Berleth E S, Kasperek E M, Grill S P, Braunscheidel J A, Graziani L A, Pickart C M
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
J Biol Chem. 1992 Aug 15;267(23):16403-11.
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). We now show that, relative to arsenite, phenylarsenoxides are 10-165-fold more potent inhibitors of protein degradation in the same system (K0.5 for inhibition by p-aminophenylarsenoxide was 3.5-20 microM, depending on the substrate). In the ubiquitin-dependent proteolytic pathway, covalent ligation of ubiquitin to protein substrates targets the latter for degradation. In certain cases, specificity in ubiquitin-substrate conjugation depends critically upon the properties of ubiquitin-protein ligase or E3. Among other effects, p-aminophenylarsenoxide decreased the steady-state level of ubiquitinated human alpha-lactalbumin; this is a substrate which is acted upon directly by ubiquitin-protein ligase-alpha (E3-alpha). This finding suggests that phenylarsenoxides (unlike arsenite) inhibit E3. Several other lines of evidence confirm this conclusion. 1) A complex of E3-alpha and the 14-kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin, with the E3 component of the complex mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated E3 (K0.5 approximately 50 microM); inhibition was readily reversed by addition of dithiothreitol (which contains a competing vicinal thiol group), but not by beta-mercaptoethylamine (a monothiol). 3) A bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly and irreversibly inactivated E3; bromoacetyl aniline, which lacks an arsenoxide moiety, did not inhibit E3. These results suggest that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site. The bifunctional phenylarsenoxide should be a useful tool for probing the relationship between structure and function in E3. As expected from prior results with arsenite, p-aminophenylarsenoxide was also a potent inhibitor of the turnover of ubiquitin-(human) alpha-lactalbumin conjugates.
三价亚砷氧化物与蛋白质的相邻巯基结合。我们之前表明，最简单的三价亚砷氧化物，即无机亚砷酸盐，能抑制兔网织红细胞裂解物中泛素依赖性蛋白质降解（Klemperer, N.S., and Pickart, C.M. (1989) J. Biol. Chem. 264, 19245 - 19242）。我们现在表明，相对于亚砷酸盐，苯亚砷氧化物在同一系统中对蛋白质降解的抑制效力要高10 - 165倍（对氨基苯亚砷氧化物抑制的K0.5为3.5 - 20 microM，取决于底物）。在泛素依赖性蛋白水解途径中，泛素与蛋白质底物的共价连接将后者靶向降解。在某些情况下，泛素 - 底物缀合的特异性关键取决于泛素 - 蛋白连接酶或E3的特性。除其他作用外，对氨基苯亚砷氧化物降低了泛素化人α - 乳白蛋白的稳态水平；这是一种直接由泛素 - 蛋白连接酶 - α（E3 - α）作用的底物。这一发现表明苯亚砷氧化物（与亚砷酸盐不同）抑制E3。其他几条证据也证实了这一结论。1）E3 - α与14 kDa泛素缀合（E2）同工酶的复合物与苯亚砷氧化物 - 琼脂糖树脂结合，复合物中的E3成分介导结合。2）对氨基苯亚砷氧化物抑制分离的E3（K0.5约为50 microM）；加入二硫苏糖醇（含有竞争性相邻巯基）可轻易逆转抑制作用，但β - 巯基乙胺（一种单巯基化合物）则不能。3）双功能苯亚砷氧化物（溴乙酰氨基苯亚砷氧化物）能快速且不可逆地使E3失活；缺乏亚砷氧化物部分的溴乙酰苯胺不抑制E3。这些结果表明E3具有必需的相邻巯基，并且在相邻巯基位点附近存在一个反应性亲核试剂。双功能苯亚砷氧化物应该是探究E3结构与功能关系的有用工具。正如之前用亚砷酸盐得到的结果所预期的那样，对氨基苯亚砷氧化物也是泛素 - （人）α - 乳白蛋白缀合物周转的有效抑制剂。
