Structural Studies in Apaf 1 mediated Apoptosis

描述（申请人的描述）：细胞凋亡在细胞凋亡中起核心作用。 后生动物的发育和体内平衡。凋亡途径的改变 与癌症和自身免疫性疾病有关凋亡 蛋白酶激活因子1（Apaf-1）通过以下途径控制细胞凋亡的起始： 促进半胱氨酸天冬氨酸蛋白酶原-9和半胱氨酸天冬氨酸蛋白酶原-3的活化。的抑制剂 细胞凋亡（IAP）蛋白家族通过抑制这些细胞凋亡抑制细胞死亡。 半胱天冬酶。最近发现的一种蛋白Smac在细胞凋亡中起着重要作用 通过解除IAP对半胱天冬酶的抑制作用。这里提出的工作 重点关注以下具体目标：（1）确定高分辨率 SMAC的结构我们获得了Smac的衍射晶体。结构 正在确定;重要的见解已经开始出现， 初步结构分析。(2)机制的生化表征 Smac的凋亡激活。与王晓东合作， 得克萨斯大学，一个体外系统已经开发出来，以研究 IAP抑制凋亡和Smac激活的机制。重要 这些发现已经开始影响我们对细胞凋亡激活的理解。 (3)Smac与IAP复合物的结构测定。救济 Smac对IAP抑制细胞凋亡的作用涉及Smac与IAP之间的直接相互作用。 这两种蛋白质。Smac与函数域之间的二元复形 人c-IAP 1已被表征。已获得小晶体。努力 以改善晶体形态。(4)测定 与IAP复合的caspase-3的结构。提供一个结构基础， 由于IAP对caspase的抑制作用，活性caspase-3的两个亚基已被 过表达、重折叠并纯化至均一作为活性复合物。一 胱天蛋白酶-3和人c-IAP 1的功能结构域之间的二元复合物将 被重组和结晶。(5)的结构确定 半胱天冬酶原-9本身以及与IAP的复合物。机构间行动计划的运作方式是， 激活的半胱天冬酶和失活的半胱天冬酶原-9。全长 半胱天冬酶原-9（C287 A）已被过度表达并纯化至同质。的 半胱氨酸天冬氨酸蛋白酶原-9晶体本身以及与抑制性c-IAP 1复合的晶体 将生成片段;这些结构将由以下任一项确定： 分子置换或多个同晶置换。

Description (applicant's description): Apoptosis plays a central role in the development and homeostasis of metazoans. Alterations in apoptotic pathways have been implicated in cancer and autoimmune diseases. The apoptotic protease-activating factor 1 (Apaf-1) controls the initiation of apoptosis by promoting the activation of procaspase-9 and procaspase-3. The Inhibitor of Apoptosis (IAP) family of proteins suppress cell death by inhibiting these caspases. A recently identified protein, Smac, plays a vital role in apoptosis by relieving the inhibitory effects of IAPs to caspases. The work proposed here focuses on the following specific aims: (1) Determination of a high-resolution structure of Smac. We have obtained diffracting crystals of Smac. The structure is being determined; and important insights have begun to emerge from preliminary structural analysis. (2) Biochemical characterization of mechanisms of apoptotic activation by Smac. In collaboration with Xiaodong Wang at the University of Texas, an in vitro system has been developed to study the mechanisms of apoptotic inhibition by IAPs and activation by Smac. Important findings have started to impact our understanding on activation of apoptosis. (3) Determination of the structure of Smac in complex with an IAP. The relief of IAP inhibition to apoptosis by Smac involves a direct interaction between these two proteins. A binary complex between Smac and a functional domain in human c-IAP1 has been characterized. Small crystals have been obtained. Efforts to improve the crystal morphology are pursued. (4) Determination of the structure of caspase-3 in complex with an IAP. To provide a structural basis of caspase inhibition by IAP, the two subunits of active caspase-3 have been over-expressed, refolded and purified to homogeneity as an active complex. A binary complex between caspase-3 and a functional domain of human c-IAP1 will be reconstituted and crystallized. (5) Determination of the structure of procaspase-9 by itself and in complex with an IAP. IAPs function by targeting both the activated caspases and the inactive procaspase-9. A full-length procaspase-9 (C287A) has been over-expressed and purified to homogeneity. The crystals of procaspase-9 by itself and in complex with an inhibitory c-IAP1 fragment will be generated; these structures will be determined by either molecular replacement or multiple isomorphous replacement.