Structural/Biochemical Analysis: Apoptosis in C. elegans

描述（由申请人提供）：凋亡在所有多细胞生物的发育和体内平衡中起着核心作用。凋亡通路的改变与包括癌症在内的许多使人衰弱的人类疾病有关。罗伯特·霍维茨（Robert Horvitz）率先进行的遗传学研究发现，在模式生物秀丽隐杆线虫（Caenorhabditis elegans）中，有四个基因控制着细胞凋亡的发生。这四个基因的蛋白产物Egl1、CED9、CED4和CED3以线性途径执行细胞死亡，构成了理解细胞凋亡的经典范式。系统的生化和x射线晶体学分析的蛋白质复合物涉及这种模式已经开始。取得了重大进展；本文提出的工作将以初步结果为基础，具体目标如下：(1)Egl1-CED9复合物的结构和功能分析。Egl1与CED9结合可触发抗凋亡蛋白CED9释放CED4。已经获得了Egl1-CED9配合物的晶体，其衍射x射线的分辨率为2.2埃。通过分子置换确定结构；改进正在进行中。与Robert Horvitz和Ding Xue的实验室合作，基于结构的功能分析正在进行中。(2)确定CED9-CED4复合物的结构。在细胞凋亡之前，CED4作为一种无活性复合物与CED9保持组成性关联。在全长CED4和大功能域CED9之间的二元配合物已经被表征。小晶体是最近获得的。结构将由多波长反常色散（MAD）来确定。(3) ced4介导的CED3活化的生化表征。(4)分离测定低聚物CED4的结构。Egl1破坏CED9-CED4异二聚体导致CED4的寡聚化，这对CED3的募集和激活至关重要。(5)确定CED4-CED3配合物的结构。CED3与低聚物CED4形成全酶。体外重建了一个CED4-CED3复合物。该配合物将结晶并确定其三维结构。

DESCRIPTION (provided by applicant): Apoptosis plays a central role in the development and homeostasis of all multi-cellular organisms. Alterations in apoptotic pathways have been implicated in many debilitating human diseases including cancer. Genetic studies pioneered by Robert Horvitz have led to the identification of four genes that control the onset of apoptosis in the model organism Caenorhabditis elegans. The protein products of these four genes, Egl1, CED9, CED4, and CED3 act in a linear pathway to execute cell death and constitute a classic paradigm for the understanding of apoptosis. Systematic biochemical and X-ray crystallographic analyses of protein complexes involved in this paradigm have been initiated. Significant progress has been achieved; the work proposed here will build on the preliminary results with the following specific aims: (1) Structural and functional analysis of an Egl1-CED9 complex. Binding of Egl1 to CED9 triggers the release of CED4 from the anti-apoptotic protein CED9. Crystals of the Egl1-CED9 complex that diffract X-rays to 2.2 Angstroms resolution have been obtained. The structure was determined by molecular replacement; refinement is in progress. In collaboration with the laboratories of Robert Horvitz and Ding Xue, structure-based functional analysis is underway. (2) Determination of the structure of a CED9-CED4 complex. CED4 remains constitutively associated with CED9 as an inactive complex prior to apoptosis. A binary complex between the full-length CED4 and a large functional domain of CED9 has been characterized. Small crystals have been obtained most recently. The structure will be determined by multi-wavelength anomalous dispersion (MAD). (3) Biochemical characterization of CED4-mediated activation of CED3. (4) Determination of the structure of oligomeric CED4 in isolation. The disruption of a CED9-CED4 hetero-dimer by Egl1 results in the oligomerization of CED4, which is essential for the recruitment and activation of CED3. (5) Determination of the structure of a CED4-CED3 complex. CED3 forms a holoenzyme with oligomeric CED4. A CED4-CED3 complex has been reconstituted in vitro. The complex will be crystallized and its three-dimensional structure will be determined.