Mechanisms of Bax Activation

描述（由申请人提供）：细胞凋亡或程序性细胞死亡是多细胞生物体正常发育和组织稳态的关键生化途径。细胞凋亡的异常抑制是人类癌症的必要因素。本申请的长期目标是阐明导致细胞凋亡的细胞内途径和分子机制。Bcl-2家族蛋白是细胞凋亡的主要调节因子。遗传和生物化学研究证实，Bcl-2家族蛋白中的两种Bax和巴克是Bcl-2依赖性细胞凋亡途径的必需介质。为了启动细胞凋亡，Bax需要经历一个激活过程，这涉及其线粒体易位，Bax同源寡聚体的形成，以及随后的线粒体损伤，这是细胞凋亡的标志。这种激活过程的机制仍然知之甚少。利用生物化学和分子生物学技术，本研究旨在确定凋亡过程中Bax激活的分子机制。目的1：探讨Bax在线粒体内的转位机制。线粒体靶向序列的作用（S），构象变化，和二聚体状态的Bax在其线粒体易位将被检查。在目标2中，将定义Bax寡聚化的关键螺旋和氨基酸残基。突变研究和生化分析将被用来研究同源寡聚化在Bax凋亡活性中的作用。在目标3中，我们将确定上游激活剂激活Bax的机制。此外，使用siRNA的功能丧失研究将与体外研究相结合，以检查其他Bcl-2家族蛋白在凋亡期间Bax激活中的作用。这些研究将确定Bax激活的分子机制，并将有助于阐明导致细胞凋亡的途径。成功完成后，这一建议不仅将提供细胞凋亡调控的基础知识，而且还可能为癌症治疗药物的设计提供新的见解。

DESCRIPTION (provided by applicant): Apoptosis, or programmed cell death, is a biochemical pathway critical to normal development and tissue homeostasis in multi-cellular organisms. Aberrant suppression of apoptosis is a requisite contributor to human cancer. The long-term objective of this application is to elucidate the intracellular pathways and the molecular mechanisms that lead to apoptosis. The Bcl-2 family proteins are major regulators of apoptosis. Genetic and biochemical studies establish that two of the Bcl-2 family proteins, Bax and Bak, are requisite mediators of the mitochondria-dependent apoptotic pathway. In order to initiate apoptosis, Bax needs to undergo an activation process, which involves its mitochondrial translocation, the formation of Bax homo-oligomers, and the subsequent damage of mitochondria, a hallmark of apoptosis. The mechanisms of this activation process remain poorly understood. Using techniques of biochemistry and molecular biology, this proposal aims at determining the molecular mechanisms of Bax activation during apoptosis. In Aim 1, we will determine the mechanism of mitochondrial translocation of Bax. The role of mitochondrial targeting sequence(s), conformational changes, and dimeric states of Bax in its mitochondrial translocation will be examined. In Aim 2, the helices and amino acid residues critical for the oligomerization of Bax will be defined. Mutational study and biochemical assays will be used to examine the role of homo-oligomerization in the apoptotic activity of Bax. In Aim 3, we will determine the mechanism of Bax activation by upstream activators. In addition, loss-of-function studies using siRNA will be combined with in vitro studies to examine the role of other Bcl-2 family proteins in the activation of Bax during apoptosis. These studies will determine the molecular mechanisms of Bax activation and will help elucidate the pathways that lead to apoptosis. Upon a successful completion, this proposal will not only provide basic knowledge on the regulation of apoptosis, but may also provide new insights on the design of therapeutic agents against cancer.