Ishii Takeshi, Sakurai Toyo, Usami Hiroko, Uchida Koji
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Biochemistry. 2005 Oct 25;44(42):13893-901. doi: 10.1021/bi051336u.
Reactive oxygen species (ROS) have the potential to damage cellular components, such as protein, resulting in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress, J. Biol. Chem. 276, 12076-12083]. In the present study, to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we analyzed the protein carbonylation on SH-SY5Y cells when these cells were submitted to an endogenous inducer of ROS production. Upon exposure of SH-SY5Y cells to this endogenous electrophile, we observed significant accumulation of protein carbonyls within the cells. Proteomic analysis of oxidation-sensitive proteins showed that the major intracellular target of protein carbonylation was one of the regulatory subunits in 26 S proteasome, S6 ATPase. Accompanied by a dramatic increase in protein carbonyls within S6 ATPase, the electrophile-induced oxidative stress exerted a significant decrease in the S6 ATPase activities and a decreased ability of the 26 S proteasome to degrade substrates. Moreover, in vitro oxidation of 26 S proteasome with a metal-catalyzed oxidation system also confirmed that S6 ATPase represents the most oxidation-sensitive subunit in the proteasome. These and the observation that down-regulation of S6 ATPase by RNA interference resulted in the enhanced accumulation of ubiquitinated proteins suggest that S6 ATPase is a molecular target of ROS under conditions of electrophile-induced oxidative stress and that oxidative modification of this regulatory subunit of proteasome may be functionally associated with the altered recognition and degradation of proteasomal substrates in the cells.
活性氧（ROS）有可能损害细胞成分，如蛋白质，导致蛋白质功能丧失和结构改变。氧化过程会影响各种侧链氨基酸基团，其中一些会转化为羰基化合物。我们之前已经表明，前列腺素D2代谢物15-脱氧-δ（12,14）-前列腺素J2（15d-PGJ2）是人类神经母细胞瘤SH-SY5Y细胞内氧化应激的有效诱导剂[近藤，M.，小谷-伊藤，T.，熊谷，T.，大泽，T.，和内田，K.（2001年）环戊烯酮前列腺素作为细胞内氧化应激的潜在诱导剂，《生物化学杂志》276，12076 - 12083]。在本研究中，为了阐明氧化应激介导的细胞退化的分子机制，我们分析了SH-SY5Y细胞在受到内源性ROS产生诱导剂作用时的蛋白质羰基化情况。当SH-SY5Y细胞暴露于这种内源性亲电试剂时，我们观察到细胞内蛋白质羰基显著积累。对氧化敏感蛋白的蛋白质组学分析表明，蛋白质羰基化的主要细胞内靶点是26S蛋白酶体的调节亚基之一，即S6 ATP酶。随着S6 ATP酶内蛋白质羰基的急剧增加，亲电试剂诱导的氧化应激使S6 ATP酶活性显著降低，26S蛋白酶体降解底物的能力下降。此外，用金属催化氧化系统对26S蛋白酶体进行体外氧化也证实，S6 ATP酶是蛋白酶体中最氧化敏感的亚基。这些以及RNA干扰导致S6 ATP酶下调从而使泛素化蛋白积累增加的观察结果表明，在亲电试剂诱导的氧化应激条件下，S6 ATP酶是ROS的分子靶点，并且蛋白酶体这个调节亚基的氧化修饰可能在功能上与细胞中蛋白酶体底物识别和降解的改变相关。
