A novel mechanism for the regulation of cellular apoptosis mediated by posttranslational N-myristoylation of cytoskeletal proteins.

蛋白质N-肉豆蔻酸化被认为是一种共翻译蛋白质修饰。最近的研究表明，蛋白质N-肉豆蔻酸化也可以发生在翻译后，例如促细胞凋亡的蛋白质Bid和细胞骨架肌动蛋白。我们先前的研究表明，肌动蛋白调节蛋白人明胶蛋白C端caspase裂解产物的N-末端9个残基可以有效地引导蛋白质N-肉豆蔻酰化。为了分析细胞凋亡过程中明胶蛋白翻译后的N-肉豆蔻酰化，我们用[~3H]肉豆蔻酸对COS-1细胞表达的明胶蛋白及其裂解产物进行了代谢标记。在COS-1细胞中表达的人明胶蛋白(TGelsolin)的C端caspase裂解产物(TGelsolin)被有效地N-肉豆蔻酰化。当瞬时转导编码全长明胶蛋白基因的COS-1细胞用凋亡诱导剂依托泊苷或星形孢子素处理时，N-肉豆蔻酰化的明胶蛋白是…基因体内代谢标记表明，更多的是被激活的。在Hela细胞的内源性明胶蛋白表达上，也观察到caspase介导的翻译后N-肉豆蔻酸化的tGelsolin在细胞凋亡过程中的产生。免疫荧光染色(结合MitoTracker染色)和亚细胞分离表明，外源表达的tGelsolin不定位于线粒体，而是广泛分布在细胞质中。为了研究这种修饰在tGelsolin抗凋亡活性中的作用，我们构建了能够同时过表达tGelsolin和EGFP的双顺反子表达载体tGelsolin-IRES-EGFP。利用tGelsolin-IRES-EGFP载体在COS-1细胞中过表达N-肉豆蔻酰化的tGelsolin可显著抑制依托泊苷诱导的细胞凋亡，而过表达非肉豆蔻酸化的tGelsolinG2a突变体不会引起细胞对凋亡的抵抗。较少

Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could also occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal 9 residues of the C-terminal caspase-cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation.To analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase-cleavage products expressed in COS-1 cells with [^3H]myristic acid was performed. It was found that the C-terminal caspase-cleavage product of human gelsolin (tGelsolin) expressed in COS-1 cells was efficiently N-myristoylated. When COS-1 cells transiently transfected with cDNA coding for full-length gelsolin were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was gener … More ated, as demonstrated by in vivo metabolic labeling. The caspase-mediated generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressd in Hela cells. Immunofluorescence staining (coupled with MitoTracker staining) and subcellular fractionation revealed that exogenously expressed tGelsolin did not localize to mitochondria, but rather was diffusely distributed in the cytoplasm.To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using the plasmid tGelsolin-IRES-EGFP significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis.These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin. Less