Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32
Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32
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DOI：
10.1038/sj.cdd.4401418
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发表时间：
2004-07-01
影响因子：
12.4
通讯作者：
Kitanaka, C
中科院分区：
文献类型：
--
作者：
Sunayama, J;Ando, Y;Kitanaka, C
促凋亡Bcl-2亚家族的Bcl-2同源结构域（BH）3-only蛋白作为Bcl-2依赖性凋亡的启动子起关键作用。到目前为止，至少有10哺乳动物BH 3-唯一的蛋白质已被确定，它是现在意识到，他们有不同的作用和机制的调节，在线粒体的凋亡信号的转导。Hrk/DP 5是哺乳动物中一种仅含BH 3的蛋白质，参与多种生理和病理性细胞凋亡，但Hrk介导的细胞凋亡的分子机制仍知之甚少。在试图确定参与Hrk介导的细胞凋亡的细胞蛋白质，我们进行了酵母双杂交筛选Hrk相互作用的蛋白质和分离的p32，线粒体蛋白，已被证明形成一个通道组成的同源三聚体。在体外结合，免疫共沉淀，以及免疫细胞化学分析验证了特定的相互作用和共定位的Hrk和p32，这两者都依赖于高度保守的C-末端区域的p32的存在。重要的是，Hrk诱导的细胞凋亡受到缺乏N-末端线粒体信号序列（p32（74-282））和保守的C-末端区域（p32（1-221））的p32突变体的表达的抑制，预期它们分别抑制Hrk与内源性p32蛋白的竞争性结合和破坏p32的通道功能。此外，小干扰RNA介导的p32基因敲低可保护Hrk诱导的细胞凋亡。总之，这些结果表明，p32可能是一个关键的分子，连接Hrk的线粒体，并在Hrk介导的细胞凋亡的调节是至关重要的。
Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32(1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.