Thiol redox systems. Chao Sun (CCK KI) Ting Jia (RBC UNL) Saeed Eshtad (MBB KI) Weishu Fan (RBC UNL)

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1 Thiol redox systems Chao Sun (CCK KI) Ting Jia (RBC UNL) Saeed Eshtad (MBB KI) Weishu Fan (RBC UNL)

2 Questions What is the function of thioredoxin?

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4 Questions What is the function of thioredoxin? How many thioredoxin isoforms are in humans and where are they localized in the cell?

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6 Questions What is the function of thioredoxin? How many thioredoxin isoforms are in humans and where are they localized in the cell? Describe the structure of thioredoxin.

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8 Questions What is the function of thioredoxin? How many thioredoxin isoforms are in humans and where are they localized in the cell? Describe the structure of thioredoxin. Name at least 3 protein targets of thioredoxin.

9 ribonucleotide reductase peroxiredoxins NFκB, AP-1, ASK-1

10 Glutaredoxins (Grxs) Electron donor Reversible glutathionylation Iron-sulfur cluster Christopher Horst Lillig et al, 2008

11 Peltoniemi, Mirva et al, 2007

12 Elke STROHER et al, 2012

13 Targets of Glutaredoxin RNR Actin Caspase 3 CH.Lillig et al, 2008; J.Wang et al,2003; S.Pan et al, 2007

14 Redox state control of the cysteinyl residues by the thioredoxin (TRX) and the glutaredoxin (GRX) systems Thioredoxin system: 1.thioredoxin (TRX) 2.thioredoxin reductase (TxR) 3.NADPH, FADH2 glutaredoxin (GRX) system: 1.glutaredoxin (GRX) 2.Glutaredoxin reductase (GxR) 3.NADPH, GSH/GSSG Lehninger principles of Biochemistry, 4 th edition 14

15 Redox cycles and reaction mechanisms of TRXs and GRXs C. Berndt et al, Am J Physiol Heart Circ Physiol.,

16 The glutaredoxin dithiol oxidoreductase mechanism A. FERNANDES and A.HOLMGREN, ANTIOXIDANTS & REDOX SIGNALING,

17 Electron flow from NADPH to substrates Name Surname 17

18 Cellular localization of TRX system Mammalian cells contain two TRX systems: 1.the cytosolic TRX1/TRXR1 2.the mitochondrial TRX2/TRXR2 TRXRs are homodimeric selenoenzymes containing a FAD Thioredoxin glutathione reductase (TGR): TrxR catalyzing reduction of both Trx and GSSG A functional overlap between Trx and GRX systems E. Arne r and A. Holmgren, Eur. J. Biochem., (2000) Name Surname 18

19 Cellular localization of GRX system Human cells contain three GRXs: 1.the cytosolic GRX1 with the active site Cys-Pro-Tyr- Cys. 2.the primary mitochondrial GRX2 3.GRX5 (a second mitochondrial GRX) Overview: Trx1 : cytosol and nucleus Trx2 : mitochondria Grx1 : cytosol Grx2:mitochondria and nucleus Grx5 :mitochondria 19

20 More detailed view of their cellular compartment E.M. Hanschmann et al., ANTIOXIDANTS & REDOX SIGNALING, 2013 Name Surname 20

21 Glutathione (GSH) *Glutathione (GSH), present in plants, animals, and some bacteria, often at high levels, can be thought of as a redox buffer. *It is a tripeptide comprised of glutamate, cysteine, and glycine, and is vital for normal cellular function. GSH: Reduced form of glutathione GSSG: Oxidized form of glutathione David L. Nelson an Michael M. Cox, Lehninger: Principles of Biochemistry, fifth edition.2008, 876. Donald Beckers et al., Redox Biochemistry. 2008, (2) 12.

22 Biosynthesis Pathway of Glutathione *Two ATP-dependent steps: *First, γ-glutamylcysteine is synthesized from L-glutamate and cysteine via the enzyme γ- glutamylcysteine synthetase. *This reaction is the rate-limiting step. *Second, glycine is added to the C-terminal of γ-glutamylcysteine via the enzyme glutathione synthetase. David L. Nelson an Michael M. Cox, Lehninger: Principles of Biochemistry, fifth edition.2008, 877

23 Maintaining the Intracellular GSH:GSSG Ratio GSSG: a disulfide derived from two glutathione molecule *Glutathione peroxidases 2GSH + H 2 O 2 GSSG + 2H 2 O 2GSH + ROOH GSSG + ROH + H 2 O GSSG is rapidly reduced by an NADPH-dependent glutathione reductase. Under normal cellular conditions, glutathione reductase can maintain a high GSH:GSSG ratio. Under extreme oxidative stress, GSSG can be rapidly exported to help preserve the GSH:GSSG ratio. Increased GSSG levels can lead to formation of mixed disulfides between GSH and protein thiols, thus lowering GSSG levels. Donald Beckers et al., Redox Biochemistry. 2008, (2) 12.

24 Concentration of GSH in Different Intracellular Compartments Compartments Concentration (mm) GSH/GSSG Endoplasmic Reticulum (ER) :1-3:1 Mitochondria :1 Cytoplasm(90%) :1-100:1 Volodymyr I. Lushchak, Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions, Journal of Amino Acids, vol Bass R, Ruddock L, Klappa P, Freedman R. (2004) A Major Fraction of Endoplasmic Reticulum-located Glutathione is Present as Mixed Disulfides with Protein. J. Biol. Chem. 279:

25 Glutathione Imported into Cells Foyer, C. H., F. L. Theodoulou, et al. (2001). "The functions of inter- and intracellular glutathione transport systems in plants." Trends in Plant Science 6(10):

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