Mechanism of Apoptosis Inhibition By Bcl-XL

Bcl-XL抑制细胞凋亡的机制

• 批准号: 6743982
• 负责人: YI-TE HSU
• 金额: $ 21.45万
• 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/6743982
• 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(申请人提供)：神经细胞因细胞凋亡而死亡 与脑缺血有关。目前，一些因素表明 影响神经细胞死亡的因素已被确认。其中， BCL-2家族代表一组蛋白质，它们作为关键的调节因子 通过促进CER存活来促进细胞凋亡，如在Bcl2和Bclxl的情况下， 或者像BAX那样的细胞死亡。促凋亡因子的上调 据报道，BAX在大脑的受影响区域，牵涉到 这种蛋白质参与促进神经细胞死亡。在健康中 活细胞，Bax主要是一种胞浆蛋白，它能够 调节性细胞死亡与其从胞浆转位到 线粒体在细胞凋亡过程中。然而，Bax的这种促凋亡活性可以 与Bcl2的促生存成员Bclxl共表达而被阻断 对神经元发育至关重要的家族，主要局限于 线粒体。 该项目的长期目标是通过以下方式定义基本机制 哪些Bax和Bclxl调节细胞凋亡以促进肿瘤的发展 神经保护性化合物。本应用程序的目标是定义 Bcl-xL抑制Bax活性的分子基础 完成远景目标。这一提议的中心假设是 Bclxl通过阻断Bax的促凋亡功能 在细胞凋亡过程中，从胞浆到线粒体的重新分布。 我们开发了一种简单的方法，通过标记Bax来跟踪它的移动 绿色荧光蛋白。这使我们能够确定存在如何 不同的BclxL突变体通过以下途径影响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是如何发挥其作用的。 效果。用我们拥有的一种独特的表位特异的非克隆抗体 针对BclXL产生的，我们已经纯化了一个主要的BclX1相关蛋白 免疫亲和层析，鉴定为三磷酸腺苷合成酶亚基。 为了实现本应用程序的目标，需要实现以下具体目标 将继续进行：1)确定BCI-XL参与的功能结构域 抑制Bax易位到线粒体，2&gt；确定特异性和 BCI-XL与ATP合酶亚基相互作用的广泛性，3)决定 Bc l-xl与ATP合酶亚基的相互作用部位，4)决定 三磷酸腺苷合酶亚基在调节Bax向线粒体重分布中的作用 和细胞死亡，以及5)确定ATP合成酶亚基是否可以调节 BcIXL的通道形成特性在本提案完成后，我们预计 对其结构/功能特性有了进一步的了解 BCI-XL及其结合蛋白及其抑制Bax的机制 在细胞凋亡过程中通过BclX1表达。