Mechanisms of Bax Activation

细胞凋亡，或程序性细胞死亡，是一种生物化学途径，对正常发育和组织 多细胞生物体内平衡。细胞凋亡的异常抑制是一个必要的贡献者， 人类癌症本申请的长期目标是阐明细胞内途径和细胞内信号转导途径。 导致细胞凋亡的分子机制。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家族中的两种蛋白Bax和巴克是必需的 介导的细胞凋亡途径。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同源的形成， 低聚物，以及随后的线粒体损伤，细胞凋亡的标志。这种机制 激活过程仍然知之甚少。利用生物化学和分子生物学技术， 该提案旨在确定凋亡过程中Bax激活的分子机制。目标1： 将决定Bax的线粒体易位机制。线粒体靶向的作用 序列，构象变化，以及Bax在其线粒体易位中的二聚体状态将是 考察在目标2中，对于Bax的寡聚化关键的螺旋和氨基酸残基将是 定义了突变研究和生化测定将用于检查同源寡聚化在以下方面的作用： Bax的凋亡活性。在目标3中，我们将确定上游Bax激活的机制。 活化剂。此外，使用siRNA的功能丧失研究将与体外研究相结合， 其他Bcl-2家族蛋白在凋亡过程中Bax活化中的作用。这些研究将决定 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，这一建议不仅将提供有关监管的基本知识， 细胞凋亡，但也可能提供新的见解设计的治疗药物对癌症。

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.