Identification of Protein-Protein Interactions and Processing Events That Traffic and Activate the Bactericidal Pore-Forming Protein Perforin-2

鉴定蛋白质-蛋白质相互作用以及运输和激活杀菌成孔蛋白 Perforin-2 的加工事件

• 批准号: 10195288
• 负责人: GEORGE Patrick MUNSON
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-18 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195288
• 关键词: Acids; Animals; Area; Bacteria; Biological Assay; Cells; Clinical; Collaborations; Communicable Diseases; Data; Deposition; Dose; EGF gene; Event; Genetic Polymorphism; Host Defense; Human; In Vitro; Individual; Infection; Innate Immune System; Integral Membrane Protein; Invaded; Investigation; Knockout Mice; Mediating; Medicine; Membrane; Microbe; Missense Mutation; Modeling; Molecular; Mus; Mycobacterium Infections; Oranges; Organ; Pathogenesis; Patients; Pattern; Peptide Hydrolases; Phagocytes; Phagocytosis; Phagolysosome; Phagosomes; Pharmaceutical Preparations; Polymers; Predisposition; Process; Proliferating; Proteins; Proteolytic Processing; Publications; Publishing; Role; Rosaniline Dyes; Rotation; Salmonella; Science; Side; Site; Stimulus; Structural Biologist; Structure; Tissues; Transmembrane Domain; Uncertainty; VDAC1 gene; antimicrobial; bactericide; base; chronic infection; clinically significant; immune function; infancy; innovation; insight; macrophage; microorganism; non-tuberculosis mycobacteria; pathogen; pathogenic bacteria; perforin 2; periplasm; polymerization; protein protein interaction; response; trafficking

The destruction of microorganisms within phagolysosomes is an essential immunological function of macrophages and other phagocytes that protects us from invading pathogens. Over the past several years we have established that Perforin-2 (PRF2), a recently described effector of the innate immune system, is pivotal for the destruction of phagocytosed bacteria. For example, we have published studies demonstrating that PRF2 knockout mice succumb to infectious doses that the majority of their wild-type littermates survive when challenged with bacterial pathogens. This is accompanied by replication and dissemination of bacteria to deeper tissues. With cell based studies we established bacteria that would normally be destroyed are able to replicate and persist within macrophages that lack PRF2. It has also recently been shown that polymorphisms within human PRF2 increase an individual's susceptibility to persistent nontuberculous mycobacterial infections. Most recently the results of our collaboration with structural biologists were published demonstrating that PRF2 polymerizes to form rings of 16 subunits. This study also revealed that the transition from pre-pore to pore is dependent upon low pH; such as would be encountered within acidifying phagosomes. Thus, our investigations spanning from the atomic to experimental mice have established that PRF2 underpins an essential function of macrophages as a pore-forming protein that permeabilizes the envelope of phagocytosed bacteria. Our working HYPOTHESIS is that PRF2-dependent killing of bacteria is a multistep process that begins with the intracellular trafficking of PRF2 as an inactive transmembrane (TM) protein in response to exogenous stimuli such as infection or pathogen- associated molecular patterns. Subsequent cleavage of PRF2 from its TM domain releases it to polymerize as a pre-pore structure on the membrane of phagocytosed bacteria. Acidification of the maturing phagosome triggers a dramatic reorganization of PRF2 that culminates in membrane penetrating pores through which other antimicrobials pass. Within this overall hypothesis are two areas of uncertainty –trafficking and proteolytic processing– that are appropriate for exploratory investigations. To redress these gaps Aim 1 will identify the protein-protein interactions that drive the intracellular trafficking of PRF2. Aim 2 will characterize the proteolytic processing events that regulate the activation of PRF2 within phagocytes. Significant impacts of this study will include a more complete understanding of macrophage mediated killing of phagocytosed bacteria and the molecular events that govern bactericidal pore formation.

吞噬体内微生物的破坏是 巨噬细胞和其他保护我们免受入侵病原体的吞噬细胞。过去几个 我们已经确定了perforin-2（prf2），这是一种先天免疫的效果 系统是破坏吞噬细菌的关键。例如，我们已经发表了 研究表明，PRF2基因敲除小鼠屈服于感染剂量的大多数 当受到细菌病原体的挑战时，野生型窝窝生存。这是由 将细菌复制和传播到更深的组织中。通过基于细胞的研究，我们建立了 通常被破坏的细菌能够复制并持续存在于巨噬细胞中 缺乏PRF2。最近还显示，人PRF2中的多态性增加 个人对持续性无结枝感染的敏感性。最近 我们与结构生物学家合作的结果发表了表明PRF2 聚合以形成16个亚基的环。这项研究还表明，从预孔到 孔取决于低pH。例如在酸化的吞噬体中遇到的。那， 我们从原子到实验小鼠进行的调查已经确定了PRF2 基础是巨噬细胞作为孔形成蛋白的基本功能，该蛋白质渗透 吞噬细菌的包膜。我们的工作假设是依赖PRF2的杀害 细菌是一个多步过程，始于PRF2的细胞内运输作为一种不活跃 响应外源刺激（例如感染或病原体）的跨膜（TM）蛋白 相关的分子模式。随后从其TM域的PRF2切割将其释放到 聚合作为吞噬细菌膜上的前孔结构。酸化 成熟的吞噬体触发了PRF2的戏剧性重组，该重组在膜上达到高潮 穿透其他抗菌素通过的毛孔。在这个总体中，有两个 不确定性的领域 - 贩运和蛋白水解处理 - 适合探索性的领域 投资。为了纠正这些差距，目标1将确定驱动蛋白质蛋白质的相互作用 PRF2的细胞内贩运。 AIM 2将表征调节的蛋白水解处理事件 吞噬细胞中PRF2的激活。这项研究的重大影响将包括更多 完全了解巨噬细胞介导的吞噬细菌和分子的杀死 控制细菌孔形成的事件。