Vesicular trafficking mechanisms regulating granulocyte function

项目摘要：细胞内囊泡运输对中性粒细胞生理和功能的各个方面都是必不可少的。 这一机制的缺陷会导致人类患病。在中性粒细胞中，囊泡运输与 胞吐、吞噬、信号传递和转运的过程；然而，分子机制 不同的中性粒细胞分泌细胞器的调节动员需要进一步阐明。我们有 确定了中性粒细胞转运的几个关键调节因子，包括小GTP酶Rab27a和它的 效应器JFC1和Munc13-4。我们还确认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。此外，我们还率先开发了系统 分析粒细胞囊泡动力学和肌动蛋白重塑的生物学方法。最后，我们有 发现了一种新的内体晚期成熟机制，涉及钙离子和钙离子之间的相互作用 感受器Munc13-4和晚期内体SNARE蛋白Synaxin 7(STX7)，调节TLR9信号和 中性粒细胞下游功能。在这里，我们使用创新的定量方法来阐明这一机制 调节囊泡转运与胞吐、吞噬和内体晚期成熟有关 中性粒细胞。我们还建议使用新型小分子抑制剂Rab27a-JFC1和Munc13-4-STX7 结合研究囊泡转运机制和阐明中性粒细胞在疾病中的功能 全身炎症的活体模型。这项资助的中心目标是阐明囊泡的运输。 控制中性粒细胞促炎过程的机制，开发翻译方法来干预 通过这些过程，并为它们用于减轻全身炎症提供临床前验证。自.以来 中性粒细胞激活失调对宿主的损害中性粒细胞分泌蛋白起基础作用 在内毒素血症、败血症和无菌炎症相关的内皮损伤中的作用，这些 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及其效应器对囊泡转运的差异调节是决定 特定的中性粒细胞功能和对侮辱的反应。我们还提出了特定的小分子调节剂 囊泡转运通路将防止中性粒细胞激活的一些有害后果 全身炎症。为了检验我们的假设，我们提出了以下具体目标：1)定义 不同的机制，调控囊泡运输，肌动蛋白依赖的推进和阻断，以及 中性粒细胞颗粒亚群的胞吐作用；2)建立调节内小体的分子机制 中性粒细胞的成熟、内体功能和核酸敏感的TLR信号；3)发展机制 以及调节中性粒细胞囊泡运输途径和迁移的翻译方法以控制 全身炎症。这项拟议的研究将阐明调控囊泡的分子机制。 中性粒细胞的转运，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.