脑特异性高表达蛋白TMEM59L诱导细胞凋亡的机制

Mechanism Underlying the Brain-specific Membrane-anchored Protein TMEM59L-mediated Apoptosis

TMEM59L为近年来新发现的脑特异性高表达蛋白,具有促凋亡效果,但其具体的凋亡机制还不清楚.构建了TMEM59L重组质粒,并在HEK293T细胞中过表达TMEM59L,用Annexin V-FITC/PI双染法确定了TMEM59L可以诱导细胞凋亡,之后用免疫印迹方法检测细胞内凋亡相关分子激活情况.结果显示,外源TMEM59L可以降低Bcl-2的蛋白表达水平,诱导细胞色素c从线粒体释放进入细胞质,并且激活caspase-9、caspase-7和caspase-3,但不激活caspase-8;活化的caspase-7和caspase-3进一步酶解死亡底物PARP,导致细胞凋亡;此外,用广谱caspase抑制剂Z-Val-Ala-Asp-FMK(Z-VAD-FMK)抑制caspase-7和caspase-3活性后,死亡底物PARP的酶解也基本被抑制.由此可见,TMEM59L是通过caspase依赖的线粒体途径诱导HEK293T细胞凋亡.

Apoptosis plays a key role in multiple biological functions and dysregulation of apoptosis leads to disease pathogenesis.Therefore,identification of new proteins mediating apoptosis and elucidation of the underlying mechanism is important not only for basic research but also for disease intervention.TMEM59L is a newly-found brain-specific anchored protein with very limited information.One study reported that TMEM59L could induce apoptosis.But the underlying mechanism remains elusive.In this paper,TMEM59L recombinant plasmid was constructed and transfected into HEK293 cells.Through Annexin V-FITC/ propidium iodide double staining and flow cytometry assay,it was confirmed that overexpression of TMEM59L could dramatically induce cell apoptosis.Through Westen blotting study,it was also found that overexpression of TMEM59L could reduce the protein level of Bcl-2,induce cytochrome c release from mitochondria to the cytosol,and activate caspase-9,caspase-3 and caspase-7,but not caspase-8.Consistently,overexpression of TMEM59L promoted the cleavage of death substrate PARP by activated caspases.While cleavage of PARP upon TMEM59L overexpression was dramatically decreased when cells were treated with a caspase inhibitor Z-Val-Ala-Asp-FMK（Z-VAD-FMK）.Together these results suggest that TMEM59L can induce apoptosis through a caspase-dependent mitochondrial pathway.Because of its high abundance in the brain,TMEM59L might participate in the physiological apoptosis during neural development and/or pathological apoptosis during neurodegenerative diseases,and these deserve further investigation.