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Inferring selective pressures for gene expression levels: G6PD and GSR in human erythrocytes

Inferring selective pressures for gene expression levels: G6PD and GSR in human erythrocytes. Armindo Salvador 1,2 and Michael A. Savageau 1 1 Department of Microbiology and Immunology The University of Michigan Medical School 2 Chemistry Department The University of Coimbra. ?.

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Inferring selective pressures for gene expression levels: G6PD and GSR in human erythrocytes

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  1. Inferring selective pressuresfor gene expression levels: G6PD and GSR in human erythrocytes Armindo Salvador1,2 and Michael A. Savageau1 1Department of Microbiology and Immunology The University of Michigan Medical School 2Chemistry Department The University of Coimbra

  2. ? Expression change Change in pathway flux Effect on cell/organism performance Superfluously expressed enzyme (eg. equilibrium enzyme): Expression change No change in pathway flux No effect on performance  Useful simplifying principle or flawed reasoning? Usual view of functionalimpact of enzyme expression

  3. Why does natural selection allow “superfluous” expressionof many enzymes? Could it be because expression is not superfluous?

  4. O2 H2O2 The hexose monophosphate shunt ATP ADP Glycolysis HK Glc G6P NADP+ GSH G6PD GL6P GSR GO6P GO6PD NADPH GSSG

  5. Superfluous(?) expression ofG6PDin erythrocytes • Capacity for NADPH supply limited by HK at <2.5% of G6PD activity • Only erythrocytes with very low residual G6PD activity are hemolyzed in vivo under unstressful conditions • Moderate G6PD deficiencies lack obvious clinical symptoms

  6. Yet… • Distribution of erythrocyte G6PD activity in human populations: • narrow mode at normal values • skewed towards high values Suggest the existence of selective pressurefor maintenance of normal G6PD activity What might these selective pressure be?

  7. Approach for pinpointing selective pressures • Set up a model • List potentially relevant criteria for good metabolic performance • Identify which performance criteria are sensitive to variations about the normal enzyme level • Evaluate evidence of design for fulfilling the relevant criteria • Seek connection of relevant performance criteria to organism fitness

  8. Model of NADPH/GSH redox cycles G6PD (+GO6PD) - NADP+bound NADP+free NADPHfree NADPHbound (X3) (X1) (X2) (X4) GSR GSH (X6) GSSG (X5)

  9. Approach for pinpointing selective pressures • Set up a model • List potentially relevant criteria for good metabolic performance • Identify which performance criteria are sensitive to variations about the normal enzyme level • Evaluate evidence of design for fulfilling the relevant criteria • Seek connection of relevant performance criteria to organism fitness

  10. Criteria for good metabolic performance • Rate of NADPH supply should be: • Sufficient at steady state • Sensitive to the oxidative load • Robust • Responsive to changing oxidative loads(same holds for GSH supply) • Concentration of NADPH should be robust • Concentration of GSSG should be low

  11. Robustness of property P: IP= sum of the absolute values of the sensitivities of P to all parameters that are not control parameters What do we mean by “robustness”? • RP=1 if P totally insensitive to every non-control parameter RP=0 if P infinitely sensitive to at least one parameter • Local measure (Savageau (1971). Nature229: 542-4)

  12. Approach for pinpointing selective pressures • Set up a model • List potentially relevant criteria for good metabolic performance • Identify which performance criteria are sensitive to variations about the normal enzyme level • Evaluate evidence of design for fulfilling the relevant criteria • Seek connection of relevant performance criteria to organism fitness

  13. Performance vs. G6PD activity t½,NADPH GSSG vsup t½,GSH RNADPH Rvsup Sdem

  14. Normal activity 10% residual activity 2% residual activity NADPH depletion underG6PD deficiency 10x increasedoxidative load

  15. Approach for pinpointing selective pressures • Set up a model • List potentially relevant criteria for good metabolic performance • Identify which performance criteria are sensitive to variations about the normal enzyme level • Evaluate evidence of design for fulfilling the relevant criteria • Seek connection of relevant performance criteria to organism fitness

  16. KM,NADP+ NADPtot kox VG6PD Effect of parameters on RNADPH

  17. Approach for pinpointing selective pressures • Set up a model • List potentially relevant criteria for good metabolic performance • Identify which performance criteria are sensitive to variations about the normal enzyme level • Evaluate evidence of design for fulfilling the relevant criteria • Seek connection of relevant performance criteria to organism fitness

  18. v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v O2- H2O2 O2 H2O2 kox t Opsonizedpathogen Complement receptor 1 Fc receptor Macrophage v Erythrocyte Adapted from Lindorfer et al. (2001) Immunol. Rev.183:10-24 Immune clearance(Humoral immunity)

  19. Clinical consequences ofmoderate G6PD deficiency • Increased susceptibility to infection-induced hemolysis • Increased susceptibility to sepsis upon severe trauma injury

  20. Normal G6PD level is necessaryto avoid strong NADPH depletionduring immune adherence to phagocytes

  21. Normal GSR level is necessaryto avoid excessive hemoglobin inactivation

  22. Closing remarks • Performance requirements other than flux capacity can drive natural selection for high expression of enzymes • A change of enzyme level can have a variety of functional effects, even if it leaves steady-state flux unchanged • Physiological values near breakpoints of performance criteria: a prevalent feature? • General framework, simple models, does not require knowledge of expression-regulating mechanisms

  23. Acknowledgements Grant to MAS from the US Public Health Service (RO1-GM30054) Fellowships to AS from Pfizer and FCT-Portugal (SFRH/BPD/9457/2002)

  24. What about glutathione reductase?

  25. Superfluous(?) expression of GSR in erythrocytes • 30-fold excess of GSR activity over erythrocyte capacity for steady NADPH supply • Only erythrocytes with very low residual GSR activity are hemolyzed in vivo • Moderate GSR deficiencies are asymptomatic • Yet, population distribution of erythrocyte GSR levels is sharply peaked

  26. Performance vs. GSR activity t½,GSH GSSG t½,NADPH RNADPH Rvsup vsup Sdem

  27. Effect of parameters on [GSSG] kox KM,GSSG VGSR NADPtot KM,NADPH

  28. Hemoglobin glutathionylation GSSG + HbSH GSH + HbSSG Poor O2 deliveryto tissues Saturation Physiologicalrange pO2

  29. 80 Harvard step test score 0 0 5 10 15 Hb (g/L) Physical performance improves with Hb concentration… Viteri & Torun (1974) Clinics Hematol.3:609-26 …so, functionally inactivating part of Hb decreases physical performance and thus fitness

  30. The natural selectionof biochemical parameters Eliminated Selected Natural selection Low fitness High fitness Bad physiologic performance Good physiologic performance Inadequate metabolic responses,waste of resources Adequate metabolic responses, conservation of resources Performancerequirements Bad parameter values of biochemical subnetwork Good parameter values of biochemical subnetwork

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