STRUCTURAL BIOLOGY OF CED-9-MEDIATED SUPPRESSION OF CED-4

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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。这四个基因的蛋白产物EGL-1、CED-9、CED-4和CED-3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。尽管有遗传学研究，但Egl1、CED9、CED4和CED3控制细胞凋亡的分子机制仍不清楚。为了实现这些目标，我们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。CED4与CED9在细胞凋亡前仍是一种不活跃的复合体。我们已经表征了CED4全长和CED9的一个大功能结构域之间的二元复合体。我们最近在两个不同的空间群中获得了这种络合物的晶体。一种晶型对X射线有微弱的衍射作用。另一种晶型的最佳晶体在国际象棋A-1站的X射线衍射率约为3.5°。我们想在CHESS A-1站收集一个本地数据集，并试图通过分子置换来解决结构问题，因为CED-9的原子坐标是可用的。这一结构将揭示CED-9对CED-4的重要调控机制，并对我们理解线虫和哺乳动物的凋亡途径具有重要影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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, EGL-1, CED-9, CED-4, and CED-3 act in a linear pathway to execute cell death and constitute a classic paradigm for the understanding of apoptosis. Despite genetic studies, the molecular mechanisms by which Egl1, CED9, CED4, and CED3 control the initiation of apoptosis remain largely unknown. Towards these goals, we have initiated systematic biochemical and X-ray crystallographic analyses of protein complexes involved in this paradigm. CED4 remains constitutively associated with CED9 as an inactive complex prior to apoptosis. We have characterized a binary complex between the full-length CED4 and a large functional domain of CED9. We recently obtained crystals of this complex in two different spacegoups. One crystal form diffracts X-ray weakly. The best crystals of the other crystal form diffract X-ray to about 3.5 ¿ at CHESS A-1 station. We would like to collect a native data set at CHESS A-1 station and attempt to solve the structure by molecular replacement, as the atomic coordinates for CED-9 are available. This structure will reveal important regulatory mechanisms of CED-4 by CED-9 and has significant impact on our understanding of the apoptotic pathway in both C.elegans and mammals.