Thiol Redox Systems

Petra Bergstrom, Xu Zhang, Aja Harris and Ben Arentson

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

Human glutaredoxin

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

Grx2

Grx2(ch1q31.2-31.3, 18 kDa)Active site: CSYC

Grx1ch5q14, 12 kDa)Active site: CPYC

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

21 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 Reductase Mechanism

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

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

Questions?