Petra Bergstrom, Xu Zhang, Aja Harris and Ben Arentson 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

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

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

Grx1 Grx2 Grx2 ch5q14, 12 kDa) Active site: CPYC Active site: CSYC Grx2

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 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

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 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

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. 102:15018-15023. 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 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: 163-180. Schulz et al (1978) Nature. 273: 120-124.

Glutathione Reductase Mechanism Oxidized native enzyme

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 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.

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

Glutathione Formation and Degradation 1 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: 335-55; Meister, Alton and Anderson, Mary. 1983. Glutathione. An. Rev. Biochem. 52: 711-60.

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. 43:883-898

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.

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. 99:9745-49

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