Overview of Research Activities - Stellenbosch...
Transcript of Overview of Research Activities - Stellenbosch...
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Overview of Research ActivitiesOverview of Research Activities
Prof Johann RohwerProf Johann RohwerLaboratory for Molecular Systems Biology
Department of BiochemistryStellenbosch University
South Africa
Biochemistry Honours Students5 March 2020
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Experiment NMR “metabolomics”
in vivo, in situ, in vitro metabolite measurements
enzyme kinetics for modelling in vivo enzyme kinetics
pH, macromolecular crowding
Theory generalised supply-demand
analysis rate equations for modelling symbolic MCA in vitro vs. in vivo kinetics
Model development of software (PySCeS, psctb, SymCA, RateChar) kinetic models of cellular systems
microbial energy metabolism
cellular redoxin networks (with Dr C Pillay, UKZN)
plant metabolism
glucocorticoid receptor dimerisation (with Prof A Louw)
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Experiment NMR “metabolomics”
in vivo, in situ, in vitro metabolite measurements
enzyme kinetics for modelling in vivo enzyme kinetics
pH, macromolecular crowding
Theory generalised supply-demand
analysis rate equations for modelling symbolic MCA in vitro vs. in vivo kinetics
Model development of software (PySCeS, psctb, SymCA, RateChar) kinetic models of cellular systems
microbial energy metabolism
cellular redoxin networks (with Dr C Pillay, UKZN)
plant metabolism
glucocorticoid receptor dimerisation (with Prof A Louw)
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Experiment: Example
Enzyme kinetics for modelling by Enzyme kinetics for modelling by NMR spectroscopyNMR spectroscopy
(“(“In vivoIn vivo enzyme kinetics”) enzyme kinetics”)
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Brief NMR introduction:Nucleus in magnetic field
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Split in energy levels
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Relaxation and Fourier Transform
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People Students who have completed their degrees
Christiaan Crous, MSc (Z. mobilis SDA) Sandra Alberts, MSc (L. lactis SDA) Justin Smith, MSc (S. cerevisiae SDA & kinetics) Johann Eicher, PhD (E. coli SDA, kinetics &
modelling) Academic staff
Prof Jacky Snoep
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Ongoing work
Question: are kinetic measurements in the test tube a realistic representation of conditions in the cell?
Investigate “in vivo” enzyme kinetics effect of macromolecular crowding
intracellular environment has very little accessible solvent space, [protein] = 250 mg/ml
effect of pH changes intracellular pH not as constant as always thought,
signal?
E. coli and S. cerevisiae glycolytic enzymes
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Current Students Z. mobilis kinetics and modelling
Theo v. Staden, PhD (Z. mobilis kinetics & modelling)
Macromolecular crowding Julian Wissing, MSc (2020)
pH effect on kinetics and modelling Tiaan Swanepoel, MSc (2018), PhD
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Effect of pH on PGI kinetics
T. SwanepoelMSc thesis (2018)
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Effect of crowding on PFK kinetics
J. WissingMSc thesis (2020)
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Model: Example
Modelling cellular redoxin Modelling cellular redoxin networksnetworks
Johann Rohwer Johann Rohwer Ché PillayChé Pillay
Laboratory for Mol. Systems Biology School of Life SciencesDept. of Biochemistry UKZNStellenbosch University PietermaritzburgSouth Africa South Africa
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Redoxin networks are essential forall living organisms
Toledano et al. (2007) FEBS Lett.
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Oxidation states of biological thiol groups
Finkel (2011) J. Cell Biol.
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Contents
Enzymes or redox couples? Kinetic characterisation of peroxiredoxin system
in S. cerevisiae Dynamics of H
2O
2 metabolism
Conclusions
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Thioredoxin system
NADP NADPH
Trx(SS)Trx(SH)2
Insulin(reduced)
Insulin(oxidised)
TR
classic Michaelis-Menten response model with mass action
Data: Holmgren 1979 JBCModel fit: Pillay et al. 2009 Biochem. J.
