Vesicular trafficking mechanisms regulating granulocyte function

描述（由申请人提供）：细胞内囊泡运输对中性粒细胞生理的几乎所有方面都是必不可少的，这种机制的缺陷导致人类疾病。在中性粒细胞中，囊泡运输与胞吐、吞噬、粘附和趋化过程直接相关；然而，在分泌、病原体内化或迁移过程中调节不同中性粒细胞分泌细胞器动员的分子机制尚不完全清楚。我们已经确定了控制中性粒细胞颗粒运输的几个关键调节因子，包括控制中性粒细胞颗粒在体外和体内的动员和分泌的小GTPase Rab27a。该功能由两个Rab27a效应子JFC1和Munc13-4调控。此外，我们最近发现了以前未被识别的中性粒细胞分泌因子，这些因子与JFC1相互作用，调节细胞骨架重塑、囊泡运输和胞外分泌，并开创了系统生物学方法的发展，以分析粒细胞中的囊泡动力学和肌动蛋白重塑。在这项应用中，我们建议使用创新的分析和定量方法来阐明中性粒细胞中与胞吐、吞噬和趋化相关的囊泡运输的调节机制。我们还建议使用rab27a效应结合的新型小分子抑制剂来询问囊泡运输机制，并通过内毒素诱导的全身炎症的体内模型阐明疾病中rab27a依赖的过程。由于中性粒细胞颗粒的毒性成分对宿主具有损伤作用，而中性粒细胞分泌蛋白在内毒素血症和脓毒症相关的内皮损伤中起着重要作用，因此这些研究具有重要的生理意义和潜在的临床应用价值。我们假设Rab27a及其效应物对水疱运输的差异调节是决定中性粒细胞特异性功能和对损伤反应的重要机制。我们还提出，囊泡运输途径的小分子调节剂将防止全身炎症期间中性粒细胞分泌的一些有害后果。为了验证我们的假设，我们提出以下具体目标：1)确定粒细胞胞外分泌过程中调节囊泡运输和肌动蛋白重塑的机制，并确定Rab27a效应因子JFC1在该机制中的作用；2)建立Munc13-4调控的与胞吐、吞噬和细菌杀伤相关的囊泡运输和囊泡融合机制；3)验证rab27a效应结合的新型小分子抑制剂调节中性粒细胞颗粒运输和胞吐，减轻炎症的假设。该研究计划的结果将揭示调节中性粒细胞囊泡运输的分子机制，并为治疗炎症性疾病提供有效的分子策略。

DESCRIPTION (provided by applicant): Intracellular vesicular transport is essential for nearly all aspects of neutrophil physiology and defects in this mechanism leads to disease in humans. In neutrophils, vesicular trafficking is directly associated with the processes of exocytosis, phagocytosis, adhesion and chemotaxis; however, the molecular mechanisms that regulate mobilization of the different neutrophil secretory organelles during secretion, pathogen internalization or migration are incompletely understood. We have identified several key regulatory factors essential for the control of granule trafficking in neutrophils including the smll GTPase Rab27a that controls the mobilization and secretion of neutrophil granules in vitro and in vivo. This function is regulated by two Rab27a effectors, JFC1 and Munc13-4. Furthermore, we have recently identified previously unrecognized neutrophil secretory factors that interact with JFC1 and regulate cytoskeleton remodeling, vesicular trafficking and exocytosis and have pioneered the development of systems biology approaches to analyze vesicular dynamics and actin remodeling in granulocytes. In this application, we propose to use innovative analytical and quantitative methods to elucidate the mechanisms regulating vesicular transport associated with exocytosis, phagocytosis and chemotaxis in neutrophils. We also propose to use novel small molecule inhibitors of Rab27a-effector binding to interrogate mechanisms of vesicular transport and elucidate Rab27a-dependent processes in disease using an in vivo model of endotoxin-induced systemic inflammation. Since the toxic content of neutrophil granules is injurious to the host and neutrophil secretory proteins play a fundamental role in the damage to endothelium associated with endotoxemia and sepsis, these studies have important physiological significance and potential clinical applications in disease. We hypothesize that the differential regulation of vesicular transport by Rab27a and its effectors is an essential mechanism to determine specific neutrophil functions and responses to insult. We also propose that small molecule modulators of vesicular transport pathways will prevent some of the deleterious consequences of neutrophil secretion during systemic inflammation. To test our hypotheses we propose the following Specific Aims: 1) To define the mechanisms that regulate vesicular trafficking and actin remodeling during exocytosis in granulocytes and establish the role of the Rab27a effector JFC1 in this mechanism; 2) To establish the mechanisms regulated by Munc13-4 in vesicular transport and vesicular fusion associated with exocytosis, phagocytosis and bacterial killing; 3) To test the hypothesis that novel small molecule inhibitors of Rab27a-effector binding modulate neutrophil granule trafficking and exocytosis and attenuate inflammation. The results of the proposed research plan should uncover the molecular mechanisms regulating vesicular transport in neutrophils and lead to effective molecular strategies for the treatment of inflammatory diseases.