Structure and Function of the Death Effector FADD

死亡效应器FADD的结构和功能

• 批准号: 6621724
• 负责人: Milton H. Werner
• 金额: $ 25.05万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621724
• 关键词: Drosophilidae; analytical ultracentrifugation; apoptosis; astrocytes; biological signal transduction; cysteine; cysteine endopeptidases; cytokine receptors; gel filtration chromatography; ligands; mitogen activated protein kinase; neoplasm /cancer; nuclear magnetic resonance spectroscopy; phosphoproteins; physical model; protein isoforms; protein protein interaction; protein structure function; receptor binding; recombinant proteins; serine; site directed mutagenesis; stoichiometry; tumor necrosis factor alpha

This proposal will investigate how a subclass of the tumor necrosis factor receptor family (TNFRs), termed death receptors (DRs), assembles a stereospecific complex at the cytoplasmic surface to initiate programmed cell death (PCD). The C-terminal domain of DRs is composed of a conserved six helix bundle known as the death domain (DD). The DD forms the nucleus of a multiprotein complex about which the enzymatic and regulatory components of the PCD machinery assemble to transduce a death stimulus into a biochemical response. DDs are present in both the receptor and its regulatory components, a.k.a. death effectors, of the death complex. The death effector through which nearly all death signals proceed is FADD. Upon ligand binding, a death receptor recruits FADD and the initiator caspase, FLICE/caspase-8, to initiate PCD. FADD is comprised of two protein interaction motifs, the DD and a second motif known as the death effector domain (DED). To define the architecture of death receptor/FADD interaction, the three dimensional structure of FADD (208 a.a.) is to be determined in solution and its binding surfaces for three death receptors-Fas, DR5 and DR3- are to be identified by site-directed mutagenesis and reconstitution of the signaling complex in vitro. The preliminary, structure and biochemical experiments demonstrate that the Fas/FADD interaction is dependent on both the DD and DED domains of FADD along a contiguous surface which traverses both domains. This surface is remarkably similar to the DD interaction surface of Drosophila Tube, a DD-containing protein unrelated to PCD. This suggests that the binding mechanism of DD-containing proteins is conserved. A novel death receptor mimic has been synthesized to reconstitute the signaling complex in vitro. This mimic will be used to identify the binding surfaces and specificity determinants for all three death receptor/FADD interactions using a combination of gel filtration, analytical centrifugation and mutagenesis. The molecular mechanism for FLICE/caspase-8 recruitment into the receptor/FADD complex is also to be determined and preliminary evidence is presented to define the FADD binding surface for FLICE/caspase-8. Finally, the action of a brain-specific antagonist of FADD function, PEA-15, will be characterized. The structure of PEA-15 and its binding mechanism to FADD in the signaling complex will be determined. The sum of these efforts will provide the first comprehensive picture of the architecture of a death-inducing signaling complex. The outcome of this study can form the basis for understanding how different death receptors use their cognate effectors to activate a specific death response.

该提案将研究肿瘤坏死因子受体家族 (TNFR) 的一个亚类（称为死亡受体 (DR)）如何在细胞质表面组装立体特异性复合物以启动程序性细胞死亡 (PCD)。 DR 的 C 端结构域由称为死亡结构域 (DD) 的保守六螺旋束组成。 DD 形成多蛋白复合物的核心，PCD 机器的酶促和调节成分在该复合物周围组装，将死亡刺激转导为生化反应。 DD 存在于死亡复合体的受体及其调节成分（又称为死亡效应器）中。 几乎所有死亡信号通过的死亡效应器是 FADD。 配体结合后，死亡受体招募 FADD 和引发剂 caspase FLICE/caspase-8，以启动 PCD。 FADD 由两个蛋白质相互作用基序组成，即 DD 和称为死亡效应结构域 (DED) 的第二个基序。 为了定义死亡受体/FADD相互作用的结构，将在溶液中测定FADD (208 a.a.)的三维结构，并通过定点鉴定其与三种死亡受体——Fas、DR5和DR3——的结合表面。体外信号复合物的诱变和重建。 初步的结构和生化实验表明，Fas/FADD 相互作用依赖于 FADD 的 DD 和 DED 结构域，沿着穿过两个结构域的连续表面。 该表面与果蝇管的 DD 相互作用表面非常相似，果蝇管是一种与 PCD 无关的含有 DD 的蛋白质。 这表明含有DD的蛋白质的结合机制是保守的。 一种新型死亡受体模拟物已被合成，可在体外重建信号复合物。 该模拟物将用于结合凝胶过滤、分析离心和诱变来识别所有三种死亡受体/FADD 相互作用的结合表面和特异性决定因素。 FLICE/caspase-8 招募到受体/FADD 复合物的分子机制也有待确定，并提供初步证据来定义 FLICE/caspase-8 的 FADD 结合表面。最后，将表征 FADD 功能的大脑特异性拮抗剂 PEA-15 的作用。 将确定 PEA-15 的结构及其与信号复合物中 FADD 的结合机制。 这些努力的总和将提供死亡诱导信号复合体架构的第一张全面图景。 这项研究的结果可以为理解不同的死亡受体如何利用其同源效应器激活特定的死亡反应奠定基础。