Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines

The reactive cysteines in H-ras are subject to oxidative modifications that potentially alter the cellular function of this protein, In this study. pu rified H-ras was modified by thiol oxidants such as hydrogen peroxide (H2O2 ), S-nitrosoglutathione. diamide, glutathione disulphide (GSSG) and cystami ne, producing as many as four charge-isomeric forms of the protein. These r esults suggest that all four reactive cysteines of H-ras are potential site s of regulatory modification reactions. S-nitrosylated and S-glutathiolated forms of H-ras were identified by protocols that depend on separation of a lkylated proteins on electrofocusing gels. S-nitrosoglutathione could S-nit rosylate H-ras on four cysteine residues, while reduced glutathione (GSH) a nd H2O2 mediate S-glutathiolation on at least one cysteine of H-ras, Either GSSG or diamide S-glutathiolated at least two cysteine residues of purifie d H-ras, Iodoacetic acid reacts with three cysteine residues. In intact NIH -3T3 cells, wild-type H-ras was S-glutathiolated by diamide. Similarly, cel ls expressing a C118S mutant or a C181S/C184S double mutant of H-ras were S -glutathiolated by diamide. These results suggest that H-ras san be S-gluta thiolated on multiple thiols in vivo and that at least one of these thiols is normally lipid-modified. In cells treated with S-nitrosocysteine, eviden ce for both S-nitrosylated and S-glutathiolated H-ras was obtained and S-ni trosylation was the predominant modification. These results show that oxida tive modification of H-ras can be extensive in vivo. that both S-nitrosylat ed and S-glutathiolated forms may be important, and that oxidation may occu r on reactive cysteines that an normally targeted for lipid-modification re actions.