Oxidative stress induces S-thiolation of specific proteins in cultured gastric mucosal cells

Abstract

Oxidative stress induces the formation of protein-mixed disulfides with low-molecular-weight thiols, especially glutathione. We analyzed this process, termed S-thiolation, in cultured gastric mucosal cells from guinea pigs by gel electrophoresis and autoradiography after radiolabeling of the intracellular glutathione pool with 35S. Hydrogen peroxide (H2O2) or diamide initiated rapid and reversible S-thiolation of specific proteins with molecular masses of 42, 30, 29, 28, and 22 kDa. Diamide caused particularly prominent S-thiolation of the 42 kDa protein. This protein was identified as actin by immunoblot analysis and actin-myosin precipitation. Fluorescence microscopy revealed that diamide caused a disappearance of normal stress fibers and a concomitant increase in actin polymerization in association with contraction of the cells. These morphological changes were completely reversible within minutes. With cells depleted of glutathione by incubation with DL-buthionine-[S,R]-sulfoximine, diamide caused severe contraction and rounding, and the cells detached from the culture plates. S-thiolation of actin could help protect gastric mucosal cells against irreversible organization of microfilaments by preserving microfilament dynamics under oxidative stress.

Oxidative imbalance in the aging inner ear

“…The accumulation of glutathionylated proteins is an indicator of protein oxidation arising from the formation of hydrogen peroxide [30,20,2]. The rise of 4-HNE signals lipid peroxidation, mainly due to the production of hydroxyl radical, and 3-NT suggests peroxynitrite reactions [26].…”

Glutathione depletion impairs transcriptional activation of heat shock genes in primary cultures of guinea pig gastric mucosal cells.

“…Male guinea pigs weighing ‫ف‬ 250 grams were purchased from Japan SLC Inc. (Shizuoka, Japan). Gastric mucosal cells were isolated aseptically from fundic glands and cultured for 2 d in RPMI 1640 containing 10% FCS, 2 mM glutamine, 100 g/ml streptomycin, and 100 U/ml penicillin, as described previously (7,17). The periodic acid-Schiff reaction showed that Ͼ 85% of the cultured cells were mucus secreting (7,17).…”

“…The contents of reduced glutathione (GSH) and its disulfide (GSSG) in cultured cells were measured as described previously (17). Briefly, acid-soluble thiols were extracted by treating the cells in 35-mm-diameter culture dishes with 200 l of 5% perchloric acid containing 2.5 mM o -phenanthroline for 30 min on ice.…”

“…However, there is some controversy about the relationship between the endogenous glutathione level and acquisition of adaptive cytoprotection in vivo (13,14). Glutathione has been shown to protect cultured gastric cells against oxygen radicals (15) and acid (16), and we have suggested that it may prevent irreversible reorganization of microfilaments of gastric epithelial cells under oxidative stress, possibly through S-thiolation of actin (17). In addition, glutathione has been suggested to mediate the initial response for acquiring tolerance to heat stress (18).…”

HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase

“…One modification of cysteine residues that takes place in cells is reversible disulphide bond formation with the cellular thiol, glutathione (termed glutathiolation) [1][2][3]. A number of studies indicate that the glutathiolation of cellular proteins (glyceraldehyde-3-phosphate dehydrogenase, BSA, haemoglobin, nuclear factor 1) can be reversed in itro in the presence of the cellular enzyme thioltransferase (glutaredoxin) [4][5][6][7][8][9].…”