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.

该建议将研究称为死亡受体（DRS）的肿瘤坏死因子受体家族（TNFR）的子类如何在细胞质表面组装一个立体特异性复合物来启动程序性细胞死亡（PCD）。 DRS的C末端结构域由一个保守的六个螺旋束组成，称为死亡结构域（DD）。 DD形成了多蛋白复合物的核，围绕PCD机械的酶促和调节成分组装，将死亡刺激转化为生化反应。 DD在受体及其调节成分（又称死亡效应者）中都存在。 几乎所有死亡信号进行的死亡效应子是FADD。 在配体结合后，死亡受体会募集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和第二个被称为死亡效应域（DED）的基序。 为了定义死亡受体/FADD相互作用的结构，应在溶液中确定FADD的三维结构（208 A.A.），其结合表面的三个死亡受体Fas-fas，Dr5和Dr3-应通过位置定向的诱变，并重新确定信号传导复合物在Vitro中。 初步，结构和生化实验表明，FAS/FADD相互作用取决于FADD的DD和DED域沿着连续的表面，该域遍布两个域。 该表面与果蝇管的DD相互作用表面非常相似，果蝇管的DD相互作用表面与PCD无关。 这表明含DD的蛋白质的结合机制是保守的。 一种新型的死亡受体模拟物已合成以重建体外信号传导复合物。 该模拟物将用于确定所有三种死亡受体/FADD相互作用的结合表面和特异性决定因素，结合凝胶过滤，分析离心和诱变。 还应确定将果/caspase-8募集到受体/FADD复合物中的分子机制，并提供初步证据，以定义FLICE/CASPASE-8的FADD结合表面。最后，将表征FADD功能的脑特异性拮抗剂PEA-15的作用。 将确定PEA-15及其与信号传导复合物中FADD的结合机理的结构。 这些努力的总和将为诱发死亡的信号复合体的结构提供第一张全面的图片。 这项研究的结果可以构成理解不同死亡受体如何使用其同源效应子激活特定死亡反应的基础。