Novel CD95 Signaling Mechanisms

新型 CD95 信号传导机制

• 批准号: 7098097
• 负责人: Marcus E. Peter
• 金额: $ 26.51万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-27 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7098097
• 关键词: CD95 molecule; actins; apoptosis; biological signal transduction; clinical research; cysteine endopeptidases; enzyme activity; enzyme induction /repression; human subject; protein protein interaction; receptor binding; receptor expression

DESCRIPTION (provided by applicant): Apoptosis is the physiological form of cell death involved in homeostasis of tissues and is dysregulated in numerous diseases. The death receptor CD95 (APO-1/Fas) induces apoptosis by forming a death-inducing signaling complex (DISC) comprised of CD95, the adaptor molecule FADD, caspases 8 and 10, and c-FLIP. In Type I cells (cells that die in a mitochondria-independent manner such as T cells) triggering CD95 with either CD95 ligand (CD95L) or an anti-CD95 antibody induces the receptor to form clusters at the cell surface. Formation of these clusters is dependent on a small amount of DISC-generated active caspase-8. We hypothesize that active caspase-8 generated at the DISC is part of a positive feedback loop and that activation of caspase-8 by other means such as by other death receptors or certain antitumor drugs can induce the clustering of CD95. We recently showed for the first time that after activation, the CD951CD95L complex is internalized through an endosomal pathway in an actin-dependent fashion. Inhibition of filamentous actin with latrunculin A in Type I cells strongly reduces DISC formation and sensitivity to CD95 apoptosis whereas Type II cells which do not internalize CD95 form very little DISC. Depending on the situation, CD95 can also activate prosurvival pathways which depend on activation of caspase-8. Induction of these protective pathways is completely abrogated when actin filaments and internalization of CD95 is blocked. We hypothesize that the act independent internalization of CD95 is required for efficient formation of the DISC and signaling through CD95 and that internalization of CD95 transports and directs signaling molecules to specific intracellular compartments activating apoptosis or prosurvival pathways depending on the cell. To test these hypotheses we propose to study the following Specific Aims using biochemical, cell biological, and molecular biological techniques: 1) Characterize the function of caspase-8 in the initiation of CD95 signaling. We will identify the caspase-8 target that regulates CD95 signal initiation and characterize its function. 2) Determine the role of the actin cytoskeleton in CD95 signaling. We will identify how CD95 is linked to the actin cytoskeleton and study the mechanism of actin regulation of CD95 signaling and internalization. 3) Determine the significance of receptor internalization for CD95 signaling. We will identify the route of internalization of the CD95/CD95L complex in apoptosing and nonapoptosing cells. We will test whether internalized DISC generates different apoptosis dependent and independent signals when compared to surface DISC. The results of this study will provide key insights into understanding how death receptors initiate death and prosurvival signals which could be the basis to interfere with death receptor mediated signaling in diseases with dysregulated apoptosis.

描述（由申请人提供）：细胞凋亡是涉及组织稳态的细胞死亡的生理形式，并且在许多疾病中失调。死亡受体 CD95 (APO-1/Fas) 通过形成由 CD95、接头分子 FADD、半胱天冬酶 8 和 10 以及 c-FLIP 组成的死亡诱导信号复合物 (DISC) 来诱导细胞凋亡。在 I 型细胞（以不依赖线粒体的方式死亡的细胞，例如 T 细胞）中，用 CD95 配体 (CD95L) 或抗 CD95 抗体触发 CD95 会诱导受体在细胞表面形成簇。这些簇的形成取决于 DISC 生成的少量活性 caspase-8。我们假设 DISC 产生的活性 caspase-8 是正反馈环的一部分，并且通过其他方式（例如其他死亡受体或某些抗肿瘤药物）激活 caspase-8 可以诱导 CD95 聚集。我们最近首次表明，激活后，CD951CD95L 复合物以肌动蛋白依赖性方式通过内体途径内化。在 I 型细胞中用 latrunculin A 抑制丝状肌动蛋白会强烈降低 DISC 的形成和对 CD95 凋亡的敏感性，而不内化 CD95 的 II 型细胞则形成很少的 DISC。根据情况，CD95还可以激活依赖于caspase-8激活的促生存途径。当肌动蛋白丝和 CD95 的内化被阻断时，这些保护途径的诱导被完全消除。我们假设 CD95 的行为独立内化对于 DISC 的有效形成和通过 CD95 的信号传导是必需的，并且 CD95 的内化将信号分子转运和引导至特定的细胞内区室，根据细胞激活细胞凋亡或促生存途径。为了检验这些假设，我们建议使用生化、细胞生物学和分子生物学技术研究以下具体目标：1) 表征 caspase-8 在 CD95 信号传导启动中的功能。我们将鉴定调节 CD95 信号起始的 caspase-8 靶点并表征其功能。 2)确定肌动蛋白细胞骨架在CD95信号传导中的作用。我们将确定 CD95 如何与肌动蛋白细胞骨架连接，并研究肌动蛋白调节 CD95 信号传导和内化的机制。 3) 确定受体内化对于 CD95 信号传导的重要性。我们将确定 CD95/CD95L 复合物在凋亡和非凋亡细胞中内化的途径。我们将测试与表面 DISC 相比，内化 DISC 是否产生不同的细胞凋亡依赖性和独立信号。这项研究的结果将为理解死亡受体如何启动死亡和促生存信号提供关键见解，这可能是干扰细胞凋亡失调疾病中死亡受体介导的信号传导的基础。