Oxidative modification of proteasome: Identification of an oxidation-sensitive subunit in 26 S proteasome
Oxidative modification of proteasome: Identification of an oxidation-sensitive subunit in 26 S proteasome
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DOI：
10.1021/bi051336u
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发表时间：
2005-10-25
期刊：
影响因子：
2.9
通讯作者：
Uchida, K
中科院分区：
文献类型：
--
作者：
Ishii, T;Sakurai, T;Uchida, K
活性氧(ROS)具有潜在的损伤细胞成分的潜能，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。我们先前已经证明，前列腺素D-2代谢产物15-脱氧-三角洲(12，14)-前列腺素J(2)(15d-PGJ(2))是人神经母细胞瘤SH-SY5Y细胞内氧化应激的有效诱导剂[Kondo，M.，Oya-Ito，T.，Kumagai，T.，Osawa，T.，and Uchida，K.(2001)Cyclopenone Prostaglandins as潜在的细胞内氧化应激诱导剂，J.Biol。化学。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细胞在内源性ROS产生诱导剂作用下蛋白质的羰化反应。将SH-SY5Y细胞暴露在这种内源性电泳液中。我们观察到细胞内蛋白质羰基的显著积累。对氧化敏感蛋白的蛋白质组学分析表明，26个S蛋白酶体S6-ATPase中的一个调节亚基是其胞内主要的蛋白质羰化靶点。伴随着S6-ATPase中蛋白质羰基的急剧增加，电泳法诱导的氧化应激显著降低了S6-ATPasc的活性，降低了26个S蛋白酶体降解底物的能力。此外，26个S蛋白酶体在金属催化氧化系统中的体外氧化也证实了S6-ATPase是蛋白酶体中氧化最敏感的亚基。这些结果以及通过RNA干扰下调S6 ATPase导致泛素化蛋白积累增加的观察表明，S6 ATPase是电泳法诱导的氧化应激条件下ROS的分子靶标，蛋白酶体这个调节亚基的氧化修饰可能在功能上与蛋白酶体底物的识别和降解改变有关。
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 D-2 metabolite, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), 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 ATPasc 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.