Structure and Function of the Death Effector FADD

本研究将探讨肿瘤坏死因子受体家族（TNFRs）的一个亚类，即死亡受体（DRs）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-末端结构域由称为死亡结构域（DD）的保守的六螺旋束组成。 DD形成多蛋白复合物的核，PCD机制的酶和调节组分围绕该复合物组装以将死亡刺激转化为生化反应。 DD存在于受体及其调节组分中，也称为死亡复合体的死亡效应子 几乎所有的死亡信号都是通过死亡效应器进行的。 在配体结合后，死亡受体募集FADD和引发剂半胱天冬酶FLICE/半胱天冬酶-8以引发PCD。 FADD由两个蛋白质相互作用基序组成，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FADD（208 a.a.）在溶液中测定，并通过体外定点诱变和信号复合物的重建来鉴定其与三种死亡受体Fas、DR 5和DR 3的结合表面。 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DD蛋白。 这表明含DD蛋白的结合机制是保守的。 合成了一种新型的死亡受体模拟物，在体外重建信号复合物。 该模拟物将用于鉴定结合表面和特异性决定簇，用于所有三种死亡受体/FADD相互作用，使用凝胶过滤、分析离心和诱变的组合。 FLICE/caspase-8招募到受体/FADD复合物的分子机制也有待确定，并提出初步证据来定义FLICE/caspase-8的FADD结合表面。最后，将表征FADD功能的脑特异性拮抗剂PEA-15的作用。 将确定PEA-15的结构及其在信号传导复合物中与FADD的结合机制。 这些努力的总和将提供第一个全面的图片结构的死亡诱导信号复合物。 这项研究的结果可以为理解不同的死亡受体如何使用其同源效应子激活特定的死亡反应奠定基础。

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.