REGULATION OF EXOCYTOSIS IN MAST CELLS

肥大细胞是专门针对免疫球蛋白E和相关过敏原的，并在引发过敏性炎症中起关键作用。它们通过分泌多种炎症介质和细胞因子来应对刺激，这些炎症介质和细胞因子可以改变血管通透性，重塑细胞外基质，并招募其他宿主防御细胞来放大炎症反应。许多分泌产物储存在细胞质内的膜结合颗粒中，它们的释放通过复合胞吐发生，这是一个涉及大多数（如果不是全部的话）储存颗粒的颗粒-质膜和颗粒-颗粒融合的巨大级联反应。虽然在了解IgE受体（FceR）的结构和早期信号传导方面取得了很大进展，但对连接刺激和复合胞吐的级联下游事件的调节和机制知之甚少。了解这些事件具有很大的医学意义，因为分层炎症反应的早期发生为开发控制哮喘、过敏性休克和其他对过敏原和活性肽的急性宿主反应的治疗提供了一个有吸引力的干预位点。形成这一建议基础的研究表明，肥大细胞中的复合胞外分泌是由一种新机制调节的，该机制涉及细胞内SNAP-23蛋白的刺激依赖性重新定位，该蛋白被认为是融合机制的一部分。SNAP-23是SNARE膜融合蛋白家族中的一员，在分泌刺激的作用下，从片足样的质膜褶皱沿着质膜和细胞内向颗粒表面迁移。SNAP-23的重新定位对复合胞外作用至关重要，据推测，它涉及动员、细胞骨架辅助重新定位和与其他SNARE蛋白结合以促进膜融合的不同步骤。本建议的总体目标是描述组成这些步骤的分子机制。Streptolysin-O通透性肥大细胞和大鼠嗜碱性白血病（RBL-2H3）细胞将用于研究SNAP-23磷酸化如何调节动员；动员前后哪些蛋白与SNAP-23相互作用；Rho家族GTPases和F-actin如何促进迁移；以及含有snap -23的SNARE复合物的组装如何与接合和分泌相关。此外，一种新发现的化合物胞吐抑制剂，一种从分泌载体膜蛋白（SCAMPs）衍生的肽，将用于分析其对SNAP-23的重新定位和功能的影响。

Mast cells are specialized for responding to immunoglobulin E and associated allergens and play a key role in initiating allergic inflammation. They respond to stimulation by secreting a variety of inflammatory mediators and cytokines that alter vascular permeability, remodel extracellular matrix, and recruit other host defense cells that amplify the inflammatory response. Many of the secretory products are stored in membrane-bounded granules within the cytoplasm, and their release occurs by compound exocytosis, a massive cascade of granule-plasma membrane and granule-granule fusions involving most if not all of the storage granules. While much progress has been made in understanding the structure and early signaling of the IgE receptor (FceR), much less is known about the regulation and mechanisms of downstream events in the cascade that links stimulation to compound exocytosis. Understanding these events is of great medical interest as early occurrence in the hierarchical inflammatory response suggests an attractive site for intervention in developing therapies that control asthma, anaphylactic shock, and other acute host reactions to allergens and active peptides. Studies forming the basis of this proposal have shown that compound exocytosis in mast cells is regulated by a novel mechanism involving stimulus-dependent relocation within the cell of the protein SNAP-23 that is thought to comprise part of the fusion machinery. SNAP-23, one of the SNARE family of membrane fusion proteins, relocates in response to secretory stimulation from lamellipodia-like plasma membrane folds along the plasma membrane and intracellularly to granule surfaces. Relocation of SNAP-23 is essential for compound exocytosis and is hypothesized to involve distinct steps of mobilization, cytoskeletally-assisted relocation, and engagement with other SNARE proteins to promote membrane fusion. The overall goal of this proposal is to characterize the molecular mechanisms comprising each of these steps. Streptolysin-O permeabilized mast cells and rat basophilic leukemia (RBL-2H3) cells will be used to address how phosphorylation of SNAP-23 regulates mobilization; what proteins interact with SNAP-23 preceding and following mobilization; how Rho family GTPases and F-actin promote relocation; and how assembly of SNAP-23-containing SNARE complexes relates to engagement and secretion. In addition, a newly discovered inhibitor of compound exocytosis, a peptide derived from one of the secretory carrier membrane proteins (SCAMPs), will be used to analyze its effects on the relocation and function of SNAP-23.