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

破坏吞噬素体内的微生物是一种基本的免疫功能 巨噬细胞和其他吞噬细胞，保护我们免受入侵的病原体。在过去的几年中 多年来，我们已经证实，穿孔素-2(PRF2)，一种最近被描述的先天性免疫的效应器 这一系统对于消灭吞噬细菌至关重要。例如，我们已经发布了 研究表明，PRF2基因敲除小鼠死于感染性剂量，而大多数小鼠 当受到细菌病原体的攻击时，野生型窝产仔存活下来。这是伴随着 细菌的复制和传播到更深层的组织。通过基于细胞的研究，我们建立了 通常会被摧毁的细菌能够在巨噬细胞内复制并持续存在 缺乏PRF2。最近还表明，人类PRF2基因的多态增加了 个体对持续性非结核分枝杆菌感染的易感性。最近的一次 我们与结构生物学家合作的结果已经发表，表明PRF2 聚合形成16个亚基的环。这项研究还揭示了从前孔到孔洞的转变 毛孔依赖于低pH值，例如在酸化吞噬体内会遇到这种情况。因此， 我们从原子到实验小鼠的研究都证明了PrF2 支持巨噬细胞作为一种穿孔蛋白的基本功能，使其通透 吞噬细菌的包膜。我们的工作假设是依赖于PRF2的杀伤 细菌是一个多步骤的过程，从PRF2作为非活性物质在细胞内的运输开始 跨膜(TM)蛋白对外部刺激的反应，如感染或病原体- 相关的分子模式。随后将PrF2从其TM结构域中切割出来，将其释放到 在吞噬细菌的膜上以预孔结构的形式聚合。水的酸化 成熟的吞噬小体触发PRF2的戏剧性重组，最终形成膜 穿透其他抗菌剂通过的毛孔。在这一总体假设中，有两个 适合探索的不确定性领域--贩运和蛋白质分解加工 调查。为了弥补这些差距，目标1将确定推动 PRF2的胞内转运。目标2将描述调节蛋白质降解过程的事件 吞噬细胞内PRF2的激活。这项研究的重大影响将包括 巨噬细胞介导的杀灭吞噬细菌及其分子机制的全面认识 控制杀菌毛孔形成的事件。

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.