Fibronectin-dependent mechanisms governing the assembly of a definitive extracellular matrix

项目总结 这项拟议的工作试图揭示管理细胞外组装的新机制 基质(ECM)蛋白纤维连接蛋白(FN)及其在指导其他ECM蛋白组装形成 确定矩阵。因为确定矩阵是关键的物理、化学和机械属性的组成部分 调节组织结构和功能，了解协调FN组装的机制 I型胶原和其他ECM蛋白是至关重要的。基质组装中的分子缺陷与 由于FN、I型胶原和其他物质的异常产生而导致的无序的ECM纤维堆积的纤维化 基质蛋白。以及纤维化、骨骼异常、肿瘤形成和其他与ECM相关的疾病 影响着全球数百万人，但在大多数情况下，人们对ECM缺陷知之甚少，而且在 很多方面，很大程度上仍无法治愈。拟议的工作将检验这样的假设，即组织和 细胞周围FN基质的不溶性控制和指导组织适当的最终基质的组装， 而FN基质的这种干扰会扰乱组织和细胞的功能，导致疾病。我们有一位将军 了解FN基质组装的主要步骤，但了解控制FN原纤维的具体机制 组织，纤维稳定性，或FN如何引导胶原蛋白的组装尚未阐明。建议数 AIMS将解决这些机制，建立在我们与以前的 工作。我们将使用我们成熟的基质组装系统来分析FN纤维的形成及其 对I型胶原蛋白组装的贡献，以确定对指导 最终的矩阵装配。目标1的目标是确定转化可逆Fn-Fn的机制 相互作用形成稳定的不容溶解的纤维。我们将测试一个新的假设，即肝素/硫酸肝素作为一种 通过与Fn结合并诱导构象变化促进强蛋白质-蛋白质的分子开关 互动。Aim 2将通过以下方式解决FN基质作为胶原纤维形成模板的假设 为胶原蛋白加工酶的本地化和激活提供平台。Aim 3将链接FN 随细胞分化而组装。利用新发现的可能与骨骼相关联的人类FN突变 不典型增生，我们将确定该突变引起的基质组装缺陷，并将应用MicroMass. 软骨形成模型系统，以了解突变的FN基质对细胞分化的影响。我们的工作 将填补我们对管理大会关键步骤的机制的理解方面的关键知识空白 一个确定的矩阵，并将提出病理过程或突变可能导致异常的途径 矩阵组织或积累。这项工作将为操纵ECM的战略产生新的想法 为了治疗或控制纤维化和其他ECM相关疾病而组装，并可能导致以ECM为基础的 改善疾病结局的治疗。

PROJECT SUMMARY The proposed work seeks to uncover novel mechanisms governing the assembly of the extracellular matrix (ECM) protein fibronectin (FN) and its role in directing assembly of other ECM proteins to form a definitive matrix. Because the definitive matrix is integral to key physical, chemical, and mechanical properties that regulate tissue structures and functions, understanding mechanisms that coordinate the assembly of FN with type I collagen and other ECM proteins is crucial. Molecular defects in matrix assembly are implicated in fibrosis in which disordered ECM fibers accumulate due to abnormal production of FN, collagen I, and other matrix proteins. Along with fibrosis, skeletal abnormalities, tumorigenesis, and other ECM-related diseases affect millions of people around the globe yet in most cases the ECM defects are poorly understood and, in many ways, still largely untreatable. The proposed work will test the hypothesis that the organization and insolubility of the pericellular FN matrix control and direct the assembly of a tissue-appropriate definitive matrix, and that perturbation of the FN matrix disrupts tissue and cell functions leading to disease. We have a general understanding of the main steps of FN matrix assembly, but specific mechanisms governing FN fibril organization, fibril stability, or how FN guides assembly of collagens have yet to be elucidated. The proposed aims will address these mechanisms, building on the foundation that we have established with our previous work. We will use our proven matrix assembly systems to analyze the formation of FN fibrils and their contributions to type I collagen assembly to determine the protein interactions that are critical for directing definitive matrix assembly. The goal of Aim 1 is to determine the mechanism that converts reversible FN-FN interactions into stable insoluble fibrils. We will test a new hypothesis that heparin/heparan sulfate acts as a molecular switch by binding to FN and inducing conformational changes that promote strong protein-protein interactions. Aim 2 will address the hypothesis that FN matrix acts as a template for collagen fibrillogenesis by providing a platform for the localization and activation of collagen processing enzymes. Aim 3 will link FN assembly with cell differentiation. Using a newly discovered human FN mutation potentially linked to a skeletal dysplasia, we will determine the matrix assembly defect caused by this mutation and will apply the micromass chondrogenesis model system to understand the effects of mutant FN matrix on cell differentiation. Our work will fill critical knowledge gaps in our understanding of the mechanisms governing key steps in the assembly of a definitive matrix, and will suggest routes by which pathological processes or mutations can cause abnormal matrix organization or accumulation. This work will generate new ideas for strategies to manipulate ECM assembly in order to treat or control fibrosis and other ECM-related diseases and may lead to ECM-based treatments to improve disease outcomes.