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 肥大细胞来源的TNF（肿瘤坏死因子）在免疫应答中起重要作用，但当在 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TNF 肥大细胞将TNF预存于细胞质颗粒中， 在特定条件下的胞吐/脱粒。肥大细胞也可以被激活以促进TNF 转录，并且新合成的TNF随时间通过组成型分泌载体释放， 高尔基体衍生的囊泡调节性和组成性TNF分泌都需要特异性相互作用 在囊泡/颗粒锚定的SNARE（v-SNARE）和锚定到血浆的SNARE之间 膜（目标膜;因此是t-SNARE），导致trans-SNARE的形成 复合物和伴随的膜融合。免疫细胞中的胞外反式SNARE复合物是 通常由VAMP（v-SNARE）、SNAP 23（t-SNARE）和突触融合蛋白（t-SNARE）组成。然而，在这方面， 强调TNF从活化的肥大细胞释放的SNARE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。 本提案的总体目标是发现受监管和监管的企业所需的缺失的v-SNARE， 来自原代肥大细胞的组成型TNF分泌。我们实验室的新证据表明， 与MVB（多泡体;一种晚期内体）内的外泌体相关，其依赖于 VAMP 7或Ykt 6（一种独特的多用途v-SNARE）与质膜融合。同时， VAMP 2最近作为介导高尔基体衍生的囊泡的胞吐作用的v-SNARE出现。 基于这些新的见解，我们假设肥大细胞利用VAMP 7/Ykt 6和VAMP 2， 释放预存和新合成的TNF分别响应不同的环境线索。 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代肥大细胞 为了实现两个具体目标，Aim 1将评估VAMP 7和Ykt 6的要求， 急性/调节性TNF胞吐作用。Aim 2将评估组成型TNF中VAMP 2的需求 胞吐作用这项研究的完成将描绘出驱动TNF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 调节肥大细胞特有的TNF释放。