Catenins, cadherins and their interactions

描述（由申请人提供）：细胞组装成组织取决于细胞间连接的形成。在粘附连接中，连环蛋白将跨膜钙粘蛋白细胞粘附分子连接到基于肌动蛋白的细胞骨架，并且在桥粒中，类似的蛋白质组装将钙粘蛋白连接到中间丝。粘附连接组装是细胞和组织结构发育的重要步骤，这些连接的丧失是转移癌细胞的标志。同样，细胞-细胞接触，如中间丝是必不可少的机械强度和完整性的组织，如皮肤和心脏，这些组件的缺陷是造成许多严重的皮肤起泡疾病。粘附连接蛋白2-连环蛋白也作为Wnt信号通路中的转录共激活因子，其控制细胞命运决定和成人组织的正常更新。在许多癌症中，由于Wnt途径组分中的突变导致的2-连环蛋白的稳定导致Wnt靶基因的不适当激活。该提案的目标是通过使用纯化的组分来重建这些分子复合物的关键部分，从而实现对细胞粘附组装体和Wnt信号传导中的2-连环蛋白的机械理解。生物化学和生物物理方法将用于确定这些相互作用的结构和亲和力。1.肌动蛋白为基础的细胞-细胞接触的组装和结构将在上皮细胞和神经元中进行研究，通过研究1-连环蛋白的上皮和神经元版本之间的相似性和差异，特别是其2-连环蛋白和肌动蛋白结合特性。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蛋白-连环蛋白复合物与肌动蛋白中的潜在作用。2.细胞粘附组件的进化起源将通过研究1-和2-连环蛋白在黏菌Dictyosteelium discoideum同源。3.桥粒钙粘蛋白与斑珠蛋白结合的结构将被确定，桥粒斑蛋白的N-和C-末端部分也将被确定，桥粒斑蛋白将钙粘蛋白-斑珠蛋白复合物连接到中间丝。4.将研究Wnt信号控制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Lrp 6的磷酸化如何抑制2-连环蛋白的磷酸化，以及磷酸化复合物如何与泛素化机制联系在一起。5. Tcf/Lef-family转录因子如何控制Wnt靶向信号传导的分子机制将通过确定与转录阻遏物和2-连环蛋白激活物复合物结合的Lef-I的结构来研究。公共卫生相关性：在发育过程中，细胞分化并与其他细胞形成特异性接触，以产生组织。这一过程中的缺陷会导致发育异常，当癌细胞从实体组织中逃逸时，细胞与细胞接触的丧失会发生在癌症转移和其他疾病中。这个基础研究项目旨在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.