Apical Ezrin Assembly the Cytoskeleton and Diarrheal Disorders

顶端埃兹蛋白组装细胞骨架和腹泻疾病

• 批准号: 8010945
• 负责人: Pedro Salas
• 金额: $ 30.12万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010945
• 关键词: Actins; Apical; Binding; Biological Assay; Brush Border; Cataloging; Catalogs; Cell Line; Cells; Chimera organism; Chloride Ion; Chlorides; Complex; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeleton; Data; Diarrhea; Disease; Dominant-Negative Mutation; ERM protein; Enterocytes; Epithelial Cells; F-Actin; Goals; Health; Immunoblotting; In Situ; In Vitro; Intermediate Filaments; Intervention; Intestines; Ion Channel; Keratin; Knockout Mice; Location; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Conformation; Mus; N Domain; Phosphorylation; Phosphotransferases; Physiologic pulse; Physiological; Play; Protein Isoforms; Proteins; Publishing; Reagent; Recombinants; Regulation; Research Personnel; Role; Signal Transduction; Site; Testing; Water; Work; apical membrane; base; cell type; deletion analysis; ezrin; in vivo; induced pluripotent stem cell; knock-down; mutant; overexpression; programs; protein protein interaction; research study; scaffold; small hairpin RNA; sodium-hydrogen exchanger regulatory factor; tissue culture

Two apical membrane proteins in intestinal epithelial cells are important effectors of cAMP-mediated signaling that result in secretory diarrheas: NHE-3 and CFTR. Both are attached via PDZ interactions to NHERF proteins, which, in turn, are attached to ezrin and actin. The integrity of this scaffold is essential for PKA/cAMP signaling on CFTR and may play a role as an apical retention signal for polarization. Previous published work in our lab has highlighted the importance of intermediate filaments (IPs) in the organization of the apical domain in simple epithelial cells, and suggest that IPs play a role in the organization of the apical actin-based scaffold in these cells. Furthermore, IPs mediate the assembly of ezrin into the above-mentioned scaffold, and PKCiota or alpha may be responsible for the activation of ezrin. The hypothesis is that binding of ezrin to apical intermediate filaments initiates the apical localization of ezrin in intestinal cells and provides a microdomain-restricted site for its activation by PKCi. The assembly of this ezrin-based scaffold is essential for the function of the effectors of secretory diarrheas (NHE-3 and CFTR). To test this hypothesis I propose to: 1) Identify the molecular interactions between ezrin and keratins and their role to localize ezrin to the apical domain of enterocytes, using T567D / T567A ezrin mutants in vitro binding assays, or GFP-ezrin constructs in vivo. 2) Identify the physiological activator of ezrin in the brushborder and the molecular interactions between PKCi or a and keratins, using recombinant PKCisoforms, expression of anti- PKCi shRNA, dominant negative PKCisoforms, pharmacological blockers, stable TET-inducible CACO-2 cell lines that knock down keratin 8, and K8 null mice. And 3) Test the functional consequences of apical ezrin complex assembly on cAMP-dependent CI"secretion, PKA localization and activation, signaling downstream of ezrin and CFTR localization, using the experimental variables and reagents created and analyzed in the previous aims in Ussing chamber experiments. The long- term goal of this project is to identify the molecular interactions responsible for the assembly of the apical ezrin- actin scaffold in intestinal cells that can be manipulated to interferewith the function of ion channels in diarrheal disorders. Lay statement: Secretory diarrheas are a severe health problem in the U.S. and worldwide. All of them operate through common effectors in the intestine that enable the secretion of chloride (and water). The machinery that supports these membrane proteins is based on cytoskeletal components and is assembled around a protein known as ezrin. This project seeks to test the hypothesisthat ezrin (and its associated chloride secreting proteins) require spatial cues from a division of the cytoskeleton,the intermediate filaments, to become assembled in the precise location to secrete water to the lumen of the intestine. The study of protein-protein interactions that result in the precise and adequate localization of ezrin and its associated proteins will provide potential points for molecular intervention on the function of this molecular complex.

肠上皮细胞中的两个顶膜蛋白是cAMP介导的信号传导的重要效应因子 这导致分泌性腹泻：NHE-3和CFTR。两者都通过PDZ相互作用连接到NHERF蛋白， 反过来，它附着在ezrin和肌动蛋白上。该脚手架的完整性对于PKA/CAMP信号至关重要 在CFTR上，可以作为极化的顶端保留信号发挥作用。以前在我们的实验室发表的作品 已经强调了中间细丝（IP）在简单中的顶端域的组织中的重要性 上皮细胞，并建议IP在基于顶端肌动蛋白的支架的组织中发挥作用 细胞。此外，IPS将Ezrin的组装介导上述脚手架，PKCiota或Alpha 可能导致埃兹林的激活。假设是埃兹林与顶端中间体结合 细丝启动丝斯蛋白在肠细胞中的顶端定位，并为微区限制位点提供 它通过pkci激活。这种基于Ezrin的支架的组装对于效应子的功能至关重要 分泌性腹泻（NHE-3和CFTR）。要检验此假设，我建议：1）确定分子 ezrin和角蛋白之间的相互作用及其在肠球菌顶端域将Ezrin定位的作用 T567D / T567A Ezrin突变体在体外结合测定中或体内GFP-综合蛋白构建体。 2）确定 Ezrin的生理激活剂在灌木丛中的生理激活剂以及PKCI或A之间的分子相互作用 角蛋白，使用重组PKCISOFORS，抗PKCI shRNA的表达，主要负PKCISOFORM， 药理学阻滞剂，稳定的TET诱导型CACO-2细胞系，可击落角蛋白8和K8无效小鼠。 3）测试根尖丝素复合物组件对CAMP依赖性CI的分泌的功能后果， PKA定位和激活，Ezrin和CFTR定位的下游信号传导，使用实验 在使用室内实验的先前目标中创建和分析的变量和试剂。长期 该项目的术语目标是确定负责顶端ezrin-组装的分子相互作用 肠细胞中的肌动蛋白支架可以操纵以干扰离子通道在腹泻中的功能 疾病。外行陈述：分泌性腹泻是美国和全球的严重健康问题。所有人 它们通过肠道中的共同效应子进行操作，使氯化物（和水）分泌。这 支持这些膜蛋白的机械基于细胞骨架成分，并组装 周围蛋白质称为ezrin。该项目旨在测试ezrin的假说（及其相关的氯化物 分泌蛋白）需要从细胞骨架的一个中间细丝的空间提示。 在精确的位置组装，将水分泌到肠腔。蛋白质蛋白的研究 导致Ezrin及其相关蛋白质的精确和适当定位的相互作用将提供 分子干预该分子复合物功能的潜在点。