Control of Apoptosis by Drosophila Cell Death Genes

果蝇细胞死亡基因对细胞凋亡的控制

• 批准号: 8018069
• 负责人: HERMANN STELLER
• 金额: $ 33.13万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8018069
• 关键词: Acquired Immunodeficiency Syndrome; Animals; Apoptosis; Apoptotic; BIRC4 gene; Basic Science; Binding; Biochemical; Biological; Biological Assay; Caspase; Caspase Inhibitor; Cell Death; Cell Survival; Cells; Cessation of life; Clinical Research; Clinical Trials; Complex; Cullin 1; Cullin Proteins; Cysteine Protease; Development; Disease; Drosophila genus; Enzyme Precursors; Evolution; Family; Genes; Genetic; Genetic Screening; Goals; Grant; Head; Health; Homeostasis; Human; In Situ; In Vitro; Induction of Apoptosis; Insecta; Knockout Mice; Laboratories; Life; Link; Malignant Neoplasms; Mammalian Cell; Mediating; Medicine; Modeling; Molecular; Molecular Genetics; Mus; Mutant Strains Mice; Mutation; Myopathy; Neurodegenerative Disorders; Pathway interactions; Play; Predisposition; Process; Property; Protein Binding Domain; Protein Family; Proteins; Regulation; Research; Role; Signal Pathway; Site-Directed Mutagenesis; Spermatogenesis; Stimulus; Stroke; Testing; Therapeutic; Time; Tissues; Tumor Suppression; Ubiquitin; Ubiquitin family; Ubiquitin-Conjugating Enzymes; Ubiquitination; Work; base; cIAP1 protein; caspase-3; cell killing; cell suicide; cullin-3; human disease; in vivo; inhibitor-of-apoptosis protein; insight; interdisciplinary approach; killings; man; mouse model; multicatalytic endopeptidase complex; neoplastic cell; novel; preclinical study; programs; protein degradation; research study; response; small molecule; sperm cell; thymocyte; tumorigenesis; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Apoptosis is a morphologically distinct form of programmed cell death that plays important roles in development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders. Apoptosis occurs by activating an intrinsic cell suicide program which is constitutively expressed in most animal cells, and key components of this program have been conserved in evolution from worms to insects to man. A central step in the execution of apoptosis is the activation of a specific class of cysteine proteases, termed caspases that are widely expressed as inactive zymogens. The overall objective of the proposed research is to gain insight into the molecular mechanisms of caspase regulation and cell death, with particular emphasis on negative regulators of caspases. The specific focus of this proposal is on a set of ubiquitin pathway proteins that play a complex but specific role in regulating the onset of apoptosis via selective protein degradation. Inhibitor of Apoptosis Proteins (IAPs) are E3-ubiquitin ligases that protect cells against unwanted apoptosis by inhibiting caspases in live cells. However, upon death-inducing stimuli, IAPs auto-ubiquitinate and self-destruct in cells that are doomed to die. One major goal of this proposal is to define the precise mechanism by which the Drosophila pro-apoptotic Reaper protein inhibits IAPs. For this purpose, we will use a combination of site-directed mutagenesis, in vitro ubiquitination assays, protein interaction analyses, in vivo expression and cell biological studies. We will also investigate the mechanism by which mouse XIAP inhibits caspases, and study the role of this protein in apoptosis and tumor suppression in vivo by taking advantage of XIAP-mutant mice that we generated during the previous project period. Finally, we will investigate the mechanism by which a cullin-3-based E3 ubiquitin ligase complex that we recently identified from a genetic screen promotes caspase activation during sperm differentiation in Drosophila. This work should significantly advance our understanding of how caspases are regulated, and how their activity can be manipulated for therapeutic purposes. PUBLIC HEALTH RELEVANCE: This proposal focuses on negative regulation of caspases, the key executioners of apoptosis (programmed cell death), by a conserved family of ubiquitin pathway proteins. Progress during past grant periods has provided the intellectual basis for developing a novel class of small-molecule cancer therapeutics that are currently being evaluated in human clinical studies, and the results from proposed mouse model studies (aim 2) will have a direct influence on guiding these clinical trials. We will take a multi-disciplinary approach that integrates cellular, molecular genetic and biochemical studies in both Drosophila and the mouse. This is a uniquely powerful approach for taking a project from basic science discoveries to pre-clinical studies. At this time we are the only laboratory in the world that uses both Drosophila and mouse genetics to investigate the regulation of cell death, and to explore how abnormal regulation of this process contributes to human diseases, with a particular focus on cancer. Therefore, this project will have profound implications for both basic science and medicine.

描述（由申请人提供）：细胞凋亡是一种形态学上独特的程序性细胞死亡形式，在发育、组织稳态和多种疾病中起重要作用，包括癌症、艾滋病、中风、肌病和各种神经退行性疾病。细胞凋亡是通过激活一个内在的细胞自杀程序而发生的，这个程序在大多数动物细胞中都是组成性表达的，这个程序的关键组成部分在从蠕虫到昆虫再到人类的进化过程中都是保守的。细胞凋亡的一个核心步骤是激活一类特定的半胱氨酸蛋白酶，称为半胱天半酶，广泛表达为无活性酶原。拟议研究的总体目标是深入了解半胱天冬酶调控和细胞死亡的分子机制，特别强调半胱天冬酶的负调控。该建议的具体重点是一组泛素途径蛋白，它们通过选择性蛋白质降解在调节细胞凋亡的发生中发挥复杂但特定的作用。凋亡抑制蛋白（IAPs）是e3 -泛素连接酶，通过抑制活细胞中的半胱天蛋白酶来保护细胞免受不必要的凋亡。然而，在诱导死亡的刺激下，iap会在注定要死亡的细胞中自动泛素化并自我毁灭。本研究的一个主要目标是确定果蝇促凋亡Reaper蛋白抑制IAPs的精确机制。为此，我们将结合位点定向诱变、体外泛素化分析、蛋白质相互作用分析、体内表达和细胞生物学研究。我们还将利用前期项目培育的XIAP突变小鼠，研究小鼠XIAP抑制caspases的机制，并在体内研究该蛋白在细胞凋亡和肿瘤抑制中的作用。最后，我们将研究基于cullin-3的E3泛素连接酶复合物在果蝇精子分化过程中促进caspase激活的机制。这项工作将极大地促进我们对半胱天冬酶是如何被调节的，以及它们的活性是如何被操纵以达到治疗目的的理解。公共卫生相关性：本提案侧重于半胱天冬酶的负调控，半胱天冬酶是细胞凋亡（程序性细胞死亡）的关键刽子手，是一个保守的泛素途径蛋白家族。过去拨款期间的进展为开发一类新型小分子癌症治疗药物提供了知识基础，目前正在人类临床研究中进行评估，而拟议的小鼠模型研究（目标2）的结果将对指导这些临床试验产生直接影响。我们将采取多学科的方法，将果蝇和小鼠的细胞、分子遗传和生化研究结合起来。这是一种独特而有力的方法，可以将一个项目从基础科学发现带到临床前研究。目前，我们是世界上唯一一个同时使用果蝇和小鼠遗传学来研究细胞死亡调控的实验室，并探索这一过程的异常调控如何导致人类疾病，特别是癌症。因此，这个项目将对基础科学和医学产生深远的影响。