Ezrin turnover and cell shape changes catalyzed by proteasome in oxidatively stressed cells
Ezrin turnover and cell shape changes catalyzed by proteasome in oxidatively stressed cells
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DOI：
10.1096/fj.02-0015com
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发表时间：
2002-10-01
期刊：
影响因子：
4.8
通讯作者：
Davies, KJA
中科院分区：
文献类型：
--
作者：
Grune, T;Reinheckel, T;Davies, KJA
我们发现埃兹蛋白（一种参与将肌动蛋白锚定到细胞膜上的细胞骨架蛋白）在氧化应激后优先降解并重新合成。使用二维凝胶和氨基末端微测序将 Ezrin 鉴定为少数几种 [S-35] 蛋氨酸预标记蛋白之一，在暴露于过氧化氢 (H2O2) 的克隆 9 大鼠肝细胞中降解。氧化应激后用[S-35]蛋氨酸对细胞蛋白进行代谢标记表明，埃兹蛋白的再合成急剧增加，但羧基末端抗埃兹蛋白单克隆抗体显示细胞内埃兹蛋白水平恒定；换句话说，降解和再合成是完全匹配的。 Ezrin 降解可被选择性蛋白酶体抑制剂（乳胞素、NLVS 和环氧霉素）和针对蛋白酶体 C2 亚基的反义寡核苷酸阻断。 H2O2 还引起细胞形状的重大变化，包括细胞直径的显着增加，这必然需要大量的细胞骨架重排。然而，过氧化物诱导的细胞直径增加被选择性蛋白酶体抑制剂乳胞素阻断。因此，埃兹蛋白的降解和再合成可能是氧化应激期间观察到的整体细胞形状变化的潜在机制。氧化应激会诱导蛋白质广泛氧化和降解，并导致细胞起泡、四舍五入和整体大小显着增加。我们的结果表明，所有这些氧化剂诱导的变化实际上可能是由蛋白酶体催化的。
We find that ezrin, a cytoskeletal protein involved in anchoring actin to the cell membrane, is preferentially degraded and resynthesized after oxidative stress. Ezrin was identified using 2-dimensional gels and amino-terminal microsequencing as one of a select few [S-35]methionine prelabeled proteins degraded in clone 9 rat liver cells exposed to hydrogen peroxide (H2O2). Metabolic labeling of cellular proteins with [S-35]methionine after oxidative stress showed that resynthesis of ezrin rose dramatically but carboxyl terminus anti-ezrin monoclonal antibodies revealed constant intracellular ezrin levels; in other words, degradation and resynthesis were exactly matched. Ezrin degradation was blocked by selective inhibitors of the proteasome (lactacystin, NLVS, and epoxomycin) and by an antisense oligonucleotide directed against the proteasome C2 subunit. H2O2 also caused major changes in cell shape, including significant increases in cell diameter, which must require substantial cytoskeletal rearrangement. Peroxide-induced increases in cell diameter were, however, blocked by the selective proteasome inhibitor lactacystin. The degradation and resynthesis of ezrin may therefore be an underlying mechanism for overall cell shape changes observed during oxidative stress. Oxidative stress induces extensive protein oxidation and degradation and significant increases in cell blebbing, rounding-up, and overall size. Our results indicate that all these oxidant-induced changes may actually be catalyzed by the proteasome.