FUNCTION OF MST4-EZRIN-ACAP4 SIGNALING IN GASTRIC PARIETAL CELL SECRETION AND HOMEOSTASIS

胃的消化功能取决于胃腔的酸化。酸分泌到 管腔是由cAMP依赖的蛋白激酶(PKA)级联激活触发的， 最终导致胃H，K-ATPase插入顶端质膜 细胞。一个偶联蛋白是Ezrin，一个80 kDa的磷酸蛋白，它被PKA在Ser66处磷酸化。 是壁细胞激活所必需的。我们早期的研究表明，幽门螺杆菌VacA扰乱了顶叶 细胞分泌，并通过钙蛋白酶诱导的Ezrin蛋白分解诱导壁细胞丢失。最新研究 证明胃化生和增生是壁细胞Ezrin丢失的结果， 提示Ezrin信号在壁细胞动态平衡和更新中的重要性。然而，几乎没有什么是 已知Ezrin信号通路在胃中作用的分子机制(S) 胃酸分泌和对幽门螺杆菌感染的反应。我们研究的长期目标是描绘出 Ezrin在胃壁细胞分泌和动态平衡中的作用 生理刺激和抗幽门螺杆菌感染。为了解决这个问题，有三个具体目标 建议：首先，我们将使用表位标签来评估MST4与ACAP4和Ezrin的相互作用 以及化学足迹的方法。这些研究将涉及对结构的详细分析 调节MST4-ACAP4直接接触的决定因素。绑定域数据将用于设计 在体外结合分析中有效和特异地干扰MST4-ACAP4相互作用的多肽。这个 这种相互作用的功能将通过多肽对酸分泌的影响来评估 通透性胃腺并将MST4相互作用缺陷的ACAP4突变体 培养ACAP4基因敲除动物的壁细胞。第二，我们将定义MST4在 调节ARF6 GTP酶在壁细胞激活过程中的时空动态。重要的是 在对幽门螺杆菌感染的生理刺激的反应中，酸分泌的这种相互作用将 然后通过功能分析和超分辨率成像分析进行评价。第三，我们计划说明 MST4-Ezrin-ACAP4信号轴协调壁细胞的分子机制 幽门螺杆菌感染时人胃器官的动态平衡。这些研究将是 通过生化和功能表征以及分泌物的光学成像来促进 新型ACAP4抑制剂MSM0601在活体壁细胞中的囊泡转运。 研究壁细胞分泌和动态平衡的分子机制具有重要意义 对了解肠道上皮细胞分泌调节的细胞生理学的意义 也有望在导致制定药物治疗策略方面大有裨益。 纠正和/或预防幽门螺杆菌感染引起的胃萎缩。

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin, an 80 kDa phosphoprotein, whose phosphorylation at Ser66 by PKA is required for parietal cell activation. Our early study demonstrated that H. pylori VacA perturbs parietal cell secretion and induces parietal cell loss via calpain-elicited ezrin proteolysis. Recent studies demonstrate that gastric metaplasia and hyperplasia are results of ezrin loss in the parietal cells, suggesting the importance of ezrin signaling in parietal cell homeostasis and renewal. However, little is known regarding the molecular mechanism(s) by which the ezrin signaling pathway operates in gastric acid secretion and responds to H. pylori infection. The long-term goal of our research is to delineate how ezrin functions in gastric parietal cell secretion and homeostasis in response to the physiological stimulation and to H. pylori infection. To address this question, three Specific Aims are proposed: first, we will evaluate how MST4 interacts with ACAP4 and ezrin using epitope-tagging and chemical footprinting approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct MST4-ACAP4 contact. Binding domain data will be used to design peptides that potently and specifically perturb MST4-ACAP4 interactions in in vitro binding assays. The function of this interaction will then be evaluated by the effects of the peptides on acid secretion using permeabilized gastric glands and by introducing MST4-interaction deficient ACAP4 mutants into cultured parietal cells from ACAP4 knock-out animals. Second, we will define the role of MST4 in regulating spatiotemporal dynamics of ARF6 GTPase during parietal cell activation. The importance of such an interaction in acid secretion in response to physiological stimulation of H. pylori infection will then be evaluated by functional assay and super-resolution imaging analysis. Third, we plan to illustrate the molecular mechanisms by which MST4-ezrin-ACAP4 signaling axis orchestrates parietal cell homeostasis in human gastric organoids in the presence of H. pylori infection. These studies will be facilitated by biochemical and functional characterization coupled with optical imaging of secretory vesicle trafficking in live parietal cells with novel ACAP4 inhibitor MSM0601. Studying the molecular mechanisms underlying parietal cell secretion and homeostasis is of great significance in understanding the cellular physiology of regulated epithelial secretion in the gut, and is also expected to be of great benefit in leading to development of pharmacological strategies for correction and/or prevention of H. pylori infection-elicited gastric atrophy.