PROTEOLYSIS IN CULTURED LIVER EPITHELIAL-CELLS DURING OXIDATIVE STRESS - ROLE OF THE MULTICATALYTIC PROTEINASE COMPLEX, PROTEASOME
PROTEOLYSIS IN CULTURED LIVER EPITHELIAL-CELLS DURING OXIDATIVE STRESS - ROLE OF THE MULTICATALYTIC PROTEINASE COMPLEX, PROTEASOME
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DOI：
10.1074/jbc.270.5.2344
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发表时间：
1995-02-03
影响因子：
4.8
通讯作者：
DAVIES, KJA
中科院分区：
文献类型：
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作者：
GRUNE, T;REINHECKEL, T;DAVIES, KJA
暴露于各种形式的轻度氧化应激显著增加了9号克隆肝细胞（正常肝上皮）培养中代谢放射性标记的“短寿命”和“长寿命”细胞蛋白的细胞内降解。连续H2O2通量；氧化还原循环醌、甲萘醌和百草枯；脂质过氧化的醛产物，4-羟基壬烯醛，丙二醛和己烯醛。一般来说，暴露于更严重的氧化应激会导致细胞内蛋白水解的浓度依赖性下降，在某些情况下会低于基线水平，当将氧化修饰的“外来”蛋白（超氧化物歧化酶和血红蛋白）添加到克隆9肝细胞的裂解物中时，与未处理的外来蛋白相比，也会选择性地降解。与细胞内蛋白水解一样，添加到细胞裂解物中的外源蛋白的降解被轻度氧化修饰大大增加，但被更严重的氧化修饰抑制。蛋白酶活性在>300-kDa细胞组分中恢复，抑制剂谱和免疫沉淀研究表明，在轻度氧化应激下观察到的多催化蛋白酶复合物蛋白酶体（proteasome）负责大部分选择性降解；每天使用针对蛋白酶体C2亚基基因起始密码子区域的反义寡脱氧核苷酸7天，严重降低了几种蛋白酶体亚基多肽的细胞内水平（通过Western blot分析），也使H2O2诱导的细胞内蛋白水解增加了约95%。没有显著影响基线蛋白水解率。广泛的研究表明，氧化应激细胞降解氧化修饰的蛋白质底物的总体能力只有少量或没有增加；这一发现得到了Western blot和Northern blot分析的支持，它们显示蛋白酶体亚基多肽或mRNA转录物的水平没有显著增加。我们得出结论，轻度氧化应激通过改变细胞蛋白，从而增加其蛋白水解敏感性，从而增加细胞内蛋白水解。相反，严重氧化应激减少细胞内蛋白水解，可能是通过产生严重受损的细胞蛋白，不易降解（例如，交联/聚集蛋白），并破坏蛋白水解酶。我们进一步得出结论，在克隆9肝细胞中，多催化蛋白酶复合物蛋白酶体负责大部分氧化修饰蛋白的识别和选择性降解。
Exposure to various forms of mild oxidative stress significantly increased the intracellular degradation of both ''short-lived'' and ''long-lived,'' metabolically radiolabeled, cell proteins in cultures of Clone 9 liver cells (normal liver epithelia), The oxidative stresses employed were bolus H2O2 addition; continuous H2O2 flux; the redox cycling quinones, menadione and paraquat; and the aldehydic products of lipid peroxidation, 4-hydroxynonenal, malonyldialdehyde, and hexenal. In general, exposure to more severe oxidative stress produced a concentration-dependent decline in intracellular proteolysis, in some cases to below baseline levels, Oxidatively modified ''foreign'' proteins (superoxide dismutase and hemoglobin) were also selectively degraded, in comparison with untreated foreign proteins, when added to lysates of Clone 9 liver cells. As with intracellular proteolysis, the degradation of foreign proteins added to cell lysates was greatly increased by mild oxidative modification, but depressed by more severe oxidative modification, The proteinase activity was recovered in >300-kDa cell fractions, and inhibitor profiles and immunoprecipitation studies indicated that the multicatalytic proteinase complex, proteasome, was responsible for most of the selective degradation observed with mild oxidative stress; up to approximately 95% for intracellular proteolysis and 65-80% for degradation of foreign modified proteins, Seven days of daily treatment with an antisense oligodeoxynucleotide, directed against the initiation codon region of the proteasome C2 subunit gene, severely depressed the intracellular levels of several proteasome subunit polypeptides (by Western blot analysis), and also depressed the H2O2 induced increase in intracellular proteolysis by approximately 95%, without significantly affecting baseline proteolytic rates. Extensive studies revealed only small or no increases in the overall capacity of oxidatively stressed cells to degrade oxidatively modified protein substrates; a finding supported by both Western blot and Northern blot analyses which revealed no significant increase in the levels of proteasome subunit polypeptides or mRNA transcripts. We conclude that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, thus increasing their proteolytic susceptibility, In contrast, severe oxidative stress diminishes intracellular proteolysis, probably by generating severely damaged cell proteins that cannot be easily degraded (e.g, cross-linked/aggregated proteins), and by damaging proteolytic enzymes, We further conclude that the multicatalytic proteinase complex proteasome is responsible for most of the recognition and selective degradation of oxidatively modified proteins in Clone 9 Liver cells.