Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Abstract

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.

Protein Expression Profiling Identifies Cyclophilin A as a Molecular Target in Fhit-Mediated Tumor Suppression

“…On the other hand, cyclophilin A is ubiquitously distributed in tissue, and the cyclophilin A/ cyclosporine A complex has numerous known activities in cell growth, differentiation, transcriptional control, and cell signaling (28). In particular, the cyclophilin A/cyclosporine A complex is known to inhibit calcineurin activity and block cell proliferation; cyclosporine A treatment alone has been reported to inhibit cell proliferation (28,31), but overexpression of cyclophilin A has been reported in human cancers by proteome analyses (32)(33)(34)(35). Indeed, cyclosporine A is unable to inhibit calcineurin activity at high concentrations of cyclophilin A; cyclosporine A treatment induced a G 1 arrest in pancreatic acinar cells, but excess cellular cyclophilin A activates calcineurin (36 *A Z score is estimated when the search result is compared against an estimated random match population.…”

Glutathionylation of chloroplast thioredoxin f is a redox signaling mechanism in plants

“…However, our results indicate that the most efficient way to glutathionylate TRX f in vitro is the use of an oxidant in the presence of GSH, as already observed for other proteins (17). Indeed, exposure of cells to ROS appears to be the more effective mechanism in vivo (15,16). Our data suggest that glutathionylation could be a way of decreasing TRXf activity under conditions of enhanced ROS production.…”

“…This modification notably occurs in response to enhanced production of reactive oxygen species (ROS) and͞or increases in oxidized glutathione (GSSG). Glutathionylation can protect proteins from irreversible oxidation and͞or modulate their activity (12)(13)(14)(15)(16)(17). Despite its theoretical appeal as a mechanism transmitting oxidative signals under stress, very little is known about glutathionylation in plants.…”

Regulation of Mitochondrial Glutathione Redox Status and Protein Glutathionylation by Respiratory Substrates

“…A number of mitochondrial proteins have been identified as being glutathionylated, including subunits of complex I (12) and complex IV (13,14), aconitase (15,16), and pyruvate dehydrogenase (17); glutathionylation of these proteins is usually associated with a decrease of their activities. Because of the disruption in protein function that GSSG can cause, cells generally pump GSSG out of the cytoplasm into the extracellular matrix.…”