THROMBOSPONDIN-4

描述（由申请人提供）：血小板反应蛋白（TSP）是一个由五种蛋白组成的家族，在组织发生和重塑期间与细胞表面和细胞外基质（ECM）之间的界面密切且短暂相关。本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。具体目标1。COMP的结构测定。对于各种TSP家族成员，结构和功能数据表明，3型重复序列和C-末端结构域形成单一复合物。该研究的目的是利用X射线晶体学和NMR来确定软骨寡聚基质蛋白（COMP）的3型重复/C-末端复合物的结构。基于分子模拟方法，我们假设COMP的C-末端折叠形成四叶β螺旋桨，并且3型重复序列形成与β螺旋桨的一个表面密切相关的环状结构。此外，我们假设3型重复序列和C-末端结构域的并列对于最佳的胶原蛋白、蛋白聚糖（PG）和整合素结合是必不可少的。具体目标2。COMP与蛋白质和PG相互作用的关键氨基酸的鉴定。COMP是一种基质细胞蛋白的假设意味着它参与ECM组装或结构，并结合细胞表面蛋白和PG来调节细胞表型。这个目标的目的是详细了解COMP与胶原蛋白II，纤连蛋白，整合素和糖胺聚糖（GAG）相互作用的分子基础。3型重复序列/C-末端复合物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中的定点诱变来鉴定。具体目标3。COMP的细胞表面受体的鉴定。初步数据表明COMP（1）与软骨细胞上的PG和整合素相互作用，（2）调节软骨形成，（3）激活ERK 1/2和p38通路以刺激血管平滑肌细胞（VSMC）迁移，（4）在乳腺肿瘤基质中由成纤维细胞高度表达。本研究的目的是确定COMP与软骨细胞、血管平滑肌细胞和成纤维细胞相互作用的分子基础。将测定各种候选受体的特异性阻断剂，通过质谱法鉴定与来自去污剂溶解细胞的COMP共免疫沉淀的蛋白质，并在COMP-琼脂糖色谱柱上对细胞提取物进行色谱分析。

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.