Novel CD95 Signaling Mechanisms

描述(申请人提供)：细胞凋亡是细胞死亡的一种生理形式，与组织的动态平衡有关，在许多疾病中调节失调。死亡受体CD95(APO-1/Fas)通过形成由CD95、接头分子FADD、caspase8和10以及c-flip组成的死亡诱导信号复合体(DISC)来诱导细胞凋亡。在I型细胞(以线粒体无关的方式死亡的细胞，如T细胞)中，触发带有CD95配体(CD95L)或抗CD95抗体的CD95会诱导受体在细胞表面形成簇。这些簇的形成依赖于少量盘状生成的活性caspase-8。我们假设，在椎间盘上产生的活性caspase-8是正反馈环路的一部分，通过其他方式激活caspase-8，如由其他死亡受体或某些抗肿瘤药物激活，可以诱导CD95的聚集。我们最近首次发现，CD951CD95L复合体在激活后，以肌动蛋白依赖的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显著减少盘状细胞的形成和对CD95凋亡的敏感性，而不内化CD95的II型细胞形成的盘状细胞很少。根据情况，CD95也可以激活依赖于caspase-8激活的生存通路。当肌动蛋白细丝和CD95的内化被阻断时，这些保护通路的诱导完全被取消。我们假设CD95的行为独立内化是有效形成视盘和通过CD95传递信号所必需的，并且CD95的内化转运和引导信号分子到特定的细胞内间隔，激活依赖于细胞的凋亡或生存途径。为了验证这些假设，我们建议使用生化、细胞生物学和分子生物学技术来研究以下特定目的：1)表征caspase-8在CD95信号起始中的功能。我们将确定调控CD95信号启动的caspase-8靶点并研究其功能。2)确定肌动蛋白细胞骨架在CD95信号转导中的作用。我们将确定CD95是如何与肌动蛋白细胞骨架相连的，并研究肌动蛋白调节CD95信号转导和内化的机制。3)确定受体内化对CD95信号转导的意义。我们将确定CD95/CD95L复合体在凋亡和非凋亡细胞中内化的途径。我们将测试内在化视盘与表面化视盘是否产生不同的凋亡依赖和独立信号。这项研究的结果将为理解死亡受体如何启动死亡和存活信号提供关键的见解，这可能是干扰死亡受体介导的信号在细胞凋亡异常的疾病中的基础。

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.