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

细胞骨架蛋白翻译后 N-肉豆蔻酰化介导的细胞凋亡调节的新机制。

基本信息

批准号：
17580080
负责人：
UTSUMI Toshihiko
金额：
$ 2.37万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

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

蛋白n -肉豆蔻酰基化已被认为是一种共翻译蛋白修饰。最近，研究表明蛋白质n -肉豆蔻酰基化也可能发生在翻译后，如促凋亡蛋白BID和细胞骨架肌动蛋白。我们之前的研究表明，作为一种肌动蛋白调节蛋白，人凝胶蛋白c端caspase裂解产物的n-末端9残基有效地指导了蛋白n-肉豆蔻酰化。为了分析细胞凋亡过程中gelsolin翻译后n -肉豆蔻酰基化，我们用[^3H]肉豆蔻酸对COS-1细胞中表达的gelsolin及其caspase-裂解产物进行了代谢标记。在COS-1细胞中表达的人凝胶蛋白（tGelsolin）的c端caspase裂解产物被有效地n -肉豆蔻酰基化。体内代谢标记表明，将编码全长gelsolin cDNA的COS-1细胞瞬时转染依托泊苷或星孢素等凋亡诱导剂后，n -肉豆荚酰基化的gelsolin生成。Hela细胞内源性gelsolin表达也观察到凋亡过程中caspase介导的翻译后n -肉豆醇化gelsolin的产生。免疫荧光染色（结合MitoTracker染色）和亚细胞分离显示外源表达的tGelsolin不局限于线粒体，而是弥漫性地分布在细胞质中。为了研究这一修饰在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的双链表达质粒tGelsolin- ires -EGFP。利用质粒tGelsolin- ires - egfp在COS-1细胞中过表达n-肉豆荚酰基化的tGelsolin可显著抑制依托opo苷诱导的细胞凋亡，而过表达非肉豆荚酰基化的tGelsolinG2A突变体则不会引起细胞凋亡抗性。这些结果表明，tGelsolin的翻译后n -肉豆肉酰化并不直接靶向线粒体，但这种修饰参与了tGelsolin的抗凋亡活性。少