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

消灭吞噬溶酶体内的微生物是免疫系统的一项基本免疫功能。 巨噬细胞和其他吞噬细胞保护我们免受入侵的病原体。过去几 多年来，我们已经确定穿孔蛋白-2（PRF 2），一种最近描述的先天性免疫的效应物， 系统，是关键的吞噬细菌的破坏。例如，我们发布了 研究表明，PRF 2基因敲除小鼠死于感染剂量， 当用细菌病原体攻击时，野生型同窝仔存活。这伴随 细菌向深层组织的复制和传播。通过基于细胞的研究， 通常会被破坏的细菌能够在巨噬细胞内复制和存活， 缺乏PRF 2。最近还表明，人PRF 2内的多态性增加了人PRF 2的表达。 个体对持续性非结核分枝杆菌感染的易感性。最近的 我们与结构生物学家合作的结果发表，表明PRF 2 聚合形成16个亚基的环。该研究还揭示了从预孔到 孔依赖于低pH值;例如在酸化吞噬体内会遇到的。因此，在本发明中， 我们从原子到实验小鼠的研究已经确定PRF 2 支持巨噬细胞作为一种孔形成蛋白的基本功能， 被吞噬的细菌的包膜。我们的工作假设是，PRF 2依赖性杀死 细菌感染是一个多步骤的过程，开始于PRF 2作为非活性蛋白的细胞内运输。 跨膜（TM）蛋白响应于外源刺激如感染或病原体- 相关的分子模式。随后从其TM结构域切割PRF 2将其释放到 噬菌体作为被吞噬细菌膜上的预孔结构。酸化 成熟的吞噬体触发PRF 2的戏剧性重组， 穿透其他抗菌剂通过的孔。在这一总体假设中， 不确定的领域-贩运和蛋白水解加工-适合探索 调查事务所为了弥补这些差距，目标1将确定蛋白质-蛋白质相互作用， PRF 2的细胞内运输。目的2将描述蛋白水解加工事件， 吞噬细胞内PRF 2的激活。这项研究的重要影响将包括更多 完全了解巨噬细胞介导的吞噬细菌的杀伤作用及其分子机制， 控制杀菌孔形成的事件。

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.