REGULATION OF EXOCYTOSIS IN MAST CELLS

肥大细胞专用于对免疫球蛋白E和相关过敏原的反应，并在启动过敏性炎症中发挥关键作用。它们通过分泌各种炎症介质和细胞因子来对刺激做出反应，这些炎症介质和细胞因子改变血管通透性，重塑细胞外基质，并招募其他放大炎症反应的宿主防御细胞。许多分泌产物储存在细胞质内的膜结合颗粒中，它们的释放是通过复合胞吐作用进行的，这是一种大规模的颗粒-质膜和颗粒-颗粒融合的级联，涉及大部分(如果不是全部)储存颗粒。虽然在了解IgE受体(FceR)的结构和早期信号方面已经取得了很大的进展，但对于将刺激与复合胞吐作用联系起来的级联反应中下游事件的调节和机制却知之甚少。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表明，在开发控制哮喘、过敏性休克和其他对变应原和活性多肽的急性宿主反应的治疗方法方面具有吸引力。形成这一建议基础的研究表明，肥大细胞中的复合胞吐受到一种新的机制的调节，该机制涉及刺激依赖的蛋白SNAP-23在细胞内的重新定位，该蛋白被认为是融合机制的一部分。Snap-23是膜融合蛋白SNARE家族中的一员，受片状脂膜样质膜折叠的分泌刺激，沿着质膜和细胞内向颗粒表面重新定位。SNAP-23的重新定位对于复合胞吐是必不可少的，并被认为涉及动员、细胞骨架辅助重新定位以及与其他SNARE蛋白接触以促进膜融合的不同步骤。这项提议的总体目标是描述组成这些步骤的分子机制的特征。将使用链溶素-O通透性肥大细胞和大鼠嗜碱性白血病(RBL-2H3)细胞来研究SNAP-23的磷酸化如何调节动员；动员前后哪些蛋白质与SNAP-23相互作用；Rho家族GTP酶和F-肌动蛋白如何促进重新定位；以及含有SNAP-23的SNARE复合体的组装如何与参与和分泌有关。此外，一种新发现的复合胞吐抑制物，一种来自分泌载体膜蛋白(SCAMPs)的多肽，将被用来分析其对SNAP-23的定位和功能的影响。

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.