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One-electron oxidation of ergothioneine and analogues investigated by pulse radiolysis: redox reaction involving ergothioneine and vitamin C.

ABSTRACT: Redox reactions of endogenous and exogenous sulphur-containing compounds are involved in protection against oxidative damage arising from the incidence and/or treatment of many diseases, including cancer. We have investigated, via pulse radiolysis, the one-electron oxidation of ergothioneine, a molecule with antioxidant properties which is detected at millimolar concentrations in certain tissues and fluids subject to oxidative stress, including erythrocytes and plasma. The spectrum of the transient species, assigned to the product of one-electron oxidation, observed after reaction of ergothioneine with the oxidizing radicals OH., N3. and CCl3O2. has a maximum absorption at 520 nm and is very similar to that obtained by oxidation of analogous molecules such as 2-mercaptoimidazole, 1-methyl-2-mercaptoimidazole, S-methyl- and S,N-dimethyl-ergothioneine. In the presence of vitamin C, the oxidized form of ergothioneine is repaired by a rapid reduction (k = 6.3 x 10(8) M(-1).s(-1)) producing ascorbyl radicals. This co-operative interaction between ergothionine and ascorbate, similar to that previously observed between vitamin E and ascorbate, may contribute to essential biological redox protection.

SUBMITTER: Asmus KD

PROVIDER: S-EPMC1217242 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Project description:The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys(18)) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys(18) by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1(C18D). We also evaluated Rac1(C18S) as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1(WT) but is resistant to thiol oxidation. In addition, Rac1(C18D), but not Rac1(C18S), shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1(C18D) in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1(C18D) in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1(WT) and the redox-insensitive Rac1(C18S) variant. Moreover, expression of Rac1(C18D) in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys(18) is a novel posttranslational modification that upregulates Rac1 activity.

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One-electron oxidation of ergothioneine and analogues investigated by pulse radiolysis: redox reaction involving ergothioneine and vitamin C.

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