Abstract
Covalent modification of cellular proteins by electrophiles is implicated to electrophilic signal transduction, dysfunction of enzymes and cytotoxicity. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a deubiquitination enzyme regulating cellular ubiquitin homeostasis, is reported to be inhibited by electrophilic modification of such as 15-deoxy-prostaglandin J2 (Li et al., Biochem Biophys Res Commun, 2004). We have recently found that glutathione (GSH) can recovered electrophilic modification of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) by exogenous electrophile such as 1,2-naphthoquinone (1,2-NQ) through S-transarylation reaction (Miura et al., Chem Res Toxicol. 2011). In this study, we examined whether or not UCH-L1 is covalently bound to 1,2-NQ and such a modified UCH-L1-NQ could undergo S-transarylation by GSH. Two-dementional SDS-PAGE with the lysate of SH-SY5Y cells pretreated with butionine sulfoximine (BSO) to deplete cellular GSH revealed that GSH appears to negatively regulate covalent modification of UCH-L1 by 1,2-NQ in the cells. Under the conditions, decrease of UCH-L1 affinity to mono-ubiquitin caused by 1,2-NQ was enhanced. With recombinat human UCH-L1, we found that UCH-L1 undergoes S-arylation of 1,2-NQ through Cys151, whereas addition of GSH caused a repression of covalent modification of 1,2-NQ to UCH-L1 and concomitant restoration of its catalytic activity. Interestingly, we also detected formation of 1,2-NQ-SG adduct in the reaction mixture. These results suggest that like GAPDH, UCH-L1 bound to 1,2-NQ readily undergoes GSH dependent S-transarylation and that GSH appears to play a role in not only scavenging electrophile prior to covalent modification of cellular proteins but also a restoration of the proteins modified by the electrophile.
