In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed site is Cys-374. Deglutathionylation G-actin leads about 6-fold increase rate polymerization.In vivo studies...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThioltransferase is a specific glutathionyl mixed-disulfide oxidoreductaseStephen A. Gravina and John J. MieyalCite this: Biochemistry 1993, 32, 13, 3368–3376Publication Date (Print):April 1, 1993Publication History Published online1 May 2002Published inissue 1 April 1993https://pubs.acs.org/doi/10.1021/bi00064a021https://doi.org/10.1021/bi00064a021research-articleACS PublicationsRequest reuse permissionsArticle Views468Altmetric-Citations258LEARN...

In murine embryonic fibroblasts, N-acetyl-l-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFκB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of p65 subunit that are responsible for inactivation. MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFκB DNA binding transactivation 2.6- 2.8-fold, respectively. NAC blocked these events without having an...

Human hemoglobin was characterized as an enzyme in a reconstituted aniline hydroxylase system containing hemoglobin, NADPH, rat liver cytochrome P-450 reductase, and atmospheric O2. This catalyzed p-aminophenol formation (turnover number 0.2 mol/min/mol of hemoglobin) with efficiency similar to that which has been reported for either microsomal or solubilized from liver. The rate the reaction linearly dependent on concentration up approximately 1 nmol hemoglobin/ml. linear range hemoenzyme...

The innate immune response constitutes the first line of defense against infections. Pattern recognition receptors recognize pathogen structures and trigger intracellular signaling pathways leading to cytokine chemokine expression. Reactive oxygen species (ROS) are emerging as an important regulator some these pathways. ROS directly interact with components or induce other post-translational modifications such S-glutathionylation, thereby altering target function. Applying live microscopy,...

Glutaredoxin (GRx, thioltransferase) is implicated in cellular redox regulation, and it known for specific efficient catalysis of reduction protein-S-S-glutathione-mixed disulfides (protein-SSG) because its remarkably low thiol pK(a) ( approximately 3.5) ability to stabilize a catalytic S-glutathionyl intermediate (GRx-SSG). These unique properties suggested that GRx might also react with glutathione-thiyl radicals (GS(.)) disulfide anion radical (GRx-SSG), thereby facilitating the...

Previous studies have suggested that the two conserved cysteines of HIV-1 protease may be involved in regulating activity. Here, we examined diglutathionylated wild type (Cys-67-SSG, Cys-95-SSG) and monoglutathionylated mutants (C67A, Cys-95-SSG C95A, Cys-67-SSG) as potential substrates for thioltransferase (glutaredoxin). Time-dependent changes extent deglutathionylation each protein were assayed by reverse phase-high performance liquid chromatography. Glutathione alone was not an effective...

In a previous study, we found that treatment of rat heart mitochondria with H(2)O(2) resulted in decline and subsequent recovery the rate state 3 NADH-linked respiration. These effects were shown to be mediated by reversible alterations NAD(P)H utilization activities specific Krebs cycle enzymes alpha-ketoglutarate dehydrogenase (KGDH) succinate dehydrogenase. The purpose current study was examine potential mechanism(s) which reversibly alters KGDH activity. We report here inactivation is...

The reversible oxidative inactivation of transcription factors has been proposed to be important in cellular responses oxidant stress and several signal transduction pathways. nuclear factor I (NFI) family is sensitive due the presence a conserved, oxidation-sensitive cysteine residue within NFI DNA-binding domain. Here we show that restoration activity oxidized NFI-C can catalyzed vitro by enzyme thioltransferase (glutaredoxin) coupled GSH GSSG reductase. To test whether GSH-dependent...

Human thioltransferase (TTase) is a 12 kDa thiol−disulfide oxidoreductase that appears to play critical role in maintaining the redox environment of cell. TTase acts as potent and specific reducing agent for protein-S-S-glutathione mixed disulfides (protein-SSG) likely formed during oxidative stress or intermediates signal transduction pathways. Accordingly, catalytic cycle itself involves covalent glutathionyl enzyme disulfide intermediate (TTase-C22-SSG). To understand molecular basis...

RNA interference is an effective method to silence specific gene expression. Its application mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression endogenous and heterogeneous genes in cell lines. However, using this method, we failed knock out proteins interest effectively. Here report the development a stable controllable for generating dsRNA...

The apparent pKa for the active site thiol of human thioltransferase (TTase) is about 3.5, but pH dependence TTase-catalyzed rates glutathione (GSH)-dependent reduction disulfide substrates displays an inflection point near 8.5. similarity pH-rate profile with titration GSH moiety suggested rate-limiting nucleophilic attack by glutathionyl thiolate species to regenerate reduced TTase from TTase-SSG intermediate. To test this hypothesis profiles dethiolation mixed bovine serum albumin...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThioltransferase in human red blood cells: purification and propertiesJohn J. Mieyal, David W. Starke, Stephen A. Gravina, Chantal Dothey, James S. ChungCite this: Biochemistry 1991, 30, 25, 6088–6097Publication Date (Print):June 1991Publication History Published online1 May 2002Published inissue 25 June 1991https://pubs.acs.org/doi/10.1021/bi00239a002https://doi.org/10.1021/bi00239a002research-articleACS PublicationsRequest reuse...

Reversible S-glutathionylation of proteins is a focal point redox signaling and cellular defense against oxidative stress. This post-translational modification alters protein function, its reversal (deglutathionylation) catalyzed specifically efficiently by glutaredoxin (GRx, thioltransferase), thioldisulfide oxidoreductase. We hypothesized that changes in might be important the development diabetic retinopathy, condition characterized Indeed, GRx activity were increased retinal homogenates...

S-glutathionylation is a reversible post-translational modification that continues to gain eminence as redox regulatory mechanism of protein activity and associated cellular functions. Many diverse proteins such transcription factors, adhesion molecules, enzymes, cytokines are reported undergo glutathionylation, although the functional impact has been less well characterized. De-glutathionylation catalyzed specifically efficiently by glutaredoxin (GRx, aka thioltransferase), facile...