Distinct domains of Bcl-XL are involved in Bax and Bad antagonism and in apoptosis inhibition.
Distinct domains of Bcl-XL are involved in Bax and Bad antagonism and in apoptosis inhibition.
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DOI：
10.1016/j.yexcr.2005.06.014
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发表时间：
2005-10
影响因子：
3.7
通讯作者：
Hui Zhou;Q. Hou;Y. Chai;Y. Hsu
中科院分区：
文献类型：
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作者：
Hui Zhou;Q. Hou;Y. Chai;Y. Hsu
促生存因子BclXL可通过两种不同的机制拮抗Bax和Bad的促凋亡作用。它可以通过阻止Bax从胞浆转位到线粒体来阻止Bax介导的细胞死亡。另一方面，Bcl-XL可以通过将Bad隔离到线粒体来中和Bad。为了定位参与抑制Bax和Bad的BclXL结构域，我们对该蛋白进行了突变分析。这是通过删除BclXL的关键结构域，包括其BH1-4结构域、柔性环、C末端疏水结构域和α5-α6发夹片段来实现的。然后用GFP-Bax或GFP-Bad共转染得到的Bc l-XL突变体。我们发现BH1-4结构域和BclXL的C末端片段对于阻断Bax在线粒体的定位是必不可少的。另一方面，只有其BH1和BH3结构域以及C端疏水片段才能将Bad隔离到线粒体上。此外，通过免疫沉淀分析，我们发现这些缺失分别影响了突变蛋白与Bax和Bad的结合能力。最后，细胞存活率分析表明，BH1-4结构域是抑制星形孢菌素诱导的细胞凋亡所需的主要结构域，提示不同的结构域参与拮抗Bax和Bad以及抑制细胞凋亡。
Pro-survival factor Bcl-XLcan antagonize the pro-apoptotic functions of Bax and Bad via two distinct mechanisms. It can block Bax-mediated cell death by preventing Bax translocation from the cytosol to mitochondria. On the other hand, Bcl-XLcan neutralize Bad by sequestering it to mitochondria. In order to map the domains of Bcl-XLinvolved in inhibiting Bax and Bad, we have carried out mutational analyses of this protein. This was done by deleting the key domains of Bcl-XL, including its BH1-4 domains, the flexible loop, the C-terminal hydrophobic domain, and segments of the α5–α6 hairpin. The resulting Bcl-XLmutant constructs were then co-transfected with either GFP-Bax or GFP-Bad. We found that the BH1-4 domains and the C-terminal segment of Bcl-XLwere essential for blocking Bax localization to mitochondria. On the other hand, only its BH1 and BH3 domains and the C-terminal hydrophobic segment were necessary for sequestering Bad to mitochondria. In addition, by immunoprecipitation analyses, we found that these deletions differentially affected the ability of the Bcl-XLmutant proteins to bind Bax and Bad. Finally, cell viability assays indicated that the BH1-4 domains of Bcl-XLwere the primary domains required for inhibiting staurosporine-induced apoptosis, suggesting that distinct domains of Bcl-XLare involved in antagonizing Bax and Bad and in apoptosis inhibition.