Nonionic detergents induce dimerization among members of the Bcl-2 family
Nonionic detergents induce dimerization among members of the Bcl-2 family
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DOI：
10.1074/jbc.272.21.13829
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发表时间：
1997-05-23
影响因子：
4.8
通讯作者：
Youle, RJ
中科院分区：
文献类型：
--
作者：
Hsu, YT;Youle, RJ
Bcl-2家族成员（包括Bcl-2、Bcl-X-L和Bax）在细胞凋亡的调节中起关键作用。这些蛋白质被认为是膜相关的，并已提出通过同源二聚化和异源二聚化来调节细胞凋亡。我们已经发现，而Bcl-2主要是膜相关的，如以前报道的，显着量的Bcl-X-L和大多数Bax蛋白是不膜相关的，因此出现在胸腺细胞和脾细胞提取物的胞质部分，这一发现允许在没有洗涤剂扰动的情况下，这些蛋白质的二聚化特性和构象的研究。对于该分析，我们已经产生了对Bax、Bcl-2和Bcl-X-L的已知表位具有特异性的单克隆抗体。针对氨基酸12和24之间的N-末端表位（α uBax 6A 7）的抗体不能结合可溶性胞质形式的Bax，表明该表位通常被掩埋。非离子去污剂改变fax构象以暴露该表位。在非离子去污剂的存在下，6A 7抗体强烈结合fax的单体形式，但不结合与Bcl-X或Bcl-2复合的Bax。相比之下，针对氨基酸3-16内的Bax的相邻表位（α mBax 5 B7）的单克隆抗体结合可溶性和去污剂改变形式的fax，并且还结合Bax.Bcl-X-L或Bax.Bcl-2复合物。令人惊讶的是，在不存在去污剂的情况下，Bax不能与Bcl-X-L形成同源二聚体或异源二聚体。这些结果表明，Bcl-2家族成员在生理条件下的一种新的构象状态，不同于形成二聚体的洗涤剂改变的状态，目前被认为是调节细胞凋亡。
Members of the Bcl-2 family (including Bcl-2, Bcl-X-L, and Bax) play key roles in the regulation of apoptosis. These proteins are believed to be membrane-associated and have been proposed to regulate apoptosis through both homodimerization and heterodimerization. We have found that whereas Bcl-2 is predominantly membrane-associated as previously reported, significant amounts of Bcl-X-L and most of the Bax proteins are not membrane-associated and thus appear in the cytosolic fraction of thymocyte and splenocyte extracts, This finding allows the study of the dimerization properties and conformation of these proteins in the absence of detergent perturbation. For this analysis, we have produced monoclonal antibodies that are specific for known epitopes of Bax, Bcl-2, and Bcl-X-L. An antibody to an N-terminal epitope (alpha uBax 6A7) between amino acids 12 and 24 fails to bind the soluble cytosolic form of Bax, indicating that this epitope is normally buried. Nonionic detergents alter the fax conformation to expose this epitope. In the presence of nonionic detergent, the 6A7 antibody avidly binds the monomeric form of fax, but not Bax complexed with either Bcl-X, or Bcl-2. In contrast, a monoclonal antibody to an adjacent epitope of Bax (alpha mBax 5B7) within amino acids 3-16 binds the soluble and detergent-altered forms of fax and also binds the Bax.Bcl-X-L or the Bax.Bcl-2 complex. Surprisingly, in the absence of detergent Bax fails to form homodimers or heterodimers with Bcl-X-L. These results demonstrate a novel conformational state of members of the Bcl-2 family under a physiological condition that is distinct from the detergent-altered state that forms dimers and is currently believed to regulate apoptosis.