thiol redox systems petra bergstrom, xu zhang, aja harris and ben arentson
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Outline
• Glutaredoxin
• Thioredoxin
• Thioredoxin Reductase and Glutathione
Reductase
• Glutathione
Glutaredoxin (Grx)(thioltransferase)
1. Reduction of protein disulfides
2. Reduction of glutathionylated
Dithiol mechanisms1. R-S2 + Grx-(SH)2 → R-(SH)2 + Grx-S2
2. Grx-S2 + 2GSH → Grx-(SH)2 + GSSH
3. R-S-SG + Grx-(SH)2 → R-(SH) + Grx-S-SG
4. Grx-S-SG + GSH → Grx-(SH)2 + GSSG
Monothiol mechanisms
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
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
hTrx1 StructureTrx fold-globular αβ sandwhich
5 β sheets4 α helices
active site: Cys32 and Cys 35
Generated from 1ERT, PDB
18 april 2023 Namn Efternamn
hTrxs Protein Targets & Protects Cells Against Stress
A.Holmgren&J.Lu, Biochemical and Biophysical Research Communication 396(2010) 120-124
hTrx as Electron Donor for RNR
FZ Avval and A Holmgren, (2009), The Journal of Biological Chemistry, 284, 8233-8240.
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
Thioredoxin Reductase• Catalyzes the reduction of oxidized Trx to its
reduced form by NADPH
• Active site - Cys-Sec-Gly-OHBiterova et al (2005) PNAS. 102:15018-15023. Sandalova et al (2001) PNAS. 98:9533-9538.
Reactions and Functions of TrxR
• TrxR1-cytosolic• TrxR2-mitochondrial
Mustacich et al (2000) Biochem J. 346:1-8.
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
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.
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
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
Intracellular Glutathione Levels
Glutathione Intracellular Concentrations
Compartment Concentration (mM) GSH:GSSG Ratio
Cytosol 5-10 30:1 – 100:1
Mitochondria 5-10 10:1
Endoplasmic Reticulum
.5 – 10 1:1 – 3:1
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: 5257-5262Kulinsky V, Kolesnichenko S (2007) Mitochondrial Glutathione Biochem. 72: 856-859.
Glutathione Formation and Degradation
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.
12
1. Glutamate Cysteine Ligase2. GSH Synthetase3. γ-Glutamyl Transpeptidase4. Dipeptidase
3
4
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 BiologyFree Radical Biology. 43:883-898
Proteins Regulated by Glutathionylation
• α-ketoglutarate dehydrogenase• Creatine kinase• HIV-1 Protease• Thioredoxin
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