Catenins, cadherins and their interactions

描述(申请人提供)：细胞组装成组织依赖于细胞间连接的形成。在粘连连接中，连接素将跨膜钙粘附素细胞黏附分子连接到基于肌动蛋白的细胞骨架，而在桥粒中，类似的蛋白质组合将钙粘附素连接到中间丝。黏附连接组装是细胞和组织结构发育的重要步骤，而这些连接的缺失是癌细胞转移的标志。同样，细胞与细胞之间的接触，如中间细丝，对于皮肤和心脏等组织的机械强度和完整性是必不可少的，这些组件中的缺陷导致了许多严重的皮肤起泡疾病。黏附连接蛋白2-连环蛋白也是Wnt信号通路中的转录共激活因子，该信号通路控制细胞命运的决定和成人组织的正常更新。在许多癌症中，由于Wnt途径组分的突变导致2-catenin的稳定，导致Wnt靶基因的不适当激活。这项提议的目的是通过使用纯化的成分来重组这些分子复合体的关键部分，从而实现对细胞黏附组装和2-连环蛋白在Wnt信号中的机制的理解。将使用生化和生物物理方法来确定这些相互作用的结构和亲和力。1.通过研究1-连环蛋白在上皮细胞和神经元中的异同，特别是其2-连环蛋白和肌动蛋白结合的特性，研究基于肌动蛋白的细胞-细胞接触的组装和结构。我们还将研究EPLIN与1-E-连环蛋白和肌动蛋白的相互作用，以评估其在连接钙粘连蛋白-连环蛋白复合体和肌动蛋白中的潜在作用。2.通过检测1-和2-连环素在黏菌盘基网柄菌中的同源物，研究细胞黏附组件的进化起源。3.将确定与蛋白结合的桥粒钙粘蛋白的结构，以及桥粒蛋白的N-端和C-端部分，它将钙粘蛋白-蛋白复合体与中间丝连接起来。4.研究Wnt信号调控2-连环蛋白破坏所需的磷酸化和泛素化的机制，特别是Wnt受体LRP6的磷酸化如何抑制2-连环蛋白的磷酸化，以及磷酸化复合体如何与泛素化机制相连。5.Tcf/Lef家族转录因子调控Wnt靶信号的分子机制将通过确定Lef-1与转录抑制因子和2-连环蛋白-激活剂复合体结合的结构来研究。与公共卫生相关：在发育过程中，细胞分化并与其他细胞形成特定的接触，以形成组织。这一过程中的缺陷会导致发育异常，在癌症转移、癌细胞从固体组织中逃逸时以及在其他疾病中会发生细胞-细胞接触的丧失。这个基础研究项目试图了解负责这些过程的分子组装。

DESCRIPTION (provided by applicant): The assembly of cells into tissues depends upon formation of intercellular junctions. In adherens junctions, the catenins link transmembrane cadherin cell adhesion molecules to the actin-based cytoskeleton, and in desmosomes an analogous protein assembly links cadherins to intermediate filaments. Adherens junction assembly is an essential step in the development of cell and tissue structure, and loss of these junctions is a hallmark of metastasizing cancer cells. Likewise, cell-cell contacts liked to intermediate filaments are essential for the mechanical strength and integrity of tissues such as the skin and heart, and defects in these assemblies are responsible for a number of severe skin blistering diseases. The adherens junction protein 2-catenin also serves as a transcriptional coactivator in the Wnt signaling pathway that controls cell fate determination and in the normal renewal of tissues in the adult. In many cancers, stabilization of 2-catenin due to mutations in Wnt pathway components results in inappropriate activation of Wnt target genes. The goal of this proposal is to achieve a mechanistic understanding of cell adhesion assemblies and of 2- catenin in Wnt signaling by using purified components to reconstitute key parts of these molecular complexes. Biochemical and biophysical methods will be used to determine the structures and affinities of these interactions. 1. The assembly and structure of actin-based cell-cell contacts will be investigated in epithelia and neurons by examining the similarities and differences between the epithelial and neuronal versions of 1-catenin, in particular its 2-catenin and actin-binding properties. We will also study the interactions of EPLIN with 1-E- catenin and actin to assess its potential role in linking the cadherin-catenin complex to actin. 2. The evolutionary origins of cell adhesion assemblies will be studied by examining homologs of 1- and 2- catenin in the slime mold Dictyostelium discoideum. 3. Structures of desmosomal cadherins bound to plakoglobin will be determined, as will the N- and C-terminal portions of desmoplakin, which links the cadherin-plakoglobin complex to intermediate filaments. 4. The mechanisms by which phosphorylation and ubiquitination required for 2-catenin destruction are controlled by Wnt signaling will be studied, in particular how phosphorylation of the Wnt receptor Lrp6 inhibits phosphorylation of 2-catenin, and how the phosphorylation complex is linked to the ubiquitination machinery. 5. The molecular mechanism of how Tcf/Lef-family transcription factors control Wnt target signaling will be investigated by determining structures of Lef-1 bound to a transcriptional repressor and to a 2-catenin-activator complex. PUBLIC HEALTH RELEVANCE: During development, cells differentiate and form specific contacts with other cells in order to create tissues. Defects in this process give rise to developmental abnormalities, and loss of cell-cell contacts occurs in cancer metastasis, when cancer cells escape from a solid tissue, and in other diseases. This basic research project seeks to understand the molecular assemblies responsible for these processes.