ZO-1 and Cytoplasmic Scaffolding of the Tight Junction

ZO-1 和紧密连接的细胞质支架

• 批准号: 6611758
• 负责人: JAMES M. ANDERSON
• 金额: $ 27.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-21 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611758
• 关键词: SDS polyacrylamide gel electrophoresis; X ray crystallography; binding proteins; cadherins; calorimetry; chimeric proteins; cytoplasm; immunocytochemistry; ligands; membrane proteins; nucleoside phosphate kinase; phosphorylation; posttranslational modifications; protein binding; protein protein interaction; protein structure function; tight junctions; tissue /cell culture; yeast two hybrid system

DESCRIPTION (provided by applicant): The long-range goal of these studies is to understand how the cytoplasmic protein ZO-1 organizes proteins at tight junction (TJ) in order to create a barrier in the paracellular pathway. ZO-1 is a member of the multi-domain MAGUK protein family (membrane-associated guanylate kinase). It binds to 3 different transmembrane proteins and at least 10 cytoplasmic proteins and actin. There is strong evidence that ZO-1 is a critical scaffold for assembling the TJ but little information about what regulates the interaction between ZO-I and its binding partners. The binding activity of other MAGUK proteins is regulated by intra- and intermolecular interactions between specific functional and structural domains. Our preliminary studies show the GUK domain of ZO-1 binds occludin, cingulin and alpha-catenin, while an adjacent acidic domain can inhibit these interacts. In Drosophila ZO-1 the acidic domain is alternatively spliced and correlates with different subcellular locations and rescue of different signaling pathways in the ZO-1 null mutant. In mammalian cultured cells, expression of ZO-1 lacking the acidic domain induces structurally aberrant ectopic TJs on the apical and lateral cell surfaces, implying the GUK-acid interaction controls a step in assembly. As junctions assemble in cultured cells the WT protein temporally precedes ZO-1 lacking the acid domain, suggesting the domain is required to respond to kinetic assembly signals. We will characterize the mechanisms by which the acidic domain regulates ligand binding to the GUK domain of ZO-1 and the functional consequences for TJ structure and function. We will characterize these protein interactions using yeast 2-hybrid and fusion protein assays and truncated fragments in cultured epithelial cells. Isothermal titration calorimetry will be used to determine the affinity of the GUK domain and its protein ligands, their regulation by the acidic domain and possible regulation by protein phosphorylation. The atomic structures and their mechanisms of regulated interaction will be visualized by solving the x-ray crystallographic structures of occludin bound to the GUK domain with and without the SH3 and acidic domains, and with and without phosphorylation. Similar studies will be performed on alpha-catenin and cingulin as time permits. These studies are the first to describe the structural basis for regulated assembly of tight junctions and will provide insight into the mechanisms controlling assembly.

描述（由申请人提供）：这些研究的长期目标是了解细胞质蛋白 ZO-1 如何在紧密连接 (TJ) 处组织蛋白质，以便在细胞旁通路中形成屏障。 ZO-1 是多结构域 MAGUK 蛋白家族（膜相关鸟苷酸激酶）的成员。它与 3 种不同的跨膜蛋白和至少 10 种细胞质蛋白和肌动蛋白结合。有强有力的证据表明 ZO-1 是组装 TJ 的关键支架，但关于调节 ZO-I 及其结合伙伴之间相互作用的信息却很少。其他 MAGUK 蛋白的结合活性受特定功能域和结构域之间分子内和分子间相互作用的调节。我们的初步研究表明，ZO-1 的 GUK 结构域结合 occludin、cingulin 和 α-catenin，而相邻的酸性结构域可以抑制这些相互作用。在果蝇 ZO-1 中，酸性结构域是选择性剪接的，并与不同的亚细胞位置相关，并拯救 ZO-1 无效突变体中的不同信号传导途径。在哺乳动物培养细胞中，缺乏酸性结构域的 ZO-1 表达会在细胞顶端和侧部表面诱导结构异常的异位 TJ，这意味着 GUK-酸相互作用控制着组装步骤。当连接在培养细胞中组装时，WT 蛋白暂时先于缺乏酸性结构域的 ZO-1，这表明需要该结构域来响应动力学组装信号。我们将描述酸性结构域调节配体与 ZO-1 GUK 结构域结合的机制以及对 TJ 结构和功能的功能影响。我们将使用酵母 2-杂交和融合蛋白测定以及培养的上皮细胞中的截短片段来表征这些蛋白质相互作用。等温滴定量热法将用于确定 GUK 结构域及其蛋白质配体的亲和力、酸性结构域对其的调节以及蛋白质磷酸化的可能调节。通过解析与 GUK 结构域结合的 occludin 的 X 射线晶体结构，无论有或没有 SH3 和酸性结构域，以及有或没有磷酸化，原子结构及其调节相互作用的机制将被可视化。如果时间允许，将对 α-连环蛋白和 cingulin 进行类似的研究。这些研究首次描述了紧密连接调节组装的结构基础，并将提供对控制组装机制的深入了解。