Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion
Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion
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DOI：
10.1074/jbc.m100142200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Szweda, LI
中科院分区：
文献类型：
--
作者：
Bulteau, AL;Lundberg, KC;Szweda, LI
缺血心肌组织血流的恢复导致氧自由基产生的增加。高活性自由基物质有可能损害细胞成分。显然，细胞活力的维持部分依赖于改变蛋白质的去除。蛋白酶体是主要的细胞内蛋白水解系统，可降解氧化和泛素化形式的蛋白质。利用体内大鼠模型，我们证明冠状动脉闭塞/再灌注导致胰凝乳蛋白酶样、肽酰谷氨酰肽水解酶和胞质提取物中蛋白酶体的胰蛋白酶样活性下降。对纯化的 20 S 蛋白酶体的分析表明，肽酶活性的下降伴随着蛋白质的氧化修饰。我们提供了确凿的证据，表明在冠状动脉闭塞/再灌注时，脂质过氧化产物 4-羟基-2-壬烯醛选择性修饰 20 S 蛋白酶体 a 样亚基 iota、C3 和 XAPC7 的同工型。蛋白酶体纯化后，闭塞/再灌注诱导的胰蛋白酶样活性下降在很大程度上得以保留。相反，在纯化后胞质提取物中观察到的胰凝乳蛋白酶样和肽基谷氨酰肽水解酶活性的损失并不明显。因此，蛋白酶体活性的降低可能是由于酶的直接氧化修饰和内源性胞质抑制蛋白和/或底物对荧光肽水解的抑制。随着蛋白酶体的抑制，氧化和泛素化蛋白质的胞质水平增加。总而言之，我们的研究结果提供了对冠状动脉闭塞/再灌注诱导的蛋白酶体失活的潜在机制以及这些事件的细胞后果的深入了解。
Restoration of blood flow to ischemic myocardial tissue results in an increase in the production of oxygen radicals. Highly reactive, free radical species have the potential to damage cellular components. Clearly, maintenance of cellular viability is dependent, in part, on the removal of altered protein. The proteasome is a major intracellular proteolytic system which degrades oxidized and ubiquitinated forms of protein. Utilizing an in vivo rat model, we demonstrate that coronary occlusion/reperfusion resulted in declines in chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like activities of the proteasome as assayed in cytosolic extracts. Analysis of purified 20 S proteasome revealed that declines in peptidase activities were accompanied by oxidative modification of the protein. We provide conclusive evidence that, upon coronary occlusion/reperfusion, the lipid peroxidation product 4-hydroxy-2-nonenal selectively modifies 20 S proteasome a-like subunits iota, C3, and an isoform of XAPC7. Occlusion/reperfusion-induced declines in trypsin-like activity were largely preserved upon proteasome purification. In contrast, loss in chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities observed in cytosolic extracts were not evident upon purification. Thus, decreases in proteasome activity are likely due to both direct oxidative modification of the enzyme and inhibition of fluorogenic peptide hydrolysis by endogenous cytosolic inhibitory protein(s) and/or substrate(s). Along with inhibition of the proteasome, increases in cytosolic levels of oxidized and ubiquitinated protein(s) were observed. Taken together, our findings provide insight into potential mechanisms of coronary occlusion/reperfusion-induced proteasome inactivation and cellular consequences of these events.