Evaluate three v-SNAREs in regulated and constitutive TNF release from mast cells

项目总结 肥大细胞来源的肿瘤坏死因子在免疫反应中发挥重要作用，但当 过量会导致炎症性疾病，包括哮喘和类风湿性关节炎。与其他肿瘤坏死因子不同 产生细胞，肥大细胞将肿瘤坏死因子预先储存在细胞质颗粒中，这可以快速地经历 在特定条件下的胞吐/脱颗粒。肥大细胞也可以被激活以促进肿瘤坏死因子 转录，新合成的肿瘤坏死因子随着时间的推移通过结构性分泌载体释放，如 高尔基体衍生的囊泡。调节性和结构性的肿瘤坏死因子分泌都需要特定的相互作用。 在囊泡/颗粒锚定的圈套(v-圈套)和固定在血浆上的圈套之间 膜(靶膜；因此t型圈套)，导致反式圈套的形成 复合体和伴随的膜融合。免疫细胞胞外反式SNARE复合体 通常由VAMP(v-SNARE)、SNAP23(t-SNARE)和Synaxin(t-SNARE)组成。然而， 突显激活的肥大细胞释放肿瘤坏死因子的陷阱的特性在很大程度上是未知的， 这阻碍了对这些细胞中刺激特异性调节肿瘤坏死因子胞吐的理解。 这项建议的总体目标是发现在受监管的和 原代肥大细胞组成性分泌肿瘤坏死因子。我们实验室的新证据表明，肿瘤坏死因子是 与微血管小体(多囊泡体；一种晚期内小体)内的外体相关，后者依赖于 VAMP7或YKT6(一种独特的、多用途的v-陷阱)与质膜融合。同时， VAMP2最近被认为是介导高尔基体小泡胞吐的v-SNARE。 基于这些新的见解，我们假设肥大细胞使用VAMP7/Ykt6和VAMP2来 根据不同的环境线索，分别释放预先储存的和新合成的肿瘤坏死因子。 为了验证这一假设，我们将利用来自KO小鼠及其野生型的原始肥大细胞 追求两个特定的目标。AIM1将在#年评估VAMP7和YKT6的要求 急性/调节性肿瘤坏死因子胞吐。AIM2将评估构成性肿瘤坏死因子对VAMP2的需求 胞吐。这项研究的完成将勾勒出驱动肿瘤坏死因子的催化机制 不同环境条件下的胞吐作用，为揭示刺激特异性奠定了基础 肥大细胞特有的肿瘤坏死因子释放的调节。

PROJECT SUMMARY Mast cell-derived TNF (tumor necrosis factor) plays important roles in immune responses, but when in excess, causes inflammatory disorders including asthma and rheumatoid arthritis. Unlike other TNF producing cells, mast cells pre-store TNF in cytoplasmic granules, which can undergo rapid exocytosis/degranulation under specific conditions. Mast cells can also be activated to promote TNF transcription, and newly synthesized TNF is released over time via constitutive secretory carriers such as Golgi-derived vesicles. Both regulated and constitutive TNF secretion require specific interactions between vesicle/granule-anchored SNAREs (v-SNAREs) and SNAREs anchored to the plasma membrane (the target membrane; hence t-SNAREs), resulting in the formation of trans-SNARE complexes and concomitant membrane fusion. Exocytic trans-SNARE complexes in immune cells are typically composed of a VAMP (v-SNARE), a SNAP23 (t-SNARE) and a syntaxin (t-SNARE). However, the identities of the SNAREs that underscore TNF release from activated mast cells are largely unknown, which has hindered the understanding of the stimulus-specific regulation of TNF exocytosis in these cells. The overall objective of this proposal is to uncover the missing v-SNAREs required in regulated and constitutive TNF secretion from primary mast cells. New evidence from our lab has suggested that TNF is associated with exosomes within MVBs (multivesicular bodies; a type of late endosomes), which rely on VAMP7 or Ykt6 (a unique, multi-purpose v-SNARE) to fuse with the plasma membrane. Meanwhile, VAMP2 has recently emerged as the v-SNARE that mediates the exocytosis of Golgi-derived vesicles. Based on these fresh insights, we hypothesize that mast cells employ VAMP7/Ykt6 and VAMP2 to release prestored and newly synthesized TNF respectively in response to different environmental cues. To test this hypothesis, we will exploit primary mast cells derived from KO mice and their wildtype littermates to pursue two specific aims. Aim1 will assess the requirement of VAMP7 and Ykt6 in acute/regulated TNF exocytosis. Aim2 will assess the requirement of VAMP2 in constitutive TNF exocytosis. The completion of this study will delineate the catalytic machineries that drive TNF exocytosis under different environmental conditions, setting a stage for revealing stimulus-specific regulation of TNF release unique to mast cells.