Structure and Function of the Death Effector FADD

本研究将研究肿瘤坏死因子受体家族（TNFRs）的一个亚类，称为死亡受体（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端结构域由一个保守的六螺旋束组成，称为死亡结构域（DD）。DD形成多蛋白复合体的细胞核，PCD机制的酶和调节成分聚集在一起，将死亡刺激转化为生化反应。DDs既存在于受体中，也存在于死亡复合体的调节成分，即死亡效应器中。几乎所有的死亡信号都是通过FADD进行的。配体结合后，死亡受体招募FADD和启动物caspase FLICE/caspase-8，启动PCD。FADD由两个蛋白质相互作用基序组成，DD和第二个基序称为死亡效应域（DED）。为了确定死亡受体/FADD相互作用的结构，需要在溶液中确定FADD （208 a.a.）的三维结构，并通过位点定向诱变和体外信号复合体重建来鉴定三种死亡受体（fas、DR5和DR3）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结构域沿两个结构域的连续表面。该表面与果蝇管（一种与PCD无关的含有DD的蛋白质）的DD相互作用表面非常相似。这表明含dd蛋白的结合机制是保守的。合成了一种新的死亡受体模拟物，在体外重建信号复合物。该模拟物将使用凝胶过滤、分析离心和诱变相结合的方法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进入受体/FADD复合物的分子机制也有待确定，并提供了初步证据来确定FLICE/caspase-8的FADD结合表面。最后，将对FADD功能的脑特异性拮抗剂PEA-15的作用进行表征。PEA-15的结构及其在信号复合体中与FADD的结合机制将被确定。这些努力的总和将提供导致死亡的信号复合体的结构的第一个全面的图片。本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器来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.