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（由申请人提供）：凋亡的神经元细胞死亡通常是 与脑缺血有关。目前，许多因素 已经确定了影响神经元细胞死亡。其中， Bcl-2家族代表了一组蛋白质，这些蛋白质是 通过促进CER生存的凋亡，例如Bcl-2和Bcl-XL， 或细胞死亡，例如Bax。促凋亡因子的上调 Bax已在大脑的受影响区域报道，这意味着 该蛋白参与促进神经元细胞死亡。健康 活细胞，Bax主要是胞质蛋白，其能力 调节细胞死亡与其从细胞质转运为 凋亡过程中的线粒体。然而，Bax的促凋亡活性可以 通过与Bcl-XL共表达Bcl-XL，BCl-XL是Bcl-2的生存成员 对神经元发展至关重要的家庭，主要局限于 线粒体。 该项目的远程目标是通过 Bax和Bcl-XL调节细胞凋亡以使 神经保护化合物。此应用的目的是定义 Bcl-XL抑制Bax活性作为迈向的第一步的分子基础 实现远程目标。该提议的中心假设是 Bcl-XL通过防止其阻止Bax的促凋亡功能 细胞凋亡过程中从细胞质重新分布。 我们已经开发了一种简单的方法来通过标记Bax的运动来跟踪Bax的运动 到绿色荧光蛋白。这使我们能够确定如何存在 各种Bcl-XL突变体的影响会影响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如何施加 影响。具有独特的表面特异性rnonoclonal抗体 针对Bcl-XL产生，我们通过 免疫亲和力色谱法并将其鉴定为ATP合酶亚基。 为了实现此应用程序的目标，以下具体目的 将追求：1）确定涉及的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通用性，3）确定 Bcl-XL和ATP合酶亚基之间相互作用的位点，4）确定 ATP合酶亚基在调节线粒体的BAX重新分布中的作用 和细胞死亡，以及5）确定ATP合酶亚基是否可以调节 该提案完成后，BCIXL的渠道形成属性，我们希望 为了进一步了解 BCI-XL及其结合蛋白及BAX抑制的潜在机制 在凋亡过程中由Bcl-X1进行。