are conducting a comprehensive analysis of the chaperone potential encoded within the genome of the metazoan C. elegans to identify key players involved in maintaining protein homeostasis. Using established C. elegans models of polyglutamine and Aβ aggregation, we are testing whether a subset of chaperones is required to maintain homeostasis regardless of the proteotoxic stress, defining a chaperone code in vivo. RNAi analysis targeted against individual chaperones suggests that only a small subset of chaperones alters homeostasis in both models. At this time, we have identified candidates belonging to the Hsp70 (HSP-1), Hsp90 (DAF-21), and Hsp60 (CCT-1) chaperone families. Using tissue and stage specific expression patterns, inducibility profiles and protein interaction data, we hope to identify commonalities within the subset to decipher the code.

POSTER 37

HYPOCHLOROUS ACID – POWERFUL ANTIMICROBIAL AND ACTIVATOR OF THE REDOX-REGULATED CHAPERONE HSP33

Wholey, W-Y., Oezcelik, D., Winter, J., Graf, P.C.F., and Jakob, U. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI Reactive oxygen species (ROS), which have the potential to oxidize and damage many cellular macromolecules including proteins, lipids and nucleic acids, are produced at very low levels during cellular metabolism. High levels of ROS such as superoxide (O2

•-), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) are generated during the host immunity defense by macrophages and neutrophils in an effort to kill invading microorganisms. Recent reports showed that the conserved dual oxidase DuOx secretes HOCl to limit bacterial colonization in mucosal barrier epithelia. This result suggested that the oxidative burst starts as early as during bacterial colonization. Not surprisingly, bacteria have evolved highly conserved anti-oxidant systems to counteract these oxidative insults. We showed that Hsp33, a highly conserved redox-regulated molecular chaperone in bacteria, plays an important role in protecting cells against oxidative stress. In vitro data demonstrated that Hsp33, which is inactive as chaperone under non-stress conditions, is rapidly activated by very low concentrations of HOCl. In vivo studies confirmed this rapid activation and revealed that Hsp33 prevents numerous cellular proteins against HOCl-induced aggregation. This finding indicated that Hsp33 is a potent chaperone that protects bacteria against HOCl-induced cell death. To investigate whether Hsp33’s protective function plays a role during bacterial colonization, we constructed an Hsp33 deletion in the pathogenic strain Vibrio cholerae. We demonstrated that the Dhsp33 V. cholerae strain was highly sensitive to HOCl treatment. A 10,000-fold decrease in cell viability was observed upon exposure to 8 µM HOCl as compared to wt V. cholerae strain. In vivo competition assays using the infant mouse model revealed that the hsp33 deletion strain of V. cholerae was more than 5-fold faster cleared from the mouse intestine than the corresponding wild type strains, indicating that Hsp33 functions as colonization factor in V. cholerae and probably other enterobacteriae. These results are in excellent agreement with earlier reports, which suggested that reactive oxygen species and particularly HOCl provide an effective antimicrobial strategy to control bacterial colonization of mucosal barrier epithelia. Our studies indicate that the molecular chaperone Hsp33 is one of the key players in the protection of bacteria against oxidative stress induced cell death.

POSTER 38

MODULATING THE ACTIVITY OF HEAT SHOCK PROTEIN 70 (HSP70) WITH DIHYDROPYRIMIDINES

Susanne Wisén and Jason Gestwicki Department of Pathology and Life Sciences Institute, University of Michigan, Ann Arbor, MI Genetic evidence implicates that the molecular chaperone Hsp70 acts as a key gatekeeper in the physiological machinery that protect against protein misfolding disorders, such as Alzheimer’s and Huntington’s diseases. Based on this evidence, we hypothesize that selective pharmacological activation of Hsp70 might be beneficial. However, very few small molecule partners for Hsp70 have been described. The Brodsky laboratory has reported that compounds with a dihydropyrimidine scaffold modify Hsp70's ATPase activity. Recently, we have developed a novel synthetic route to dihydropyrimidines using microwave techniques, and we have synthesized an exploratory series of approximately 40 compounds. In addition to these compounds, we have added ~200 dihydropyrimidines from the University of Pittsburgh’s Center for Chemical Methodologies and Library Development (UPCMLD). Within this relatively small number of compounds, we have uncovered candidates that bind Hsp70 and modify both its ATPase and refolding activites, as well as enhance its anti-aggregation activity against amyloids in vitro. Moreover, some of these molecules possess interesting biological activity in cells; for example, one of our lead compounds blocks polyglutamine (polyQ) self-assembly in yeast models of Huntington’s disease and yields a phenotype that is reminiscent of cells that express high levels of Hsp70. Because of the dearth of comparable reagents, we envision that these compounds could find use in the exploration of chaperone biology.

POSTER 39

NON-CANONICAL WALKER A MOTIF IN TORSINA DEFINES A NOVEL SUB-FAMILY OF AAA+ ATPASES WITH DISTINCT BIOCHEMICAL PROPERTIES

Zhonghua Liu*‡, Hui-Chuan Wu*, Maria Nagy*, Sabina Kedzierska†, and Michal Zolkiewski*§ *Department of Biochemistry, Kansas State University, Manhattan, KS †Department of Biochemistry, University of Gdansk, Gdansk, Poland ‡ Present address: Department of Embryology, Carnegie Institution, Baltimore, MD Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ sequence modules contain the nucleotide-binding Walker A motif: GxxGxGK(T/S). We identified a sub-family of AAA+ sequences that contain Asn in Walker A instead of Thr or Ser. This non-canonical sub-family includes torsinA, an ER protein linked to human dystonia. Mammalian sequences homologous to torsinA can be divided into four distinct groups: one with the canonical Walker A motif and three with the non-canonical sequence. The purified AAA+ module of torsinA did not form hexamers and showed low ATPase activity when compared to a canonical AAA+ ATPase ClpB. Replacement of the Walker A Thr in ClpB with Asn inhibited the ClpB ATPase and its chaperone activity in vivo and in vitro, induced preferential binding of ADP vs. ATP, and uncoupled the linkage between the ATP-bound conformation and the high-affinity substrate binding. In agreement with the latter result, trapping the AAA+ module of torsinA in the ATP-bound state with a Walker B mutation inhibited interactions of torsinA with other cellular components and stimulated its secretion from yeast cells. In contrast, introduction of the canonical Walker A sequence into torsinA inhibited the protein’s secretion. In summary, AAA+ ATPases with the non-canonical Walker A motif display significantly different biochemical properties from the canonical AAA+ ATPases and may utilize distinct mechanisms to link the ATPase cycle and interactions with other macromolecules. We postulate that the non-canonical AAA+ ATPases might not function as hexameric machines generating conformational work, but may serve as nucleotide-driven protein-interaction switches responding to physiological signals.