Studies on the Mechanism of Oxidative Modification of Human Glyceraldehyde-3-phosphate Dehydrogenase by Glutathione: Catalysis by Glutaredoxin

Lind, Christina; Gerdes, Robert; Schuppe-Koistinen, Ina; Cotgreave, Ian A.

doi:10.1006/bbrc.1998.8695

Cited by 100 publications

(68 citation statements)

References 24 publications

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“…To selectively detect glutathionylated proteins, a recombinant mutant E. coli glutaredoxin (GrxC-C14S-C65Y), engineered for enhanced and selective deglutathionylation of glutathione-protein mixed disulfides, was used as the reductant in step 2 ( Fig. 1A) (16,25,26). To induce oxidative stress within the ER, we overexpressed a hyperactive allele of the Ero1 protein (Ero1*), which we have characterized previously as an effective means to facilitate ER oxidative stress (7,9).…”

Section: Resultsmentioning

confidence: 99%

Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress

Wang

Sevier

2016

Journal of Biological Chemistry

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Redox fluctuations within cells can be detrimental to cell function. To gain insight into how cells normally buffer against redox changes to maintain cell function, we have focused on elucidating the signaling pathways that serve to sense and respond to oxidative redox stress within the endoplasmic reticulum (ER) using yeast as a model system. Previously, we have shown that a cysteine in the molecular chaperone BiP, a Hsp70 molecular chaperone within the ER, is susceptible to oxidation by peroxide during ER-derived oxidative stress, forming a sulfenic acid (؊SOH) moiety. Here, we demonstrate that this same conserved BiP cysteine is susceptible also to glutathione modification (؊SSG). Glutathionylated BiP is detected both as a consequence of enhanced levels of cellular peroxide and also as a by-product of increased levels of oxidized glutathione (GSSG). Similar to sulfenylation, we observe glutathionylation decouples BiP ATPase and peptide binding activities, turning BiP from an ATP-dependent foldase into an ATP-independent holdase. We show glutathionylation enhances cell proliferation during oxidative stress, which we suggest relates to modified BiP's increased ability to limit polypeptide aggregation. We propose the susceptibility of BiP to modification with glutathione may serve also to prevent irreversible oxidation of BiP by peroxide.Cell homeostasis and numerous vital cell functions rely on the maintenance of a proper intracellular redox balance. Oxidative folding in the endoplasmic reticulum (ER) 2 is one intracellular system that is prone to disruption upon alterations in the redox poise. Insufficient oxidizing capacity in the ER leads to the accumulation of proteins in reduced non-native states (1, 2). Alternatively, an overly oxidizing ER results in cellular toxicity, likely caused in part by the mis-pairing of protein cysteine residues resulting from a decreased capacity to reduce nonnative disulfides (3, 4). Crucial for maintaining a continual flux of folding polypeptides through the ER are the systems that buffer against fluctuations in the ER redox environment.Recently, several proteins have been demonstrated to act as sensors of redox fluctuations in the ER. In a common theme, these proteins sense changes in the ER redox environment and respond with beneficial alterations in their enzymatic activities. The enzyme Ero1 is a major source of oxidizing equivalents in the ER, and Ero1 activity is coupled to the ER redox environment. When the ER balance shifts to overly oxidizing conditions, regulatory cysteine residues in Ero1 become oxidized to disulfides, which decreases Ero1 oxidase activity to help restore redox balance (5-7). In addition, sensors that cope with the potential for protein folding defects have emerged. These redox-dependent chaperones do not directly impact the flux of oxidizing equivalents, like Ero1, but rather are activated to help limit polypeptide aggregation during unfavorable redox conditions. Oxidation of active site cysteines in the oxidoreductase PDI has been demonstrated to...

