Mechanism of Apoptosis Inhibition By Bcl-XL

Bcl-XL抑制细胞凋亡的机制

• 批准号: 6370337
• 负责人: YI-TE HSU
• 金额: $ 26.45万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6370337
• 关键词: BCL2 gene /protein; Bax gene /protein; Rodentias; apoptosis; confocal scanning microscopy; enzyme structure; green fluorescent proteins; hydrogen transporting ATP synthase; mitochondria; monoclonal antibody; neurons; protein localization; protein protein interaction; protein purification; protein structure function; tissue /cell culture; transfection; western blottings

DESCRIPTION(provided by applicant): Neuronal cell death by apoptosis is often associated with cerebral ischemia. Currently, a number of factors that influence neuronal cell death have been identified. Among them, members of the Bcl-2 family represent a group of proteins that serve as key regulators of apoptosis by promoting either ceR survival as in the case of Bcl-2 and Bcl-XL, or cell death as in the case of Bax. Upregulation of the pro-apoptotic factor Bax has been reported in the affected area of the brain, implicating the participation of this protein in promoting neuronal cell death. In healthy living cells, Bax is predominantly a cytosolic protein and its ability to modulate cell death is associated with its translocation from the cytosol to mitochondria during apoptosis. This pro-apoptotic activity of Bax however, can be blocked by coexpression with Bcl-XL, a pro-survival member of the Bcl-2 family that is essential for neuronal development and is localized mainly to mitochondria. The long-range goal of this project is to define the underlying mechanisms by which Bax and Bcl-XL regulate apoptosis to enable the development of neuroprotective compounds. The objective of this application is to define the molecular basis by which Bcl-XL inhibits Bax activity as a first step towards accomplishing the long-range goal. The central hypothesis for this proposal is that Bcl-XL blocks the pro-apoptotic function of Bax by preventing its redistribution from the cytosol to mitochondria during apoptosis. We have developed a simple method of tracking the movement of Bax by tagging it to the green fluorescent protein. This enables us to determine how the presence of various Bcl-XL mutants affects the intracellular distribution of Bax by confocal microscopy. In addition, we have begun to study how Bcl-XL exerts its effect. With a unique epitopespecific rnonoclonal antibody that we have generated against Bcl-XL, we have purified a major Bcl-X1 associated protein by immunoaffinity chromatography and identified it as ATP synthase subunit. To accomplish the objective of this application, the following specific aims will be pursued: 1) determine the functional domains of BCI-XL involved in inhibiting Bax translocation to mitochondria, 2> determine the specificity and universality of BCI-XL interaction with ATP synthase subunit, 3) determine the sites of interaction between Bcl-XL and ATP synthase subunit, 4) determine the role of ATP synthase subunit in regulating Bax redistribution to mitochondria and cell death, and 5) determine whether ATP synthase subunit can regulate the channel forming properties of BcIXL Upon completion of this proposal, we expect to have gained further understanding of the structural/functional properties of BCI-XL and its binding protein and the underlying mechanism of Bax inhibition by Bcl-X1 during apoptosis.

描述（由申请人提供）：神经细胞凋亡导致的神经细胞死亡通常是 与脑缺血有关。目前，一些因素， 影响神经元细胞死亡。其中， Bcl-2家族代表了一组蛋白质，其作为细胞凋亡的关键调节因子， 细胞凋亡通过促进如Bcl-2和Bcl-XL的情况中的ceR存活， 或者像Bax那样的细胞死亡。促凋亡因子的上调 据报道，Bax存在于大脑的受影响区域， 该蛋白参与促进神经元细胞死亡。健康 在活细胞中，Bax主要是一种胞质蛋白， 调节细胞死亡与其从胞质溶胶转移到 线粒体凋亡过程中。然而，Bax的这种促凋亡活性可以 通过与Bcl-2的促存活成员Bcl-XL共表达而被阻断。 一个对神经元发育至关重要的家族，主要定位于 线粒体 该项目的长期目标是通过以下方式确定基本机制： Bax和Bcl-XL调节细胞凋亡， 神经保护化合物本申请的目的是定义 Bcl-XL抑制Bax活性的分子基础， 实现长期目标。这一提议的核心假设是 Bcl-XL通过阻止Bax的促凋亡功能， 在凋亡过程中从细胞质到线粒体的再分布。 我们已经开发了一种简单的方法，通过标记来跟踪Bax的运动 到绿色荧光蛋白。这使我们能够确定 各种Bcl-XL突变体的表达通过以下方式影响Bax的细胞内分布： 共聚焦显微镜此外，我们已经开始研究Bcl-XL如何发挥其 效果用一种独特的表位特异性单克隆抗体 针对Bcl-XL产生，我们已经纯化了一个主要的Bcl-X1相关蛋白， 免疫亲和层析鉴定为ATP合酶亚基。 为了实现本申请的目的，以下具体目标 将追求：1）确定参与的BCI-XL的功能域， 抑制Bax易位到线粒体，2>确定特异性和 BCI-XL与ATP合酶亚基相互作用的普遍性，3）确定 Bcl-XL和ATP合酶亚基之间相互作用的位点，4）决定 ATP合成酶亚基在调节Bax向线粒体再分布中的作用 和细胞死亡，以及5）确定ATP合酶亚基是否可以调节 BcIXL的通道形成特性在完成本提案后，我们预计 进一步了解结构/功能特性， BCI-XL及其结合蛋白与Bax抑制机制 Bcl-X1在细胞凋亡中的作用