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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。细胞凋亡在所有多细胞生物体的发育和稳态中起着核心作用。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。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。这四个基因EGL-1、CED-9、CED-4和CED-3的蛋白产物以线性途径作用以执行细胞死亡，并且构成理解细胞凋亡的经典范例。尽管进行了遗传学研究，但Egl 1、CED 9、CED 4和CED 3控制细胞凋亡起始的分子机制在很大程度上仍然未知。为了实现这些目标，我们已经启动了系统的生化和X射线晶体学分析的蛋白质复合物参与这一范例。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。我们已经表征了全长CED 4和CED 9的大功能域之间的二元复合物。我们最近在两个不同的空间群中获得了这种复合物的晶体。一种晶型弱衍射X射线。另一种晶型的最佳晶体在CHESS A-1站的X射线衍射值约为3.5 <$s。我们希望在CHESS A-1站收集一个天然数据集，并尝试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。该结构将揭示CED-4通过CED-9的重要调控机制，并且对我们理解秀丽隐杆线虫和哺乳动物中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.