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

评估肥大细胞调节和组成型 TNF 释放中的三种 v-SNARE

• 批准号: 10432701
• 负责人: Hao Xu
• 金额: $ 7.4万
• 依托单位: UNIVERSITY OF SOUTHERN MISSISSIPPI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10432701
• 关键词: Acute; Allergens; Allergic inflammation; Animals; Asthma; Biological Assay; Bone Marrow; Breeding; Cell Line; Cell membrane; Cell surface; Cells; Complex; Confocal Microscopy; Cues; Cytoplasmic Granules; Development; Disease; Double-Stranded RNA; Endosomes; Endotoxins; Enzyme-Linked Immunosorbent Assay; Exocytosis; Fluorescence; Golgi Apparatus; Harvest; IgE; Immune; Immune response; Inflammatory; Innate Immune Response; Investigation; Knock-out; Knockout Mice; Lead; Length; Light; Measures; Mediating; Membrane; Membrane Fusion; Methods; Molecular; Multivesicular Body; Mus; Natural Immunity; Nature; Pathway interactions; Pattern; Peritoneal; Play; Poly I-C; Proteolysis; Recycling; Regulation; Rheumatoid Arthritis; Role; SNAP receptor; SNAP23 gene; Signal Transduction; Stimulus; TNF gene; Testing; The Jackson Laboratory; Time; Tumor-Derived; Vesicle; Viral; Work; adaptive immunity; anti-IgE; base; cellubrevin; combat; exosome; granule cell; insight; late endosome; mast cell; novel therapeutic intervention; pathogen; release factor; response; syntaxin A; transcription factor; vesicular SNARE proteins

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.

项目摘要 肥大细胞衍生的TNF（肿瘤坏死因子）在免疫反应中起着重要作用，但是在 过量，引起炎症性疾病，包括哮喘和类风湿关节炎。与其他TNF不同 产生细胞的细胞，肥大细胞在细胞质颗粒中预储存的TNF，可以快速进行 在特定条件下的胞吐/脱粒。肥大细胞也可以激活以促进TNF 转录和新合成的TNF随时间推移通过构成分泌载体（例如 高尔基体囊泡。受监管和本构的TNF分泌都需要特定的相互作用 在囊泡/颗粒锚定的幼虫（V-SNARES）和固定在等离子体上的小网之间 膜（靶膜；因此T-snares），导致跨环的形成 复合物和伴有膜融合。免疫细胞中的外旋转式鼻nare复合物是 通常由鞋面（V-SNARE），SNAP23（T-SNARE）和语法（T-SNARE）组成。然而， 强调从活化肥大细胞释放TNF的网罗的身份在很大程度上是未知的， 这阻碍了对这些细胞中TNF胞吐作用的刺激特异性调节的理解。 该提案的总体目的是揭示受监管和 原代肥大细胞的组成型TNF分泌。我们实验室的新证据表明TNF是 与MVB中的外泌体相关（多囊体；一种晚期内体），依赖于 VAMP7或YKT6（一种独特的多功能V-SNARE）与质膜融合。同时， VAMP2最近成为介导高尔基体囊泡的胞吐作用的V-SNARE。 基于这些新的见解，我们假设肥大细胞采用VAMP7/YKT6和VAMP2到 响应不同的环境提示，分别释放预测和新合成的TNF。 为了检验该假设，我们将利用源自KO小鼠及其野生型的原代肥大细胞 同窝官追求两个具体目标。 AIM1将评估VAMP7和YKT6的要求 急性/调节的TNF胞吐作用。 AIM2将评估构成型TNF中VAMP2的需求 胞吞作用。这项研究的完成将描绘驱动TNF的催化机器 在不同的环境条件下胞吐作用，为揭示特异性刺激的阶段 TNF释放的调节是肥大细胞独有的。