Vesicular trafficking mechanisms regulating granulocyte function

项目概述：细胞内囊泡转运对于中性粒细胞生理学的各个方面都是必不可少的， 这种机制的缺陷会导致人类疾病。在中性粒细胞中，囊泡运输与 胞吐作用、吞噬作用、信号传导和迁移的过程;然而， 调节不同中性粒细胞分泌细胞器的动员需要进一步阐明。我们有 确定了中性粒细胞颗粒运输的几个关键调节因子，包括小的GTTRab 27 a及其 效应器JFC 1和Munc 13 -4。我们还确定WASH是一种调节细胞骨架的中性粒细胞因子 重塑、囊泡运输和胞吐作用。此外，我们还率先开发了 生物学方法来分析粒细胞中的囊泡动力学和肌动蛋白重塑。我们终于有 确定了一种新的内体晚期成熟机制，涉及钙离子与核内体之间的相互作用。 传感器Munc 13 -4和晚期内体SNARE蛋白突触融合蛋白7（STX 7），以调节TLR 9信号传导， 下游中性粒细胞功能。在这里，我们使用创新的定量方法来阐明机制 调节与胞吐、吞噬和晚期内体成熟相关的囊泡转运， 中性粒细胞我们还建议使用Rab 27 a-JFC 1和Munc 13 -4-STX 7的新型小分子抑制剂 结合研究囊泡转运机制和阐明疾病中中性粒细胞的功能， 全身性炎症的体内模型。这项资助的中心目标是阐明 控制中性粒细胞促炎过程的机制，开发翻译方法来干扰 并为它们用于减轻全身性炎症提供临床前验证。以来 中性粒细胞活化失调对宿主有害，中性粒细胞分泌蛋白起着重要作用。 在与内毒素血症、败血症和无菌性炎症相关的内皮损伤中的作用，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 Rab 27 a及其效应物对囊泡转运的差异调节是确定 特异性中性粒细胞功能和对损伤的反应。我们还提出，小分子调节剂的具体 囊泡运输途径将防止在运输过程中中性粒细胞活化的一些有害后果。 全身炎症。为了验证我们的假设，我们提出了以下具体目标：1）定义 差异调节囊泡运输、肌动蛋白依赖性推进和阻断的机制，以及 中性粒细胞颗粒亚群的胞吐作用; 2）建立调节内体 中性粒细胞中的成熟、内体功能和核酸敏感TLR信号传导; 3）发展机制 和翻译的方法来调节中性粒细胞囊泡运输途径和迁移， 全身性炎症这项研究将阐明调控囊泡的分子机制。 在中性粒细胞中的转运，并导致治疗炎症性疾病的新策略。

Project Summary: Intracellular vesicular transport is essential for all aspects of neutrophil physiology and defects in this mechanism leads to disease in humans. In neutrophils, vesicular trafficking is associated with the processes of exocytosis, phagocytosis, signaling and transmigration; however, the molecular mechanisms that regulate mobilization of the different neutrophil secretory organelles require further elucidation. We have identified several key regulators of neutrophil granule trafficking, including the small GTPase Rab27a and its effectors JFC1 and Munc13-4. We have also identified WASH as a neutrophil factor that regulates cytoskeleton remodeling, vesicular trafficking and exocytosis. Furthermore, we have pioneered the development of systems biology approaches to analyze vesicular dynamics and actin remodeling in granulocytes. Finally, we have identified a novel mechanism of late endosomal maturation that involves the interaction between the calcium sensor Munc13-4 and the late endosomal SNARE protein syntaxin 7 (STX7), to regulate TLR9 signaling and downstream neutrophil functions. Here, we use innovative quantitative methods to elucidate the mechanisms regulating vesicular transport associated with exocytosis, phagocytosis and late endosomal maturation in neutrophils. We also propose to use novel small-molecule inhibitors of Rab27a-JFC1 and Munc13-4-STX7 binding to investigate mechanisms of vesicular transport and to elucidate neutrophil function in disease using in vivo models of systemic inflammation. The central goal of this grant is to elucidate the vesicular transport mechanisms that govern neutrophil pro-inflammatory processes, develop translational approaches to interfere with these processes and provide preclinical validation for their use to attenuate systemic inflammation. Since dysregulated neutrophil activation is injurious to the host and neutrophil secretory proteins play fundamental roles in the damage to the endothelium associated with endotoxemia, sepsis and sterile inflammation, these studies have important physiological significance and potential clinical applications. 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 specific vesicular transport pathways will prevent some of the deleterious consequences of neutrophil activation during systemic inflammation. To test our hypotheses we propose the following Specific Aims: 1) Define the mechanisms that differentially regulate vesicular trafficking, actin-dependent propulsion and blockage, and exocytosis of neutrophil granule subsets; 2) Establish the molecular mechanisms regulating endosomal maturation, endosomal function and nucleic acid-sensing TLR-signaling in neutrophils; 3) Develop mechanistic and translational approaches to regulate neutrophil vesicular trafficking pathways and migration for the control of systemic inflammation. The proposed research will elucidate the molecular mechanisms regulating vesicular transport in neutrophils and lead to new strategies to treat inflammatory diseases.