Mitochrondrial Regulation of Apoptosis

细胞凋亡的线粒体调控

• 批准号: 8246121
• 负责人: TOMOMI KUWANA
• 金额: $ 24.32万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8246121
• 关键词: Mitochrondrial; Regulation; Apoptosis

DESCRIPTION (provided by applicant): The mitochondrial pathway of apoptosis is the major mechanism whereby cells in vertebrate organisms undergo programmed cell death. It is vital for homeostasis of the immune system, tumor suppression and the regulation of cell number in development. Mitochondrial outer membrane permeabilization (MOMP) and release of intermembrane space proteins such as cytochrome C are central to this process. MOMP is controlled by the Bcl-2 family of proteins. Pro-apoptotic members, Bax and Bak, are activated by another subgroup of the Bcl-2 family, BH3-only proteins, and induce the release of intermembrane space proteins. Anti-apoptotic members of the Bcl-2 family inhibit this process. Based on the finding that BH3-only proteins bind to anti-apoptotic Bcl-2 family members to induce apoptosis, drugs that mimic this effect were developed and are being used in cancer therapy. Better understanding of how Bcl-2 family proteins interact with the MOM and induce permeabilization will allow generation of new or even better drugs. The central hypothesis of this proposal is that there are MOM proteins that co- operate with pro-apoptotic Bcl-2 family proteins to generate lipidic pores in the MOM. The object of this application is to identify these molecules and to determine how they contribute to Bax and Bak activation followed by pore formation. Our Specific Aims are: Aim 1. Identify MOM proteins that co-operate with Bid for induction of MOMP. Unknown MOM proteins assist Bid in Bax mediated-permeabilization of membranes. MOM proteins will be fractionated and reconstituted in proteo-liposomes to identify the active fraction by the dextran release assay. We will investigate whether the identified MOM protein knockdown has the same inhibitory effect as Bid deficiency in mouse embryonic fibroblast (MEF) lines. Aim 2. Determine how Bax activation is regulated prior to MOMP. Using a dextran release kinetics assay, we will determine the time course of Bax conformational change and oligomerization in relation to membrane permeabilization. We will also determine the mechanism of Bcl-xL inhibition by analysis of its effect on release kinetics. We will follow up these experiments by examining cytochrome C release kinetics using mitochondria isolated from cytochrome C-GFP expressing cells in time lapse fluorescent microscopy. Aim 3. Demonstrate lipidic pores induced by Bax and Bak and determine whether Bax localizes to these pores. Our cryo-EM data suggest that Bax generates lipidic pores in the membrane. We will use high-pressure freezing/freeze substitution-EM to demonstrate the existence of these pores in OMVs and mitochondria both in isolation and in cells. This technique will allow us to detect pores with immunogold or nanogold labeled Bax. Mapping the distribution of Bax along the edge of the pores will enable us to determine whether Bax pores are mainly lipidic or proteinaceous. PUBLIC HEALTH RELEVANCE: Apoptosis is a fundamental biological process whereby cells execute their own death upon receiving apoptotic signals. Mitochondria, an organelle that sustains life, is also found to play a major role in apoptosis by releasing apoptogenic proteins, which is regulated by a group of proteins in the Bcl-2 family. The proposed study will increase our understanding of molecular mechanisms that underlie mitochondrial outer membrane permeabilization, and enable us to develop more effective therapeutic strategies for diseases such as cancer.

描述（由申请人提供）：凋亡的线粒体途径是脊椎动物生物体中细胞经历程序性细胞死亡的主要机制。这对于免疫系统的稳态，肿瘤抑制和发育中细胞数的调节至关重要。线粒体外膜透化（MOMP）和膜间空间蛋白（例如细胞色素c）的释放是该过程的核心。 MOMP由BCL-2蛋白质家族控制。促凋亡的成员Bax和Bak被BCl-2家族的另一个亚组，仅BH3蛋白激活，并诱导膜间空间蛋白的释放。 Bcl-2家族的抗凋亡成员抑制了这一过程。基于以下发现，仅BH3蛋白与抗凋亡Bcl-2家族成员结合以诱导细胞凋亡，因此开发了这种作用并用于癌症治疗中。更好地了解Bcl-2家族蛋白如何与妈妈相互作用并诱导透化将允许产生新的甚至更好的药物。该提议的中心假设是，有一些与培养基Bcl-2家族蛋白共同运行的MOM蛋白质在MOM中产生脂质毛孔。该应用的目的是识别这些分子，并确定它们如何促进Bax和Bak激活，然后是孔形成。我们的具体目的是：目标1。确定与竞标MOMP合作的MOM蛋白质。未知的MOM蛋白有助于竞标膜的Bax介导的渗透性。 MOM蛋白将在蛋白 - 脂质体中分离并重构，以通过葡聚糖释放测定法识别活性分数。我们将研究所鉴定的MOM蛋白敲低是否具有与小鼠胚胎成纤维细胞（MEF）系中的出价缺乏相同的抑制作用。 AIM 2。确定在MOMP之前如何调节BAX激活。使用葡聚糖释放动力学测定，我们将确定与膜通透性相关的BAX构象变化和低聚的时间过程。我们还将通过分析其对释放动力学的影响来确定BCL-XL抑制的机制。我们将通过使用从细胞色素c-GFP中分离出的线粒体在延时荧光显微镜下从细胞色素c-GFP中分离出的线粒体来跟踪这些实验。 AIM 3。证明由Bax和Bak诱导的脂质孔，并确定Bax是否定位在这些孔中。我们的冷冻EM数据表明，BAX在膜上产生脂质孔。我们将使用高压冻结/冻结替代-EM来证明这些孔在OMV和线粒体中的存在和细胞中的存在。这项技术将使我们能够检测具有免疫金或标记为Bax的纳米质量的毛孔。映射沿孔边缘的BAX的分布将使我们能够确定Bax孔是否主要是脂质或蛋白质。 公共卫生相关性：细胞凋亡是一个基本的生物学过程，细胞在接受凋亡信号后会执行自己的死亡。线粒体是一种维持生命的细胞器，还发现通过释放凋亡蛋白在凋亡中起主要作用，该蛋白质受Bcl-2家族中的一组蛋白质调节。拟议的研究将增加我们对线粒体外膜透化基础的分子机制的理解，并使我们能够为癌症等疾病制定更有效的治疗策略。