Demasi Marilene, Silva Gustavo Monteiro, Netto Luis Eduardo Soares
Departmento de Biologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277 São Paulo, São Paulo 05508-900, Brazil.
J Biol Chem. 2003 Jan 3;278(1):679-85. doi: 10.1074/jbc.M209282200. Epub 2002 Oct 29.
The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.
从酿酒酵母中纯化的20 S蛋白酶体核心被还原型谷胱甘肽（GSH）、半胱氨酸（Cys）或GSH前体γ-谷氨酰半胱氨酸抑制。胰凝乳蛋白酶样活性比胰蛋白酶样活性受GSH的影响更大，而肽基谷氨酰水解活性（半胱天冬酶样）不受GSH抑制。通过Cys-S(O)-4-硝基苯并-2-恶唑-1,3-二唑加合物的光谱表征证明了20 S核心中Cys-亚磺酸的形成，表明20 S蛋白酶体Cys残基可能通过氧化的Cys-亚磺酸形式与还原巯基（GSH、Cys和γ-谷氨酰半胱氨酸）反应。通过GSH-生物素掺入和单溴代双马来酰亚胺烷基化减少，在体外证明了20 S核心的S-谷胱甘肽化。诸如N-乙基马来酰亚胺（-S-巯基H烷基化剂）、二甲基酮（-SO亚磺酸H反应物）或7-氯-4-硝基苯并-2-恶唑-1,3-二唑（-SH或-SOH反应物）等化合物高度抑制蛋白酶体胰凝乳蛋白酶样活性。体内实验表明，从H₂O₂处理的细胞中提取的20 S蛋白酶体显示胰凝乳蛋白酶样活性降低，并伴有S-谷胱甘肽化，这通过用NaBH₄还原后20 S核心释放GSH来证明。此外，用H₂O₂预处理的细胞通过测定GSH/氧化型谷胱甘肽比率评估的还原能力降低，蛋白质羰基水平升高。目前的结果表明，在生理水平上，酵母20 S蛋白酶体受其巯基含量调节，从而将细胞内氧化还原信号与蛋白酶体介导的蛋白水解偶联起来。
