Novel CD95 Signaling Mechanisms

描述（由申请人提供）：细胞凋亡是参与组织稳态的细胞死亡的生理形式，在许多疾病中失调。死亡受体CD 95（APO-1/Fas）通过形成由CD 95、衔接分子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（以非依赖性方式死亡的细胞，如T细胞）中，用CD 95配体（CD 95 L）或抗CD 95抗体触发CD 95诱导受体在细胞表面形成簇。这些簇的形成依赖于少量DISC产生的活性半胱天冬酶-8。我们假设在DISC产生的活性半胱天冬酶-8是正反馈环的一部分，并且通过其他方式如通过其他死亡受体或某些抗肿瘤药物激活半胱天冬酶-8可以诱导CD 95的聚集。我们最近首次表明，激活后，CD 951 CD 95 L复合物是通过内体途径在肌动蛋白依赖的方式内化。在I型细胞中用latrunculin A抑制丝状肌动蛋白强烈降低DISC形成和对CD 95凋亡的敏感性，而不内化CD 95的II型细胞形成非常少的DISC。根据情况，CD 95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CD 95的内化被阻断时，这些保护途径的诱导被完全废除。我们假设CD 95的行为独立内化是有效形成DISC和通过CD 95进行信号传导所必需的，并且CD 95的内化将信号传导分子转运并引导至特定的细胞内隔室，激活细胞凋亡或促生存途径，这取决于细胞。为了验证这些假设，我们建议使用生物化学、细胞生物学和分子生物学技术研究以下具体目的：1）表征caspase-8在CD 95信号传导起始中的功能。我们将鉴定调节CD 95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CD 95信号传导中的作用。我们将确定CD 95是如何连接到肌动蛋白细胞骨架，并研究肌动蛋白调节CD 95信号转导和内化的机制。3)确定受体内化对CD 95信号传导的意义。我们将确定CD 95/CD 95 L复合物在趋化和非趋化细胞中的内化途径。我们将测试与表面DISC相比，内化的DISC是否产生不同的凋亡依赖性和独立信号。本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，这可能是干扰凋亡失调疾病中死亡受体介导的信号传导的基础。

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.