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细胞自我毁灭的内在信号诱导过程。它是一种高度调控的机制，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。尽管细胞死亡途径的许多进化上保守的成分已被鉴定，但参与细胞凋亡调节的分子机制仍然很大程度上未知。由于宿主细胞凋亡可以限制病毒的增殖，因此许多病毒已经进化出多种策略来调节细胞死亡途径。介导这种病毒干预的蛋白质为了解细胞死亡程序提供了重要的见解。该提案的长期目标是通过研究三种杆状病毒编码的细胞凋亡调节因子：P35、P49 和 IAP，确定细胞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强大而方便的系统来对细胞凋亡的诱导和抑制进行分子分析。基于细胞凋亡领域的最新进展，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。我们通过定义两种不可逆抑制剂的 caspase 选择性的分子决定因素，重点关注 P49 抑制对 pancaspase 抑制剂 P35 具有抗性的起始 caspase 的新能力。利用最近发现的杆状病毒向培养的果蝇细胞有效传递凋亡调节因子的能力，我们确定了 P49 和 P35 的体内靶标，并定义了该模型生物中的 caspase 级联。我们通过表征混合 IAP 和细胞凋亡效应器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，我们还通过使用新型显性失活抑制剂研究寡聚化对于病毒和细胞 IAP 的功能意义。总的来说，这些研究有望提供有关病毒与宿主相互作用以及动物程序性细胞死亡调节的新的基本信息。这些知识将有助于开发细胞凋亡相关疾病的治疗策略。