转运蛋白颗粒复合物TRAPPC在气道粘蛋白爆发式分泌行为中的关隘效应

Airway mucin explosive exocytosis in response to various secretagogue stimulations is a complicated and sophisticated process. Although an increasing researches have been focused on the mechanisms of airway mucin exocytosis, few crucial regulatory pionts for these stimulations-induced secretory responses have been reported. The exocytosis of mucin can be divided into the following steps: coating, translocation, tethering, docking, fusion and exocytosis. But so far the tethering step is just the missing link in the continuous process. The discovery of transport protein particle complex (TRAPPC) which has a dual-function of tethering protein and guanine nucleotide exchanger factor has demonstrated an underlying molecular mechanism for vesicle tethered on the targeted membrane. Furthermore, the tethering step, to a certain extent, plays a “gated point” effect, leading to the pathological explosive secretory process. In the present study, we determine the role of neutrophil elastase on mucin5AC (MUC5AC) secretion in vivo and in vitro. We also analyse the intracellular location and expression of TRAPPC subunit, and subsequent its gated-effect on MUC5AC explosive exocytosis by transfection with the mutant of TRAPPC subunit. We further assess the function of Rab protein, and its interaction with TRAPPC subunit in the MUC5AC excessive secretion induced by TRAPPC. Thus, we explore the hypothesis that tethering protein complex TRAPPC is responsible for the enhanced excessive exocytosis of MUC5AC, and this identification might aid in the discovery of new therapeutic approaches to mitigate the mucin explosive secretion.

气道黏液高分泌可由诸多因素导致，但这些因素纷繁复杂，且下游途径多有交错，难以凝炼出有效的干预靶点。粘蛋白分泌大致可分为包被、转运、拴系、锚定、膜融合和出胞等步骤，但目前对拴系结构基础及作用机制尚不明确。据信具备拴系和鸟嘌呤核苷酸交换因子双重功能的转运蛋白颗粒复合物（TRAPPC）与其调控蛋白Rab的相互作用是分泌囊泡拴系于靶标膜的关键，而此步骤恰恰是具有殊途同归特征的关隘节点，具有理想的可干预性。本研究借在体和离体实验，观察中性粒细胞弹力酶刺激下粘蛋白5AC的爆发式分泌特点，并通过构建TRAPPC突变体，揭示TRAPPC的胞内定位、表达及在粘蛋白爆发式分泌过程中的拴系关隘效应，从而明确其角色功能。同时观察调控蛋白Rab及其突变体与TRAPPC的相互作用，阐明Rab对TRAPPC调控特点，以期深入诠释气道粘蛋白爆发式出胞方式的关隘节点，并为防治气道粘蛋白爆发式分泌提供有效的干预靶点。

气道黏液高分泌是慢性气道炎症性疾病进展、恶化的独立危险因素。诸多因素可诱导气道黏液高分泌的发生和发展，但这些因素纷繁复杂，且其下游途径多有交集，难以凝炼出根本性的可干预靶点。气道黏液的主要有型成分粘蛋白5AC（MUC5AC）分泌大致可分为分泌囊泡的包被、胞内膜性细胞器之间的定向转运、分泌囊泡与靶标膜的拴系、锚定、膜融合和出胞等步骤，但目前对其出胞时与靶标膜的拴系耦联机制尚不明确。据信，转运蛋白颗粒复合物（TRAPPC）与其调控蛋白Rab/Rho蛋白的相互作用是分泌囊泡拴系于靶标膜的关键，而此步骤恰恰属关隘出口，是理想的干预节点。本研究借助在体动物实验和离体细胞实验，观察中性粒细胞弹力酶刺激下粘蛋白5AC的高分泌，并通过观察TRAPPC及其突变体的分布特征，揭示TRAPPC的胞内定位、表达及在粘蛋白出胞过程中的拴系耦联模式，从而明确其在此过程中的功用。同时观察调控蛋白Rab/Rho蛋白与TRAPPC的相互作用，阐明Rab/Rho蛋白对TRAPPC介导效应的调控特点，以期深入阐述气道粘蛋白MUC5AC的出胞拴系模式和关键位点，以期为气道黏液高分泌我提供可能的干预靶点。

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