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Thiol Redox Systems

Thiol Redox Systems. Petra Bergstrom, Xu Zhang, Aja Harris and Ben Arentson. Outline. Glutaredoxin Thioredoxin Thioredoxin Reductase and Glutathione Reductase Glutathione. Glutaredoxin (Grx) (thioltransferase). Reduction of protein disulfides 2. Reduction of glutathionylated.

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Thiol Redox Systems

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  1. Thiol Redox Systems Petra Bergstrom, Xu Zhang, Aja Harris and Ben Arentson

  2. Outline • Glutaredoxin • Thioredoxin • Thioredoxin Reductase and Glutathione Reductase • Glutathione

  3. Glutaredoxin (Grx)(thioltransferase) • Reduction of protein disulfides 2. Reduction of glutathionylated

  4. Dithiol mechanisms • R-S2 + Grx-(SH)2 → R-(SH)2 + Grx-S2 • Grx-S2 + 2GSH → Grx-(SH)2 + GSSH • R-S-SG + Grx-(SH)2 → R-(SH) + Grx-S-SG • Grx-S-SG + GSH → Grx-(SH)2 + GSSG Monothiolmechanisms

  5. Human glutaredoxin Sun et al. (1998) J. Mol. Biol. 280, 687-701.

  6. Grx1 ch5q14, 12 kDa) Active site: CPYC Grx2 (ch1q31.2-31.3, 18 kDa) Active site: CSYC Grx2

  7. Three protein targets of glutaredoxin. • De-glutathionylation of Actin-SSG • De-glutathionylation of NF1 • De-glutathionylation of ASK-1 and Akt Shelton et al. 2005

  8. Human Thioredoxin (hTrx) & Isoforms • 12 kDa • Conserved active site sequence- • Cys-Gly-Pro-Cys • hTrx1-cytosol and nucleus • hTrx2-mitochondria • Separate gene • Both are essential

  9. hTrx1 Structure Trx fold -globular αβ sandwhich 5 β sheets 4 α helices active site: Cys32 and Cys 35 Generated from 1ERT, PDB 25 september 2014 Namn Efternamn

  10. hTrxs Protein Targets & Protects Cells Against Stress A.Holmgren&J.Lu, Biochemical and Biophysical Research Communication 396(2010) 120-124

  11. hTrx as Electron Donor for RNR FZ Avval and A Holmgren, (2009), The Journal of Biological Chemistry, 284, 8233-8240.

  12. Thioredoxin • Maintains a reduced environment in cytosol of cells with a low redox potential • Regenerate reduced forms of Msrs and Prxs • Stress inducible antioxidant factor

  13. Thioredoxin Reductase • Catalyzes the reduction of oxidized Trx to its reduced form by NADPH • Active site - Cys-Sec-Gly-OH Biterova et al (2005) PNAS. 102:15018-15023. Sandalova et al (2001) PNAS. 98:9533-9538.

  14. Reactions and Functions of TrxR • TrxR1-cytosolic • TrxR2-mitochondrial Mustacich et al (2000) Biochem J. 346:1-8.

  15. Glutaredoxin • Catalyzes reduction of proteins that are thiolated by GSH • Recycled to GSH via recycling system of NADPH and GR • 2 isoforms in mammals- Grx1 (cytosolic) and Grx2 (mitochondrial/nuclear) • GR reduces GSSG to GSH at the expense of NADPH

  16. Glutathione Reductase Structure • GR activities found in mitochondrial and cytoplasm Karplus et al (1989) J.Mol.Biol. 210: 163-180. Schulz et al (1978) Nature. 273: 120-124.

  17. Glutathione Reductase Mechanism

  18. Koharyova et al (2008) Gen. Physiol. Biophys. 27:71-84.

  19. Glutathione (GSH) • A tripeptide composed of glutamate, cysteine, and glycine • Found primarily in eukaryotes and gram-negative bacteria • ~90% of intracellular glutathione is found in cytoplasm • Remaining 10% is split between mitochondria, endoplasmic reticulum, and nucleus

  20. GSH Continued • Primary function is maintenance of intracellular redox homeostasis via protection versus ROS and RNS http://bcn.boulder.co.us/health/rmeha/glut11.gif

  21. Intracellular Glutathione Levels 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: 5257-5262 Kulinsky V, Kolesnichenko S (2007) Mitochondrial Glutathione Biochem. 72: 856-859.

  22. Glutathione Formation and Degradation 1 2 3 4 • Glutamate Cysteine Ligase • GSH Synthetase • γ-Glutamyl Transpeptidase • Dipeptidase Wang, W. and Ballatori N. (1998) Endogenous Glutathione Conjugates: Occurrence and Biological Functions. Pharm. Reviews. 50: 335-55; Meister, Alton and Anderson, Mary. 1983. Glutathione. An. Rev. Biochem. 52: 711-60.

  23. Glutathionylation • Post-translational modification where GSH is attached to protein via disulfide bond • Involved in regulation of a variety of regulatory, structural, and metabolic proteins Dalle-Donne I, Rossi R, Giustarini D, Colombo R, Milzani A (2007) S-glutathionylation in Protein Redox Regulation. Free Radical Biology. 43:883-898

  24. Proteins Regulated by Glutathionylation • α-ketoglutarate dehydrogenase • Creatine kinase • HIV-1 Protease • Thioredoxin

  25. Glutathionylation of Thiroredoxin Casagrande S, et al. (2002) Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thiroedoxin systems. PNAS. 99:9745-49

  26. Questions?

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