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: 7035381
• 负责人: JAMES M. ANDERSON
• 金额: $ 27.93万
• 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/7035381
• 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）：这些研究的远距离目标是了解细胞质蛋白ZO-1如何在紧密连接（TJ）中组织蛋白质，以在副细胞途径中产生屏障。 ZO-1是多域MAGUK蛋白家族（膜相关的鸟苷酸激酶）的成员。它与3种不同的跨膜蛋白和至少10个细胞质蛋白和肌动蛋白结合。有充分的证据表明，ZO-1是组装TJ的关键支架，但几乎没有关于调节ZO-I与其结合伙伴之间相互作用的信息。其他MAGUK蛋白的结合活性受特定功能和结构结构域之间的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结构结合了occludin，Cingulin和Alpha-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，酸性结构域是剪接的，并与不同的亚细胞位置相关，并拯救了ZO-1 NULL突变体中不同信号通路。在哺乳动物培养的细胞中，缺乏酸性结构域的ZO-1表达在顶端和侧细胞表面上诱导了结构异常的异位TJ，这表明GUK-ACID相互作用控制了组装中的一步。当连接组装在培养细胞中时，WT蛋白在时间上之前缺乏酸性结构域，这表明该结构域需要响应动力学组装信号。我们将表征酸性结构域调节配体与ZO-1的GUK结构域的结合以及TJ结构和功能的功能后果的机制。我们将使用酵母2-杂交和融合蛋白测定和截短的上皮细胞中的碎片来表征这些蛋白质相互作用。等温滴定量热法将用于确定古克结构域及其蛋白质配体的亲和力，通过酸性结构域调节，并通过蛋白质磷酸化调节。通过有或没有SH3和酸性结构域的X射线结构，有或没有磷酸化，可以通过解决与GUK结构域结合的X射线晶体学结构，并具有和不具有磷酸化的情况，从而可视化原子结构及其机制。随着时间的允许，将对α-catenin和cingulin进行类似的研究。这些研究是第一个描述调节紧密连接组装的结构基础的研究，并将洞悉控制组装的机制。