REGULATION OF VIRUS-INDUCED PROGRAMMED CELL DEATH

病毒诱导的程序性细胞死亡的调节

• 批准号: 6572521
• 负责人: PAUL D FRIESEN
• 金额: $ 25.21万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6572521
• 关键词: Baculoviridae; apoptosis; endopeptidases; gene mutation; protease inhibitor; recombinant proteins; site directed mutagenesis; tissue /cell culture; transfection; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): Programmed cell death, or apoptosis, is a built-in, signal-induced process by which a cell self-destructs. It is a highly regulated mechanism that is critical for normal development, tissue homeostasis, and the elimination of pathogen-infected cells. In humans, misregulated programmed cell death is associated with tumorigenesis, neurodegenerative diseases, immunodeficiency, and viral pathogenesis. Although many evolutionarily conserved components of the cell death pathway have been identified, the molecular mechanisms involved in cellular regulation of apoptosis are still largely unknown. Since host cell apoptosis can limit virus multiplication, many viruses have evolved diverse strategies to regulate the cell death pathway. The proteins that mediate such viral intervention have provided key insight into the cell death program. The long term objective of this proposal is to define the molecular mechanisms by which apoptosis is regulated through the study of three baculovirus-encoded apoptotic regulators: P35, P49, and IAP. Our approach focuses on the use of baculovirus-infected insect cells as a powerful yet convenient system for molecular analysis of both the induction and suppression of apoptosis. Building on recent advances in the apoptosis field, we use integrated approaches in biochemistry, genetics, and cell biology to determine the molecular mechanism of P49 and IAP anti-apoptotic activity. We focus on P49's novel ability to inhibit an initiator caspase resistant to the pancaspase inhibitor P35 by defining the molecular determinants of caspase selectivity by both irreversible inhibitors. Utilizing the recently discovered capacity of baculoviruses to efficiently deliver apoptotic regulators to cultured Drosophila melanogaster cells, we identify the in vivo targets of P49 and P35 and define the caspase cascade in this model organism. We determine the molecular mechanism of virus IAP anti-apoptotic activity by characterizing the interactions between hybrid IAPs and cellular apoptotic effectors. In concert, we also investigate the functional significance of oligomerization for both viral and cellular IAPs by using novel dominant negative inhibitors. Collectively, these studies are expected to provide new and fundamental information on virus-host interactions and the regulation of programmed cell death in animals. Such knowledge will contribute to the development of therapeutic strategies for apoptosis-associated diseases.

描述（由申请人提供）：程序性细胞死亡或细胞凋亡是一种内在的信号诱导过程，细胞通过该过程自毁。它是一种高度调节的机制，对于正常发育、组织稳态和消除病原体感染的细胞至关重要。在人类中，失调的程序性细胞死亡与肿瘤发生、神经退行性疾病、免疫缺陷和病毒发病机制有关。虽然许多进化上保守的细胞死亡途径的组成部分已被确定，参与细胞凋亡的细胞调控的分子机制仍然是未知的。由于宿主细胞凋亡可以限制病毒增殖，许多病毒已经进化出不同的策略来调节细胞死亡途径。介导这种病毒干预的蛋白质为细胞死亡程序提供了关键的见解。本提案的长期目标是通过研究三种杆状病毒编码的凋亡调节因子：P35，P49和IAP来确定凋亡调节的分子机制。我们的方法侧重于使用杆状病毒感染的昆虫细胞作为一个强大而方便的系统，用于细胞凋亡的诱导和抑制的分子分析。基于细胞凋亡领域的最新进展，我们使用生物化学，遗传学和细胞生物学的综合方法来确定P49和IAP抗凋亡活性的分子机制。我们专注于P49的新的能力，抑制引发剂半胱天冬酶的pancaspase抑制剂P35通过定义两个不可逆抑制剂的半胱天冬酶的选择性的分子决定因素。利用最近发现的杆状病毒的能力，有效地提供细胞凋亡调节剂培养的果蝇细胞，我们确定在体内的P49和P35的目标，并定义在这个模型生物体的caspase级联。我们确定的病毒IAP抗凋亡活性的分子机制，通过表征杂交IAP和细胞凋亡效应之间的相互作用。在演唱会上，我们还调查了寡聚化的功能意义，为病毒和细胞的IAP使用新的显性负抑制剂。总的来说，这些研究有望提供关于病毒-宿主相互作用和动物程序性细胞死亡调控的新的基本信息。这些知识将有助于发展糖尿病相关疾病的治疗策略。