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Section: Resultsmentioning

confidence: 99%

Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress

Wang

Sevier

2016

Journal of Biological Chemistry

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“…Grxs are highly specific for the reduction of mixed disulfides between protein thiols and GSH (32,33), suggesting a common mechanism of redox regulation with proteins being S-glutathionylated upon oxidation of the GSH pool and deglutathionylated by Grxs. Glutathionylation alters the biological function of several important processes and proteins such as actin polymerization (34), GAPDH (35), protein tyrosine phosphatase 1B (36), creatine kinase (37), c-Jun (38), NF-B subunit p50 (39), caspase-3 (40), HIV protease (41), and cAMP-dependent protein kinase (42). In many cases Grxs have been shown to restore the biological activity (34)(35)(36)41).…”

Section: Discussionmentioning

confidence: 99%

“…Glutathionylation alters the biological function of several important processes and proteins such as actin polymerization (34), GAPDH (35), protein tyrosine phosphatase 1B (36), creatine kinase (37), c-Jun (38), NF-B subunit p50 (39), caspase-3 (40), HIV protease (41), and cAMP-dependent protein kinase (42). In many cases Grxs have been shown to restore the biological activity (34)(35)(36)41). Although Grx2 has a particular high affinity for protein GSH disulfides (20), we were unable to detect a general increase in total protein glutathionylation in the knockdown cells.…”

Section: Discussionmentioning

confidence: 99%

Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells toward doxorubicin and phenylarsine oxide

Lillig

Lönn

Enoksson

et al. 2004

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

140

100

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Glutaredoxin (Grx) belongs to the thioredoxin fold superfamily and catalyzes glutathione-dependent oxidoreductions. The recently discovered mitochondrial and nuclear Grx (Grx2) differs from the more abundant cytosolic Grx (Grx1) by its higher affinity toward S-glutathionylated proteins and by being a substrate for thioredoxin reductase. Here, we have successfully established a method to silence the expression of Grx2 in HeLa cells by using short interfering RNA to study its role in the cell. Cells with levels of Grx2 <3% of the control were dramatically sensitized to cell death induced by doxorubicin͞adriamycin and phenylarsine oxide but did not show signs of a general increase in oxidative damage with respect to carbonylation and glutathionylation. The ED 50 for doxorubicin dropped from 40 to 0.7 M and for phenylarsine oxide from 200 to 5 nM. However, no differences were detected after treatment with cadmium, a known inhibitor of Grx1. These results indicate a crucial role of Grx2 in the regulation of the mitochondrial redox status and regulation of cell death at the mitochondrial checkpoint.

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“…This will result in an oxidative stress, increasing GSSG by the glutathione peroxidase reaction, and may lead to glutathionylation of proteins as a signaling mechanism. Glutaredoxin is known to be highly effective in regenerating glutathionylated proteins (58,59) and may therefore be central in signal transduction. The mitochondria are a major source of reactive oxygen species in the cell, due to electron leakage from the respiratory chain (60 -62).…”

Section: Fig 6 Multiple Tissue Northern Blot Analysis Of Human Grx1mentioning

confidence: 99%

Cloning and Expression of a Novel Human Glutaredoxin (Grx2) with Mitochondrial and Nuclear Isoforms

Lundberg¹,

Johansson²,

Chandra³

et al. 2001

Journal of Biological Chemistry

301

260

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Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase. Today glutaredoxins are known as a multifunctional family of GSHdisulfide-oxidoreductases belonging to the thioredoxin fold superfamily. In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. The human Grx2 gene, located at chromosome 1q31.2-31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms that differed in their 5 region were identified. These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively. The 125-residue Grx domain and the two full-length variants were expressed in E. coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria. We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal.

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Studies on the Mechanism of Oxidative Modification of Human Glyceraldehyde-3-phosphate Dehydrogenase by Glutathione: Catalysis by Glutaredoxin

Cited by 100 publications

References 24 publications

Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress

Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress

Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells toward doxorubicin and phenylarsine oxide

Cloning and Expression of a Novel Human Glutaredoxin (Grx2) with Mitochondrial and Nuclear Isoforms

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