Dimerization properties of human BAD -: Identification of a BH-3 domain and analysis of its binding to mutant BCL-2 and BCL-XL proteins
Dimerization properties of human BAD -: Identification of a BH-3 domain and analysis of its binding to mutant BCL-2 and BCL-XL proteins
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DOI：
10.1074/jbc.272.49.30866
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发表时间：
1997-12-05
影响因子：
4.8
通讯作者：
Oltersdorf, T
中科院分区：
文献类型：
--
作者：
Ottilie, S;Diaz, JL;Oltersdorf, T
Bad是一种程序性细胞死亡诱导剂，最近通过其与抗凋亡蛋白BCL-2结合的能力从小鼠cDNA文库中分离出来。序列分析表明Bad是编码程序性细胞死亡诱导因子和抑制因子的BCL-2基因家族的成员。为了进一步分析BAD在BCL-2家族形成的同源和异源二聚体网络中的作用，我们克隆了BAD的人类同源物，并评估了它的生物活性及其与野生型和突变型BCL-2家族蛋白的相互作用。我们的研究结果表明，人类BAD蛋白和其小鼠同源物一样，在转染到哺乳动物细胞中时能够诱导细胞凋亡。此外，在酵母双杂交实验和体外相互作用定量实验中，人类Bad与BCL-2和BCL-X-L相互作用。人类BAD的序列比对发现，与其他BCL-2家族蛋白一样，存在一个BH-3同源结构域，从该结构域衍生的肽能够完全抑制BAD与BCL-X-L的二聚化。因此，正如先前在BAX、BAK、BCL-2和BCL-X-L中所示，BAD的BH3结构域是其与其他BCL-2家族蛋白二聚化所必需的。我们进一步分析了BAD与各种失去BAX和BAK结合能力的BCL-2和BCL-X-L突变体的结合能力，其中一些突变体保留了生物活性，而另一些则没有。令人惊讶的是，所有突变的BCL-2和BCL-X-L蛋白都与人类BAD强烈相互作用。因此，我们的数据表明，BCL-2和BCL-X-L的突变可以不同地影响不同促死蛋白的异二聚体结合，并可能涉及异二聚体与生物活性之间的关系。
Bad, an inducer of programmed cell death, was recently isolated from a mouse cDNA library by its ability to bind to the anti-apoptotic protein BCL-2. Sequence analysis suggested that Bad was a member of the BCL-2 gene family that encodes both inducers and inhibitors of programmed cell death. To further analyze the role of BAD in the network of homo- and heterodimers formed by the BCL-2 family, we have cloned the human homologue of BAD and assessed its biological activity and its interactions with wild type and mutant BCL-2 family proteins. Our results indicate that the human BAD protein, like its mouse homologue, is able to induce apoptosis when transfected into mammalian cells. Furthermore, in yeast two-hybrid assays as well as quantitative in vitro interaction assays, human Bad interacted with BCL-2 and BCL-X-L. Sequence alignments of human BAD revealed the presence of a BH-3 homology domain as seen in other BCL-2 family proteins, Peptides derived from this domain were able to completely inhibit the dimerization of BAD with BCL-X-L. Thus, as previously shown for BAX, BAK, BCL-2, and BCL-X-L, the BH3 domain of BAD is required for its dimerization with other BCL-2 family proteins. BAD was further analyzed for its ability to bind to various mutants of BCL-2 and BCL-X-L that have lost the ability to bind BAX and BAK, some of which retain biological activity and some of which do not. Surprisingly, all of the mutated BCL-2 and BCL-X-L proteins analyzed strongly interacted with human BAD. Our data thus indicate that mutations in BCL-2 and BCL-X-L can differentially affect the heterodimeric binding of different death-promoting proteins and have implications concerning the relationship between heterodimerization and biological activity.