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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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