Inhibition of ubiquitin-protein ligase (E3) by mono- and bifunctional phenylarsenoxides. Evidence for essential vicinal thiols and a proximal nucleophile.

摘要: Trivalent arsenoxides bind to vicinal thiol groups of proteins. We showed previously that the simplest trivalent arsenoxide, inorganic arsenite, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (Klemperer, N.S., and Pickart, C.M. (1989) J. Biol. Chem. 264, 19245-19242). now show that, relative phenylarsenoxides are 10-165-fold more potent inhibitors same system (K0.5 for inhibition by p-aminophenylarsenoxide was 3.5-20 microM, depending on substrate). In proteolytic pathway, covalent ligation ubiquitin substrates targets latter degradation. certain cases, specificity ubiquitin-substrate conjugation depends critically upon properties ubiquitin-protein ligase or E3. Among other effects, decreased steady-state level ubiquitinated human alpha-lactalbumin; this is a substrate which acted directly ligase-alpha (E3-alpha). This finding suggests (unlike arsenite) inhibit Several lines evidence confirm conclusion. 1) A complex E3-alpha 14-kDa ubiquitin-conjugating (E2) isozyme binds phenylarsenoxide-Sepharose resin, with E3 component mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated approximately 50 microM); readily reversed addition dithiothreitol (which contains competing group), but not beta-mercaptoethylamine (a monothiol). 3) bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly irreversibly inactivated E3; bromoacetyl aniline, lacks an arsenoxide moiety, did These results suggest possesses essential there reactive nucleophile proximal site. The should be useful tool probing relationship between structure function As expected from prior also inhibitor turnover ubiquitin-(human) alpha-lactalbumin conjugates.