THROMBOSPONDIN-4

描述(申请人提供)：凝血酶原蛋白(TSPs)是一个由五种蛋白质组成的家族，在组织发生和重塑过程中，它们与细胞表面和细胞外基质(ECM)之间的界面密切相关。该项目的长期目标是阐明TSP基因家族成员的结构和功能。下一个支助期的具体重点将放在以下领域。特定目标1.CoMP的结构测定。对于不同的TSP家族成员，结构和功能数据表明类型3重复，C-末端结构域形成单个复合体。本研究的目的是利用X射线结晶学和核磁共振技术确定软骨寡聚基质蛋白(COMP)的3型重复/C末端复合体的结构。基于分子模拟的方法，我们假设COMP的C-末端折叠形成一个四叶片的贝塔螺旋桨，而类型3重复形成的环状结构与贝塔螺旋桨的一个表面密切相关。此外，我们假设3型重复和C-末端结构域的并列对于最佳的胶原、蛋白多糖(PG)和整合素结合是必不可少的。具体目标2.确定COMP与蛋白质和PGs相互作用的关键氨基酸。Comp是一种基质细胞蛋白的假说意味着它参与ECM的组装或结构，并与细胞表面蛋白和PGs结合来调节细胞表型。这一目标的目的是详细了解COMP与II型胶原、纤维连接蛋白、整合素和糖胺多聚糖(GAG)相互作用的分子基础。在3型重复/C末端复合体中，这些相互作用的关键氨基酸将通过在完整分子和单个结构域的背景下进行定点突变来鉴定。特定目的3.COMP细胞表面受体的鉴定。初步数据表明，comp(1)与软骨细胞上的PGs和整合素相互作用，(2)调节软骨形成，(3)激活ERK1/2和p38途径刺激血管平滑肌细胞(VSMC)迁移，(4)在乳腺肿瘤间质中成纤维细胞高表达。其目的是确定COMP与软骨细胞、VSMCs和成纤维细胞相互作用的分子基础。不同候选受体的特异性封闭试剂将被检测，与洗涤剂溶解的细胞中的CoMP共免疫沉淀的蛋白质将被质谱学鉴定，细胞提取物将被CoMP-Sepharose柱层析。

DESCRIPTION (provided by applicant): The thrombospondins (TSPs) are a family of five proteins that are closely and transiently associated with the interface between the cell surface and extracellular matrix (ECM) during tissue genesis and remodeling. The long-term goal of this project is to elucidate the structure and function of the members of the TSP gene family. Specific focus for the next period of support will be on the following areas. Specific Aim1. Determination of the structure of COMP. For various TSP family members, structural and functional data indicate that the type 3 repeats and the C-terminal domain form a single complex. The goal of the proposed study is to use X-ray crystallography and NMR to determine the structure of the type 3 repeat/C-terminal complex of cartilage oligomeric matrix protein (COMP). Based on molecular modeling approaches, we hypothesize that the C-terminal of COMP folds to form a four-bladed beta propeller and the type 3 repeats form looped structures that are closely associated with one surface of the beta propeller. Furthermore, we hypothesize that the juxtaposition of the type 3 repeats and the C-terminal domain is essential for optimal collagen, proteoglycan (PG) and integrin binding. Specific Aim 2. Identification of key amino acids for the interaction of COMP with proteins and PGs. The hypothesis that COMP is a matricellular protein implies that it participates in ECM assembly or structure and binds to cell surface proteins and PGs to modulate cellular phenotype. The goal of this aim is a detailed understanding of the molecular basis for the interaction of COMP with collagen II, fibronectin, integrins and glycosaminoglycans (GAGs). Key amino acids for these interactions within the type 3 repeat/C-terminal complex will be identified by site-directed mutagenesis within the context of the intact molecule and in the individual domains. Specific Aim 3. Identification of cell surface receptors for COMP. Preliminary data indicate that COMP (1) interacts with PGs and integrins on chondrocytes, (2) modulates chondrogenesis, (3) activates ERK 1/2 and p38 pathways to stimulate vascular smooth muscle cell (VSMC) migration, and (4) is highly expressed by fibroblasts in the stroma of mammary tumors. The goal of this aim is to determine the molecular basis for the interaction of COMP with chondrocytes, VSMCs and fibroblasts. Specific blocking reagents for various candidate receptors will be assayed, proteins that coimmunoprecipitate with COMP from detergent solubilized cells will be identified by mass spectrometry and cell extracts will be chromatographed on COMP-Sepharose columns.