STRUCTURE-FUNCTION ANALYSIS OF BCL-2 PROTEIN IDENTIFICATION OF CONSERVED DOMAINS IMPORTANT FOR HOMODIMERIZATION WITH BCL-2 AND HETERODIMERIZATION WITH BAX
STRUCTURE-FUNCTION ANALYSIS OF BCL-2 PROTEIN IDENTIFICATION OF CONSERVED DOMAINS IMPORTANT FOR HOMODIMERIZATION WITH BCL-2 AND HETERODIMERIZATION WITH BAX
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DOI：
10.1074/jbc.270.20.11962
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发表时间：
1995-05-19
影响因子：
4.8
通讯作者：
REED, JC
中科院分区：
文献类型：
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作者：
HANADA, M;AIMESEMPE, C;REED, JC
Bcl-2 蛋白是程序性细胞死亡的抑制因子，它与自身形成同二聚体，并与同源蛋白 Bax（细胞死亡启动子）形成异二聚体。 Bax 在酿酒酵母中作为膜结合融合蛋白的表达导致致命表型，该表型可通过 Bcl-2 的共表达来抑制。对酵母中人 Bcl-2 缺失突变体的功能分析表明，存在至少三个抑制 Bar 介导的细胞毒性所需的保守结构域，称为结构域 A（氨基酸 11-33）、B（氨基酸 138-154）和 C（氨基酸 188-196）。使用GST-Bcl-2融合蛋白的体外结合实验表明，Bcl-2(Delta B)和Bcl-2(Delta C)缺失突变体与Bax异二聚化的能力明显受损，但保留了与野生型Bcl-2同二聚化的能力。相比之下，Bcl-a(Delta A) 和 NH2 末端缺失突变体 Bcl-2(Delta 1-82) 在体外保留了 Bax 结合活性，但未能抑制酵母中 Bar 介导的细胞毒性。 197-218 区域中结构域 C 下游的序列也被证明是体外 Bax 结合和酵母抗死亡功能所必需的。使用体外结合测定以及酵母双杂交方法对 Bcl-2/Bcl-2 同二聚化进行的分析提供了支持 Bcl-2/Bcl-2 同二聚化的头尾模型的证据，并揭示了 NH2 末端 A 结构域内的序列与需要同时存在羧基 B 和 C 结构域的结构相互作用。除了进一步描述 Bcl-2 内同源二聚化所需的结构特征外，本文报告的结果支持 Bcl-2 通过直接结合 Bax 来促进细胞存活的假设，但表明结合 Bax 的能力可能不足以发挥抗细胞死亡功能。
The Bcl-2 protein is a suppressor of programmed cell death that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of cell death. Expression of Bax in Saccharomyces cerevisiae as a membrane-bound fusion protein results in a lethal phenotype that is suppressible by co-expression of Bcl-2. Functional analysis of deletion mutants of human Bcl-2 in yeast demonstrated the presence of at least three conserved domains that are required to suppress Bar-mediated cytotoxicity, termed domains A (amino acids 11-33), B (amino acids 138-154), and C (amino acids 188-196). In vitro binding experiments using GST-Bcl-2 fusion proteins demonstrated that Bcl-2(Delta B) and Bcl-2(Delta C) deletion mutants had a markedly impaired ability to heterodimerize with Bax but retained the ability to homodimerize with wild-type Bcl-2. In contrast, Bcl-a(Delta A) and an NH2-terminal deletion mutant Bcl-2(Delta 1-82) retained Bax binding activity in vitro but failed to suppress Bar-mediated cytotoxicity in yeast. Sequences downstream of domain C in the region 197-218 also were shown to be required for Bax-binding in vitro and anti-death function in yeast. Analysis of Bcl-2/Bcl-2 homodimerization using both in vitro binding assays as well as a yeast two-hybrid method provided evidence in support of a head-to-tail model for Bcl-2/Bcl-2 homodimerization and revealed that sequences within the NH2-terminal A domain interact with a structure that requires the presence of both the carboxyl B and C domains in combination. In addition to further delineating structural features within Bcl-2 that are required for homo-dimerization, the findings reported here support the hypothesis that Bcl-2 promotes cell survival by binding directly to Bax but suggest that ability to bind Bax can be insufficient for anti-cell death function.