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Core model
B A
MM′
DC
k1
k-1
k2
k-2
dm 'dt
=k 1am−k−1bm '−k 2cm' +k−2dm
differential equation
solve for m subject to steady state
calculate v2
irreversible case
\
m=mt (k−1 b+k 2c )
k 1a+k−2d+k−1b+k 2c
v 2=
k 1 k 2mt ac (1− ΓK eq )
k 1a+k−2d+k−1b+k 2c
v 2=(k 1a mt)c
k1
k 2
a+c
Apparent V max : k 1amt
Apparent K m: k 1
k 2
a
k cat
K m
=k 2
Pillay et al. (2009) Biochem. J.
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Changes in apparent Michaelis-Menten parameters
Model Parameters Apparent M-M Parameters
VTR
k2
[Trx]t NADPH K
mV
maxk
catk
cat/K
m
1 1 2 1 0.186 0.386 0.193 1.035
10 1 2 1 2.183 4.378 2.189 1.003
100 1 2 1 21.925 43.860 21.930 1.000
1 10 2 1 0.012 0.348 0.174 14.620
1 100 2 1 0.001 0.333 0.167 263.158
1 1 50 1 0.001 0.496 0.010 8.333
1 1 0.1 1 0.462 0.046 0.463 1.002
1 1 2 10 0.356 0.724 0.362 1.018
1 1 2 0.1 0.026 0.065 0.032 1.236
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Contents
Enzymes or redox couples? Kinetic characterisation of peroxiredoxin system
in S. cerevisiae Dynamics of H
2O
2 metabolism
Conclusions
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Peroxiredoxin system in S. cerevisiae
Fit kcat
and KTrxSS
M. BadenhorstMSc thesis (2020)
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Peroxiredoxin system in S. cerevisiae
Fit k2
M. BadenhorstMSc thesis (2020)
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Contents
Enzymes or redox couples? Kinetic characterisation of peroxiredoxin system
in S. cerevisiae Dynamics of H
2O
2 metabolism
Conclusions
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Response of E. coli model to oscillating H2O
2
M. Badenhorst (2017)
0.1 µM midpoint25% amplitude
0.2 µM midpoint99% amplitude
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Pulsed H2O
2 in S. cerevisiae model
M. BadenhorstMSc thesis (2020)
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Work in progress...
comparative analysis of redoxin networks Generalised Supply-Demand Analysis
quantitative analysis of redox signalling develop kinetic models of
peroxiredoxins dynamics of H
2O
2
effect of Prx decamerisation
Mycobacterium tuberculosis redoxin network
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Conclusions
Why should I model redoxin networks? detect kinetic motifs fit parameters from experimental data explore untested scenarios quantify control parameters unravel mechanism
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People
University of KwaZulu-Natal (Pietermaritzburg) Dr Ché Pillay Diane Lind (MSc) Erin Blom (MSc) Nolyn John (PhD)
Stellenbosch University Chris Barry (PhD 2017-) Melinda Badenhorst (MSc 2018-2020)
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Modelling
Software tools Software tools developed and useddeveloped and used
(Python programming language)(Python programming language)
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PyscesToolbox
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Tools and Techniques
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Experiment NMR “metabolomics”
in vivo, in situ, in vitro metabolite measurements
enzyme kinetics for modelling in vivo enzyme kinetics
pH, macromolecular crowding
Theory generalised supply-demand
analysis rate equations for modelling symbolic MCA in vitro vs. in vivo kinetics
Model development of software (PySCeS, psctb, SymCA, RateChar) kinetic models of cellular systems
microbial energy metabolism
cellular redoxin networks (with Dr C Pillay, UKZN)
plant metabolism
glucocorticoid receptor dimerisation (with Prof A Louw)
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Interested in joining the lab?
Contact me any time to discuss options! [email protected] or 021-808-5843