1. Petra Bergstrom, Xu Zhang, Aja Harris and Ben Arentson
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 Monothiol 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
Monothiol mechanisms

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

6. Grx1 Grx2 Grx2 ch5q14, 12 kDa) Active site: CPYC
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
17 april 2017
Namn Efternamn

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

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

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
In human cells, complexes between
reduced thioredoxin and ASK1 (apotosis signaling kinase) prevent this mitogen activated
kinase kinase kinase from signaling the apoptosis response; however ASK1 is released from
the complex when thioredoxin becomes oxidized.
Search pubmed for thioredoxin localization

13. Thioredoxin Reductase
Catalyzes the reduction of oxidized Trx to its reduced form by NADPH
Active site - Cys-Sec-Gly-OH
Overall structure of the mTrxR2 holoenzyme
3 domains– yellow=FAD binding domain, green=NADPH binding domain, blue=interface domain
Very different from prokayrotic TrxRs
In mammals—encoded by 3 separate genes, large in size and have broad substrate specificity, also selenoenzymes
Active site sequence- Cys-Sec-Gly-OH
Biterova et al (2005) PNAS. 102: Sandalova et al (2001) PNAS. 98:

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
Dimeric
Each subunit consists of NADP+ domain, FAD domain and an interface domain
GSH is bound to FAD domain of one subunit and interface domain of another
Karplus et al (1989) J.Mol.Biol. 210:
Schulz et al (1978) Nature. 273:

17. Glutathione Reductase Mechanism
Oxidized native enzyme

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
Glutathione is a tripeptide composed of glutamate, cysteine, and glycine (gammaglutamylcysteineylglycine.
Gram positive bacteria have been shown to utilize glutathione.
Biosynthesis is thought to mainly occur in cytosol. Total cellular concentrations are thought to be between .5 and 10 mM, which most of the GSH in the reduced form
Ratio of GSH and GSSG is often used to express redox state of a cell---GSH:GSSG ration is larger than 10:1 in the cytosol and mito and as low as 1:1 in the ER. The lower the GSH:GSSG in the ER favors formation of disulfide bons during folding of secretory and membrane proteins.

20. GSH Continued
Primary function is maintenance of intracellular redox homeostasis via protection versus ROS and RNS
This figure is to show how the oxidationi/reduction thiol cycle of GSH works. Hre glutathione peroxidase, a selenoprotein is the enzyme that uses electros from GSH to

21. Intracellular Glutathione Levels
Glutathione Intracellular Concentrations
Compartment
Concentration (mM)
GSH:GSSG Ratio
Cytosol
5-10
30:1 – 100:1
Mitochondria
10:1
Endoplasmic Reticulum
.5 – 10
1:1 – 3:1
GSH can only be imported into ER
10-15% of total Glutathione found in mito, but concentration is as hih as cyhtosol. Concentration is so high b/c of defence of mitochondrial organelle from ROS. GSH cannot go backwards into cytosol. Without GSH in mito, damage occurs. It is preventable of death b/c of ascorbate and GSH esters, which function to defend mito.
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:
Kulinsky V, Kolesnichenko S (2007) Mitochondrial Glutathione Biochem. 72:

22. Glutathione Formation and Degradation1 2 3
GSH is made intracellularly in all mammallian cells but the liver is a major biosynthesis. Synthesis occurs from threee amino acid precursos l glutamate, l cysteine, and glycine occurs in the cytosol. First step is catalyzed by Glutamate Cysteine Ligase = Glutamate + Cysteine, and the secon d is catalyzed by GSH Synthetase. First step is controlled by negative feedback from end product GSH, also availability of cysteine can regulate GSH formation.
Biodegration occurs outside the cell– The breakdown is catalyzed by gamma GT and dipeptidase on the surface of epithelial tissues. Gamma GT removes gamm glutamyl from GSH and dipeptidase removes glycine. AA can then be reabsorbed.
Glutamate cysteine ligase—catalyzes committed step in GSH biosynthesis. This happens most likely due to a phosphorylation of the gamma carboxylate of glutamate This enzyme conjugates glutamate via its gamma carboxylate to cysteine. Amino group of cysteine serves as a nucleophile and attacks the gamma gluamyl phospahte intermediate to yield gammaglutamylcysteine. This step is tightly regualted, as it is the committed step to production. GSH is limited by free cysteine, and GSL is feedback regulated by end product GSH. Additional measures of regulation include transcriptional regulation, post-translational modification, presense of modifier subunit to enzyme (addition of modifier subunit decreases Km for glutamate
Glutathione synthetase couples glycine to gamma glutamylcysteine. Works by an acylphosphate intermediate is generated by the transfer of the gamma phosphate of ATP to carboyxlate of gamma glutamylcysteine. The alpha amino gropu of glycine can then attack this phosphorylated intermediate, displacing the inorganic phosphate to form GSH. This enzyme rapidly converts reactant to GSH in vivo . Liver is primarily where GSH is made. GS activity is several fold higher than GCL
GGT is gamma glutamyl transpeptidase 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: ;
Meister, Alton and Anderson, Mary Glutathione. An. Rev. Biochem. 52:

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
Protein which is modified by the attachment of a glutathione molecule by a disulfide bond. Only low PKA cysteinyl residues will be modified
Many proteins contain free thiols that can be modified by the reversible formation of mixed disulfides with glutathione. Protein glutathionylation is of significance for defense against oxidative damage and in redox signaling. Here we outline the mechanisms and possible significance of protein glutathionylation
Occurs mostly due to oxidatve stress as a possible protection mecanism for cysteine.
Dalle-Donne I, Rossi R, Giustarini D, Colombo R, Milzani A (2007) S-glutathionylation in Protein Redox Regulation. Free Radical Biology. 43:

24. Proteins Regulated by Glutathionylation
α-ketoglutarate dehydrogenase
Creatine kinase
HIV-1 Protease
Thioredoxin
Alpha keto dehy takes alpha ketoglutarate to succinyl co a in krebs cycle.
Creatine kinase takes creatine to phosphocreatine
Hiv 1 protease is responsible for cleaving proteins at the right sight so hiv can prosper.
Most aer glutathionylated are inactivated.

25. Glutathionylation of Thiroredoxin
Trx can undergo glutathionylation in t cells exposed to oxidative stress
Done by labeling intracellular GSH of t-cell blasts with S35 cys and analyzed cell lysate by 2D electorphoresis under non reducing conditions and saw a spot with an IP/M corresponding to TRX
Left figure shows mass spec resluts of TRX when GSSG not added and when GSSG added. Weight of GSH is 305 daltons. It is clearly glutathiolyated. Next they looked at activty when purified protein was incubated with 5 mM GSSG for 2 hours. Activity of insulin disulfide reductase was followed by following oxidation of NADPH. Open circles are with GSSG and closed are without GS attached. Activty is much lower in those without GSH attached meaning glutathione was attached to cys when GSSG present.
They also looked at how incubation with GSSG effets the activity of TRx coupled to NADPH as a substrate for TRX reductase, which shows that TRX can undergo glutathyolation, which can regulate activity and function!!!
Casagrande S, et al. (2002) Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thiroedoxin systems. PNAS. 99:

26. Questions?