Structural Studies of Apoptosome Assembly in Apoptosis

细胞凋亡中凋亡体组装的结构研究

• 批准号: 6875043
• 负责人: YIGONG SHI
• 金额: $ 28.12万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-22 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6875043
• 关键词: Baculoviridae; X ray crystallography; adenosine triphosphate; apoptosis; calorimetry; chemical kinetics; conformation; crystallization; cysteine endopeptidases; cytochrome c; enzyme structure; enzymes; isomorphous substitution; molecular assembly /self assembly; protein binding; protein protein interaction; recombinant proteins; stoichiometry; structural biology

Apoptosis plays a central role in the development and homeostasis of all multi-cellular organisms. Alterations in apoptotic pathways have been implicated in cancer and autoimmune diseases. The formation of a 1.4 MDa apoptosome holoenzyme, involving Apaf-1, cytochrome c, dATP, and caspase-9, is essential to many important forms of apoptosis in mammals. The work proposed here focuses on the following specific aims: (1) Biochemical analysis of the formation of an apoptosome holoenzyme. The cloning, expression, and purification of all three protein components involved in the formation of an apoptosome holoenzyme have been successfully completed. Preliminary characterization of the apoptosome holoenzyme has been performed, verifying that all recombinant proteins are biochemically active. Systematic characterization of the formation of the apoptosome holoenzyme will be pursued. (2) Determination of the structures of a mini-apoptosome involving the CARD domain of Apaf-1 in complex with the activated caspase- 9. The CARD domain of Apaf-1 mediates the formation of a 440-kDa mini-apoptosome involving active caspase-9. Large amounts of this mini-apoptosome have been assembled and initial crystallization trials have been performed. The determination of the three-dimensional structure of this mini-apoptosome will be pursued. (3) Determination of the structures of the full-length Apaf-1 by itself and in complex with cytochrome c. Apaf-1 by itself exists in an inhibitory conformation. Upon binding to cytochrome c, Apaf-1 undergoes conformational change that allows binding to dATP or ATP. These structures will be determined by multiple isomorphous replacement. (4) Determination of the structure of the complete apoptosome holoenzyme. The feasibility of this project has been demonstrated by the reconstitution of the complete and functional apoptosome holoenzyme in vitro. This 1.4 MDa complex will be crystallized. The structure will be determined by either molecular replacement or by multiple isomorphous replacement.

凋亡在所有多细胞生物的发育和稳态中起着核心作用。 凋亡途径的改变与癌症和自身免疫性疾病有关。 涉及APAF-1，细胞色素C，DATP和CASPASE-9的1.4 MDA凋亡组全酶的形成对于哺乳动物中许多重要形式的凋亡形式至关重要。 此处提出的工作重点介绍了以下特定目的：（1）息肉全酶形成的生化分析。 所有三种蛋白质成分的克隆，表达和纯化已成功完成。已经进行了圆顶体全酶的初步表征，以验证所有重组蛋白具有生化活性。 将追求圆顶体全酶形成的系统表征。 （2）确定与激活的caspase-9中涉及APAF-1的卡片域的迷你座域的结构。 已经组装了大量的这个小型跨斜体，并进行了初始结晶试验。 将追求该迷你座小体的三维结构的确定。 （3）按本身以及与细胞色素c复杂的全长APAF-1的结构确定。 APAF-1本身存在于抑制构象中。 与细胞色素C结合后，APAF-1经历构象变化，允许与DATP或ATP结合。 这些结构将由多种同构置换确定。 （4）确定完整圆顶体全酶的结构。 该项目的可行性通过体外的完整和功能性全酶的重构来证明。 这种1.4 MDA复合物将结晶。 结构将通过分子置换或多个同构置换来确定。