Giulivi C, Davies K J
Institute for Toxicology, University of Southern California, Los Angeles 90033.
J Biol Chem. 1993 Apr 25;268(12):8752-9.
Cells exposed to oxidative stress have been shown previously to exhibit both protein oxidation and increased proteolysis. Experiments conducted with purified proteins in vitro have indicated that oxidatively modified proteins may be selectively degraded by intracellular proteases, but a definitive cause-and-effect relationship has not been demonstrated previously in intact cells. Several investigators have proposed that oxidatively modified proteins are selectively degraded within cells, but the possibility that oxidants may activate intracellular proteases (directly or indirectly) to catalyze the indiscriminate degradation of undamaged proteins has not been discounted. Armed with the knowledge that dityrosine is a specific product of protein oxidation, we undertook a series of experiments to test the hypothesis that oxidized proteins undergo selective intracellular degradation. Our results demonstrate that dityrosine is produced in the hemoglobin molecule when red blood cells are exposed to a continuous flux of hydrogen peroxide (H2O2). The dityrosine so produced is only released from the hemoglobin by proteolysis and is stable to prolonged incubation with cell extracts. Inhibitors of proteolysis have no effect on dityrosine production but do effectively prevent dityrosine release. Proteasome (the 670-kDa multicatalytic proteinase complex, that we have previously called macroxyproteinase or MOP (Pacifici, R. E., Salo, D. C., and Davies, K. J. A. (1989) Free Radical Biol. & Med. 7, 521-526; Salo, D. C., Pacifici, R. E., Lin, S. W., Giulivi, C., and Davies, K. J. A. (1990) J. Biol. Chem. 265, 11919-11927; Pacifici, R. E., and Davies, K. J. A. (1991) Gerontology 37, 166-180) appears responsible for dityrosine release during the selective degradation of oxidatively modified proteins in red blood cells and red cell extracts. We conclude that the elevated rates of proteolysis observed in response to oxidative stress do, indeed, reflect selective degradation of oxidatively modified (damaged) proteins. Despite a relatively low production rate, dityrosine has a high fluorometric quantum yield and is, of course, a specific product of protein oxidation. As an apparently stable metabolic end product, dityrosine may prove to be an extremely valuable (cellular or urinary) marker or index of organismal oxidative stress.
先前已表明，暴露于氧化应激的细胞会同时出现蛋白质氧化和蛋白水解增加的情况。体外使用纯化蛋白质进行的实验表明，氧化修饰的蛋白质可能会被细胞内蛋白酶选择性降解，但此前在完整细胞中尚未证实明确的因果关系。几位研究人员提出，氧化修饰的蛋白质在细胞内被选择性降解，但氧化剂可能（直接或间接）激活细胞内蛋白酶以催化未受损蛋白质的非特异性降解这种可能性并未被排除。基于二酪氨酸是蛋白质氧化的特定产物这一认知，我们开展了一系列实验来验证氧化蛋白质在细胞内进行选择性降解的假说。我们的结果表明，当红细胞暴露于持续的过氧化氢（H₂O₂）流时，血红蛋白分子中会产生二酪氨酸。如此产生的二酪氨酸仅通过蛋白水解从血红蛋白中释放出来，并且在与细胞提取物长时间孵育时是稳定的。蛋白水解抑制剂对二酪氨酸的产生没有影响，但确实能有效阻止二酪氨酸的释放。蛋白酶体（670 kDa的多催化蛋白酶复合物，我们之前称之为大氧化蛋白酶或MOP（帕西菲奇，R. E.，萨洛，D. C.，和戴维斯，K. J. A.（1989年）《自由基生物学与医学》7，521 - 526；萨洛，D. C.，帕西菲奇，R. E.，林，S. W.，朱利维，C.，和戴维斯，K. J. A.（1990年）《生物化学杂志》265，11919 - 11927；帕西菲奇，R. E.，和戴维斯，K. J. A.（1991年）《老年医学》37，166 - 180）似乎负责红细胞和红细胞提取物中氧化修饰蛋白质选择性降解过程中二酪氨酸的释放。我们得出结论，在氧化应激反应中观察到的蛋白水解速率升高确实反映了氧化修饰（受损）蛋白质的选择性降解。尽管二酪氨酸的产生速率相对较低，但它具有较高的荧光量子产率，并且当然是蛋白质氧化的特定产物。作为一种明显稳定的代谢终产物，二酪氨酸可能被证明是一种极其有价值的（细胞或尿液）生物氧化应激标志物或指标。
