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动态平衡中起着核心作用。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。由Robert Horvitz开创的遗传学研究已经确定了四个控制模式生物秀丽线虫细胞凋亡开始的基因。这四个基因的蛋白质产物Egl1、CED9、CED4和CED3以线性方式作用于细胞死亡，构成了理解细胞凋亡的经典范例。已经开始对这一范例中涉及的蛋白质复合体进行系统的生化和X射线结晶学分析。已经取得了重大进展；这里提出的工作将以初步结果为基础，具体目标如下：(1)Egl1-CED9复合体的结构和功能分析。Egl1与CED9的结合触发抗凋亡蛋白CED9释放CED4。获得了X射线衍射分辨率为2.2埃的Egl1-CED9络合物晶体。结构是通过分子置换确定的；精细化正在进行中。与罗伯特·霍维茨和丁雪的实验室合作，基于结构的功能分析正在进行中。(2)CED9-CED4络合物的结构测定。CED4与CED9在细胞凋亡前仍是一种不活跃的复合体。研究了CED4全长与CED9大功能结构域之间的二元复合体。最近已经获得了小晶体。结构将由多波长反常色散(MAD)决定。(3)CED4介导的CED3激活的生化特性。(4)分离鉴定低聚体CED4的结构。Egl1对CED9-CED4异源二聚体的破坏导致CED4的寡聚，这是CED3募集和激活所必需的。(5)CED4-CED3络合物的结构测定。CED3与寡聚体CED4形成全酶。CED4-CED3络合物已在体外重组。该络合物将被结晶，其三维结构将被确定。

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.