Analysis of proapoptotic functions of BH3-only protein BAD

大多数Bcl-2家族成员通过其羧基末端疏水结构域靶向线粒体。促细胞凋亡的B113蛋白BAD靶向线粒体，尽管它没有疏水尾。线粒体易位被认为是BAD介导的细胞凋亡的关键事件，但其靶向机制尚不清楚。在这个项目中，我们证明了外源性GFP-BAD的细胞内定位在转染的细胞系中是不同的，即，细胞质定位或线粒体积累。有趣的是，虽然GFP-BAD分散在整个细胞质和细胞核中，但与Bcl-xL共转染将其募集到线粒体中，而没有任何凋亡信号。相反，GFP-BAD与缺少疏水C末端的突变体Bcl-xL（Bcl-xLΔC）的共转染不能将BAD募集到线粒体。Bcl-xL和14-3-3大量捕获的BAD相关蛋白的TOF-质量分析。更重要的是，内源性BAD蛋白在人口腔鳞状细胞癌SAS细胞中主要保留在线粒体上，而Bcl-xLΔC的过表达强烈地干扰了这一点。这些数据表明Bcl-xL可以将BAD募集到线粒体，并且该过程似乎独立于细胞凋亡信号而发生。

Most Bcl-2 family members are targeted to the mitochondria through their carboxyl terminal hydrophobic domain. A pro-apoptotic B113-only protein BAD is targeted to the mitochondria, although it has no hydrophobic tail. Mitochondrial translocation is believed to be a crucial event of BAD-mediated apoptosis, but the targeting mechanism is less clear. In this project, we demonstrate that intracellular localization of extrinsic GFP-BAD varied in transfected cell lines, i.e., cytoplasmic localization or mitochondrial accumulation. Interestingly, though GFP-BAD was dispersed throughout the cytoplasm and the nucleus, co-transfection with Bcl-xL recruited it to the mitochondria without any apoptotic signals. In contrast, co-transfection of GFP-BAD with the mutant Bcl-xL lacking hydrophobic c-terminus (Bcl-xLΔC) could not recruit BAD to the mitochondria. TOF-mass analysis of BAD-associating proteins fished Bcl-xL and 14-3-3 abundantly. Importantly, endogeneous BAD proteins mostly retained at the mitochondria in human oral squamous cell carcinoma SAS cells, and that was firmly interfered by the Bcl-xLΔC overexpression. These data indicate that Bcl-xL can recruit BAD to the mitochondria, and this process appears to occur independently of apoptotic signals.