ZO-1 and Cytoplasmic Scaffolding of the Tight Junction

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，以便在细胞旁途径中形成屏障。ZO-1是多结构域MAGUK蛋白家族（膜相关鸟苷酸激酶）的成员。它结合3种不同的跨膜蛋白和至少10种细胞质蛋白和肌动蛋白。有强有力的证据表明ZO-1是组装TJ的关键支架，但关于什么调节ZO-1与其结合伴侣之间的相互作用的信息很少。其他MAGUK蛋白的结合活性由特定功能和结构域之间的分子内和分子间相互作用调节。我们的初步研究表明ZO-1的GUK结构域结合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，酸性结构域是选择性剪接的，并且与ZO-1无效突变体中不同的亚细胞位置和不同信号通路的拯救相关。在哺乳动物培养的细胞中，ZO-1缺乏酸性结构域的表达诱导顶端和侧面细胞表面上的结构异常的异位TJ，这意味着GUK-酸相互作用控制组装中的一个步骤。当连接在培养的细胞中组装时，WT蛋白在时间上先于缺乏酸性结构域的ZO-1，表明该结构域是响应动力学组装信号所必需的。我们将表征酸性结构域调节配体与ZO-1的GUK结构域结合的机制，以及TJ结构和功能的功能后果。我们将使用酵母双杂交和融合蛋白测定和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，它们通过酸性结构域的调节以及通过蛋白质磷酸化的可能调节。原子结构及其相互作用的调节机制将通过求解结合到具有和不具有SH 3和酸性结构域的GUK结构域以及具有和不具有磷酸化的occludin的X射线晶体学结构来可视化。如果时间允许，将对α-连环蛋白和扣带蛋白进行类似的研究。这些研究是第一个描述的结构基础，调节组装的紧密连接，并将提供深入了解控制组装的机制。

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.