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，或称细胞凋亡，是一种内在的、信号诱导的过程，细胞通过这种过程自毁。这是一种高度调控的机制，对正常发育、组织动态平衡和消除病原体感染的细胞至关重要。在人类中，调节不当的程序性细胞死亡与肿瘤发生、神经退行性疾病、免疫缺陷和病毒致病有关。尽管已经鉴定出许多进化保守的细胞死亡途径成分，但参与细胞凋亡调控的分子机制在很大程度上仍不清楚。由于宿主细胞的凋亡可以限制病毒的增殖，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提供了对细胞死亡程序的关键洞察。这项提案的长期目标是通过对三种杆状病毒编码的凋亡调节因子P35、P49和IAP的研究，确定调控细胞凋亡的分子机制。我们的方法侧重于使用杆状病毒感染的昆虫细胞作为一种强大而方便的系统，用于诱导和抑制细胞凋亡的分子分析。在细胞凋亡领域最新进展的基础上，我们利用生物化学、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。我们重点研究了P49‘S通过定义两种不可逆抑制物对caspase选择性的分子决定因素来抑制对pancaspase抑制剂P35具有抗性的启动子caspase的新能力。利用最近发现的杆状病毒有效地向培养的果蝇黑腹细胞运送凋亡调节剂的能力，我们确定了P49和P35的体内靶点，并确定了这个模式生物中的caspase级联。我们通过鉴定杂合IAP和细胞凋亡效应分子之间的相互作用来确定病毒IAP抗凋亡活性的分子机制。同时，我们还通过使用新的显性负性抑制物来研究病毒和细胞IAP寡聚的功能意义。总的来说，这些研究有望提供有关病毒-宿主相互作用和动物细胞程序性死亡调控的新的基本信息。这些知识将有助于制定与细胞凋亡相关的疾病的治疗策略。