THE ROLE OF MITOCHONDRIAL CHANNELS IN CELL DEATH

线粒体通道在细胞死亡中的作用

• 批准号: 7192504
• 负责人: KATHLEEN W. KINNALLY
• 金额: $ 32.48万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7192504
• 关键词: Apoptosis; Apoptotic; BCL-2 Protein; BCL2 gene; Bax protein; Biological; Cell Death; Cell Line; Cell physiology; Cells; Characteristics; Data; Degenerative Disorder; Development; Disease regression; Electrophysiology (science); Enzyme-Linked Immunosorbent Assay; Eukaryota; Eukaryotic Cell; Family; Family member; Goals; Hela Cells; Hematopoietic; Homeostasis; Immunoprecipitation; Knock-out; Knowledge; Lead; Localized; Malignant Neoplasms; Mediator of activation protein; Membrane; Methodology; Microscopic; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Myocardial Infarction; Nature; Numbers; Organogenesis; Outer Mitochondrial Membrane; Pathologic Processes; Pathology, Other; Pathway interactions; Permeability; Physiological; Play; Process; Property; Protein Family; Proteins; Protocols documentation; Regulation; Research Personnel; Rest; Role; Serum; Signal Transduction; Signaling Protein; Staurosporine; Stroke; Techniques; Testing; Tissues; Withdrawal; chemotherapy; cytochrome c; insight; member; membrane model; mitochondrial membrane; novel; novel therapeutics; patch clamp; prevent; programs; reconstitution; research study; response; therapeutic target; tumor

DESCRIPTION (provided by applicant): The release of cytochrome c from mitochondria is often recognized as the commitment step of apoptosis. The mitochondrial apoptosis-induced channel (MAC) forms early in apoptosis in hematopoietic cells, concurrent with Bax translocation into mitochondria. Our data show MAC could provide a pathway for cytochrome c to exit mitochondria and over-expression of anti-apoptotic Bcl-2 suppresses this channel activity. There is a gap in knowledge defining the relationship between BCL-2 family proteins and MAC, e.g. are Bak and Bax components of MAC? The hypothesis of this proposal is that MAC provides a pathway for cytochrome c, and perhaps other proteins in the mitochondrial intermembrane space, to exit the mitochondria during apoptosis and MAC is a critical target for regulation of the mitochondrial apoptotic response by members of the BCL-2 family of proteins. This proposal is aimed at testing this hypothesis using a combination of electrophysiological, molecular biological and microscopic techniques. Aim 1 is to systematically characterize the channel properties of MAC and its permeability to cytochrome c, thereby testing whether MAC can permit the release of cytochrome c from mitochondria early in apoptosis. We plan to determine if cytochrome c transports through MAC using electrophysiology and ELISA. Aim 2 is to determine if pro-apoptotic Bax and Bak are integral, structural components of MAC, and will be tested, in part, by assessing MAC activity in mitochondria of cells that are knockouts for Bax and/or Bak, which will also determine if there is more than one kind of MAC activity. Aim 3 is to determine the mechanism(s) by which pro-apoptotic BH3-only and anti-apoptotic Bcl-2/Bcl-xL proteins regulate MAC formation. We hypothesize that pro- and anti-apoptotic proteins act by antagonistically regulating formation of MAC. We plan to determine if BH3-only proteins promote the formation of MAC and if anti-apoptotic BCL2 family members can inhibit the formation or activity of MAC. One approach will be to assess the development of MAC activity while patch-clamping mitochondria in the presence of Bcl-2/Bcl-xL and/or representative BH3-only proteins. These experiments will provide fundamental insights into the nature of mitochondrial involvement in apoptosis and the regulation by BCL-2 family proteins. These experiments may identify novel therapeutic targets, e.g., MAC, to modulate apoptosis in cancer and other pathologies, e.g., myocardial infarction.

描述（由申请人提供）：细胞色素c从线粒体的释放通常被认为是细胞凋亡的承诺步骤。线粒体凋亡诱导通道（MAC）在造血细胞凋亡早期形成，与Bax易位进入线粒体同时发生。我们的数据显示MAC可以提供细胞色素c退出线粒体的途径，并且抗凋亡Bcl-2的过表达抑制了该通道的活性。在定义BCL-2家族蛋白和MAC之间的关系方面存在知识空白，例如MAC的巴克和Bax组分是吗？该提议的假设是MAC为细胞色素c以及线粒体膜间隙中的可能其他蛋白质提供了在细胞凋亡期间离开线粒体的途径，并且MAC是BCL-2蛋白质家族成员调节线粒体细胞凋亡反应的关键靶标。该建议旨在使用电生理学、分子生物学和显微镜技术相结合来检验这一假设。目的1是系统地表征MAC的通道特性及其对细胞色素c的渗透性，从而测试MAC是否可以允许细胞色素c在细胞凋亡早期从线粒体释放。我们计划使用电生理学和ELISA来确定细胞色素c是否通过MAC转运。目的2是确定促细胞凋亡Bax和巴克是否是MAC的完整的结构组分，并且将部分地通过评估Bax和/或巴克敲除的细胞的线粒体中的MAC活性来测试，这也将确定是否存在多于一种MAC活性。目的3是确定促凋亡BH 3-only和抗凋亡Bcl-2/Bcl-xL蛋白调节MAC形成的机制。我们推测，促凋亡和抗凋亡蛋白的作用，拮抗调节MAC的形成。我们计划确定BH 3-only蛋白是否促进MAC的形成，以及抗凋亡BCL 2家族成员是否可以抑制MAC的形成或活性。一种方法是在存在Bcl-2/Bcl-xL和/或代表性仅含BH 3的蛋白质的情况下，在膜片钳线粒体时评估MAC活性的发展。这些实验将提供基本的见解线粒体参与细胞凋亡的性质和BCL-2家族蛋白的调节。这些实验可以鉴定新的治疗靶点，例如，MAC，以调节癌症和其他病理学中的细胞凋亡，例如，心肌梗死