Structural/Biochemical Analysis: Apoptosis in C. elegans

描述（由申请人提供）：细胞凋亡在所有多细胞生物体的发育和体内平衡中起核心作用。细胞凋亡途径的改变与包括癌症在内的许多使人衰弱的疾病有关。罗伯特·霍维茨（Robert Horvitz）开创的遗传学研究已经鉴定出四种控制模式生物秀丽隐杆线虫（Caushabditis elegans）细胞凋亡发生的基因。Egl 1、CED 9、CED 4和CED 3这四个基因的蛋白产物以线性途径作用以执行细胞死亡，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参与这一范例已经启动。已经取得了重大进展，这里提出的工作将建立在以下具体目标的初步结果：（1）Egl 1-CED 9复合物的结构和功能分析。Egll与CED 9的结合触发CED 4从抗凋亡蛋白CED 9释放。已经获得了Egl 1-CED 9复合物的晶体，其将X射线衍射至2.2埃的分辨率。通过分子置换确定了结构;正在进行改进。与Robert Horvitz和Ding Xue的实验室合作，正在进行基于结构的功能分析。(2)CED 9-CED 4复合物的结构的测定。CED 4在细胞凋亡之前作为非活性复合物与CED 9保持组成性相关。已经表征了全长CED 4和CED 9的大功能结构域之间的二元复合物。最近获得了小晶体。结构将由多波长反常色散（MAD）确定。(3)CED 4介导的CED 3活化的生物化学表征。(4)分离的寡聚CED 4的结构的测定。Egll对CED 9-CED 4异源二聚体的破坏导致CED 4的寡聚化，这对于CED 3的募集和活化是必需的。(5)CED 4-CED 3复合物的结构的测定。CED 3与寡聚CED 4形成全酶。CED 4-CED 3复合物已在体外重构。配合物将被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.