Mechanisms of Bax Activation

细胞凋亡，或程序性细胞死亡，是一种对正常发育和组织至关重要的生化途径 多细胞生物体的动态平衡。细胞凋亡的异常抑制是一种必不可少的因素 人类癌症。这一应用的长期目标是阐明细胞内途径和 导致细胞凋亡的分子机制。Bcl2家族蛋白是细胞凋亡的主要调节因子。 遗传学和生物化学研究证实，Bcl2家族中的两种蛋白Bax和Bak是必需的 线粒体依赖的细胞凋亡途径的中介物。为了启动细胞凋亡，BAX需要 经历一个激活过程，这涉及到它的线粒体易位，形成Bax同源基因. 低聚物，以及随后的线粒体损伤，这是细胞凋亡的标志。这其中的机制 激活过程仍然知之甚少。利用生物化学和分子生物学的技术，这 该提案旨在确定Bax在细胞凋亡过程中激活的分子机制。在目标1中，我们 将决定Bax的线粒体易位机制。线粒体靶向的作用 Bax在其线粒体易位中的序列(S)、构象变化和二聚体状态将是 检查过了。在目标2中，对Bax齐聚至关重要的螺旋和氨基酸残基将是 已定义。突变研究和生化分析将被用来检验同种异构化在 Bax的细胞凋亡活性。在目标3中，我们将确定bax的上游激活机制。 激活剂。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Bcl2家族蛋白在细胞凋亡过程中激活Bax的作用。这些研究将确定 Bax激活的分子机制，将有助于阐明导致细胞凋亡的途径。 在成功完成后，这项建议不仅将提供有关监管的基本知识 细胞凋亡，但也可能为抗癌治疗剂的设计提供新的见解。

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-oliomerization in the apoptotic activity of Bax. In Aim 3, we will determine the mechanism of Bax activation by upstream activators. In addition, loss-of-functionstudies 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.