Crystal structure of <i>S</i>‐glutathiolated carbonic anhydrase III

Mallis, Robert J.; Poland, Bradley W.; Chatterjee, Tapan K.; Fisher, Rory A.; Darmawan, Steven; Honzatko, Richard B.; Thomas, James A.

doi:10.1016/s0014-5793(00)02022-6

Cited by 82 publications

(71 citation statements)

References 30 publications

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“…Thus, in the case of Trx, susceptibility to glutathionylation is mainly due to the three-dimensional structure rather than to the chemical-physical properties of the amino acids in the vicinity, as has been suggested for other proteins (26). A glutathione adduct on Cys-72 also might be favored by the primary structure, however, as this amino acid is next to a basic one, Lys-71, which might stabilize the adduct by interacting electrostatically with the ␥-glutamyl group of GSH (27).…”

Section: Discussionmentioning

confidence: 91%

Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems

Casagrande

Bonetto

Fratelli

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

323

203

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To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with 35 S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrixassisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to deglutathionylate itself. These data suggest that the intracellular glutathione͞glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions. T hioredoxin (Trx; ref. 1), a ubiquitous redox protein, is an essential cofactor electron donor for ribonucleotide reductase, but also has many other cellular functions, including regulation of transcription factors, apoptosis, and antioxidant activity and can act exogenously as a redox active growth factor (1, 2). The catalytic activity of Trx resides in its active site where the two redox active Cyss (Cys-31 and Cys-34 in human Trx) undergo reversible oxidation͞reduction. In addition to the conserved Cys residues in the active site, three additional structural Cys residues (Cys-61, Cys-68, and Cys-72) are present in the structure of human Trx.The present paper describes the experiments that led us to the conclusion that Trx can undergo glutathionylation in T cells exposed to oxidative stress. Protein glutathionylation, the formation of a disulfide between a Cys in a protein and the Cys in the tripeptide glutathione (GSH), is a modification that can be induced in cells by oxidative stress. Glutathionylation can occur by direct oxidation of a protein and GSH, by a thiol-disulfide exchange between a protein Cys and oxidized glutathione (GSSG), and also with the intermediacy of S-nitrosoglutathione (GSNO; refs. 3 and 4). Glutathionylation of proteins is reversible, as those proteins can be reduced by glutaredoxins (5, 6), and the process serves to regulate protein functions by the redox state of the cell (i.e., by the GSSG͞GSH ratio; refs. 7-9).To demonstrate the glutathionylation of Trx, we first labeled the intracellular GSH pool of the T-cell blasts with [ 35 S]Cys and analyzed the cell lysate by two dimensional electrophoresis under nonreducing conditions. A spot was found in the autoradiographic protein map with IP͞M r corresponding to those of Trx. In the second part of the study, we incubated recombinant human Trx in the presence or ...

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Section: Discussionmentioning

confidence: 91%

Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems

Casagrande

Bonetto

Fratelli

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

323

203

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“…This occurs when the reversible oxidation or reduction of a protein thiol allows it to change its function in response to alterations in the GSH/GSSG ratio. Protein activity can change following formation of an intraprotein disulfide, as happens for the transcription factor OxyR (25), or by formation of a protein-glutathione mixed disulfide, as occurs with carbonic anhydrase (44). However, for these changes in protein activity to function as redox switches, the ratio of oxidatively modified to unmodified protein must change appropriately in response to an altered GSH/GSSG ratio (2)(3)(4).…”

Section: Discussionmentioning

confidence: 99%

Glutaredoxin 2 Catalyzes the Reversible Oxidation and Glutathionylation of Mitochondrial Membrane Thiol Proteins

Beer

Taylor

Brown

et al. 2004

Journal of Biological Chemistry

372

350

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The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.Oxidative damage and redox signaling can regulate protein thiol redox state (1-4). A major way in which this occurs is through the response of protein thiols to changes in the glutathione (GSH) to glutathione disulfide (GSSG) ratio (2, 5, 6). The intracellular GSH/GSSG ratio is usually kept high (Ͼ99% reduced) through reduction of GSSG to GSH by glutathione reductase, enabling GSH to act as an antioxidant (2, 6). However, during oxidative stress or redox signaling reactive oxygen species (ROS) 1 oxidize GSH to GSSG directly, or catalyzed by glutathione peroxidases. Protein thiols respond to the decreased GSH/GSSG ratio by forming mixed disulfides with glutathione through thiol-disulfide exchange between the thiolate anion and GSSG (protein thiols typically have pK a values of ϳ8 -9, but these can vary widely depending on the local e...

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“…All of these post-translational modifications can potentially act as 'redox switches' (Cabiscol and Levine, 1996;Mallis et al, 2000;Schafer and Buettner, 2001;Zheng et al, 1998), altering the function of a protein and, thereby, enabling it to respond sensitively to the reduction potential of a particular redox couple or to the production of a particular ROS. Although structural alterations brought about by these modifications can potentially have a major effect on protein function, in only a few cases have detailed structural analyses shown clearly how this occurs.…”

Section: Post-translational Protein Modification By H 2 O 2 and Nomentioning

confidence: 99%

Mitochondrial redox signalling at a glance

Collins

Chouchani

James

et al. 2012

Journal of Cell Science

233

152

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Crystal structure of S‐glutathiolated carbonic anhydrase III

Cited by 82 publications

References 30 publications

Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems

Glutathionylation of human thioredoxin: A possible crosstalk between the glutathione and thioredoxin systems

Glutaredoxin 2 Catalyzes the Reversible Oxidation and Glutathionylation of Mitochondrial Membrane Thiol Proteins

Mitochondrial redox signalling at a glance

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