Analysis of proapoptotic functions of BH3-only protein BAD

大多数Bc l-2家族成员通过其羧基末端疏水结构域靶向线粒体。一种仅与B113结合的促凋亡蛋白BAD定位于线粒体，尽管它没有疏水的尾巴。线粒体易位被认为是BAD介导的细胞凋亡的关键事件，但靶向机制尚不清楚。在这个项目中，我们证明了外源GFP-BAD在细胞内的定位在不同的细胞系中是不同的，即细胞质定位或线粒体聚集。有趣的是，尽管GFP-BAD分散在细胞质和细胞核中，但与Bcl-XL共转染后，GFP-BAD被招募到线粒体，而没有任何凋亡信号。相反，绿色荧光蛋白-BAD与缺失疏水C末端的突变体BclXL(ΔC)共转染不能向线粒体募集BAD。不良结合蛋白的TOF-MS分析丰富地捕获了Bclxl和14-3-3。重要的是，内源性Bad蛋白主要保留在口腔鳞状细胞癌细胞的线粒体中，并受到BclxlΔC过表达的强烈干扰。这些数据表明，Bcl-xl可以向线粒体募集BAD，并且这一过程似乎不依赖于凋亡信号而发生。

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.