Granule exocytosis and neutrophil activation

描述(由申请人提供)： 胞吐作用在介导与中性粒细胞激活相关的各种功能反应中的假定作用尚未得到直接测试。我们将使用一种新的方法：产生包含不同SNARE蛋白卷曲卷曲结构域的TAT-融合蛋白；检验阻断不同颗粒亚群的胞吐将防止特定的中性粒细胞表型变化的假设，该策略可能被用于防止体内中性粒细胞介导的组织损伤。该提案指导阶段的具体目标是：1)确定包含SNAP-23、Synaxins和VAMPS的SNARE结构域的TAT融合蛋白抑制四个中性粒细胞亚群的胞吐的能力。这一目标的培训目标是发展分子生物学和细胞生物学技能，使申请者能够在新的水平上解决实验问题，并将其应用于本提案的独立阶段。2)确定体内应用TAT-SNARE融合蛋白的最有效途径，并观察其对大鼠急性中性粒细胞依赖炎症模型的影响。这一目标的训练目标是学习中性粒细胞依赖炎症的动物模型，并建立TAT融合蛋白在体内抑制中性粒细胞胞吐的能力。3)通过蛋白质组学分析确定与SNARE蛋白相互作用的其他蛋白质。这一目标的培训目标是发展进行蛋白质组分析的能力。在指导阶段获得的最先进的技术将极大地增加专业知识，使PI成为一名成功的独立调查员。对于独立的阶段，具体目标是：1)确定中性粒细胞胞吐作用调节的特定功能反应；2)确定体内抑制中性粒细胞胞吐作用阻断炎症反应的机制；3)研究刺激中性粒细胞胞吐作用的分子机制。申请者将接受的培训将使她在经验、兴趣和尖端技术的结合方面独树一帜，并将最大限度地发挥她成为NIH资助的独立调查员的潜力。(摘要结束)

DESCRIPTION (provided by applicant): The postulated role of exocytosis in mediating various functional responses associated with neutrophil activation has not been directly tested. We will use a novel approach; generating TAT-fusion proteins containing the coiled-coil domains of different SNARE proteins; to test the hypothesis that blocking exocytosis of distinct granule subsets will prevent specific neutrophil phenotypic changes and this strategy may be used to prevent neutrophil-mediated tissue damage in vivo. The specific objectives of the mentored phase of this proposal are: 1) to determine the ability of TAT-fusion proteins containing the SNARE domains of SNAP-23, syntaxins, and VAMPs to inhibit exocytosis of each of the four neutrophil granule subsets. The training goal of this aim is to develop molecular biology and cell biology skills that will allow the applicant to address experimental questions at new levels and apply them in the independent phase of this proposal. 2) To determine the most effective way to administer the TAT-SNARE fusion proteins in vivo and to determine the effect of these fusion proteins on models of acute neutrophil-dependent inflammation in rats. The training goals of this aim are to learn animal models of neutrophil-dependent inflammation, and to establish the ability of TAT-fusion proteins to inhibit neutrophil exocytosis in vivo. 3) Define other proteins that interact with SNARE proteins by proteomic analysis. The training goal of this aim is to develop the ability to perform proteomic analysis. The state-of-the-art techniques acquired during the mentored phase will greatly increase the expertise that will allow the PI to become a successful independent investigator. For the independent phase the specific objectives are: 1) To determine the specific functional responses that are regulated by neutrophil granule exocytosis; 2) To define the mechanisms by which inhibition of neutrophil exocytosis in vivo blocks the inflammatory response; and 3) To investigate the molecular mechanisms by which exocytosis is stimulated. The training the applicant will receive will make her unique in the combination of experience, interest, and state-of-the-art technology and will maximize her potential to become an independent NIH-funded investigator. (End of Abstract)