Thrombospondin 4 A1

血小板反应蛋白 4 A1

• 批准号: 7650755
• 负责人: John W LAWLER
• 金额: $ 71.11万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7650755
• 关键词: 3-Dimensional; Adhesions; Affect; Alanine; Amino Acids; Apoptosis; Area; Arthritis; Aspartic Acid; Behavior; Binding; Binding Sites; C-terminal; Calcium; Calcium Binding; Cartilage; Cell Adhesion; Cell Surface Proteins; Cell physiology; Cell surface; Cells; Cellular Structures; Chondrocytes; Coin; Collagen; Collagen Fibril; Comprehension; Cues; Data; Development; Disease Progression; Epiphysial cartilage; Extracellular Matrix; Family; Fibroblasts; Fibronectins; Gene Family; Genes; Glycosaminoglycans; Goals; Individual; Inflammation; Integrin Binding; Integrins; Ions; Knockout Mice; Knowledge; Lead; Mediating; Metals; Molecular; Mutagenesis; Mutate; Mutation; Peptide Hydrolases; Peptide Library; Phenotype; Physiological; Process; Protein Binding; Proteins; Proteoglycan; Pseudoachondroplastic spondyloepiphyseal dysplasia syndrome; Publishing; Recombinants; Regulation; Role; Series; Signal Pathway; Site; Site-Directed Mutagenesis; Specificity; Stimulus; Structure; System; THBS1 gene; Thrombospondin 1; Thrombospondins; Tissues; Wound Healing; X-Ray Crystallography; Zinc; aggrecan; angiogenesis; base; design; disease-causing mutation; insight; mammalian COMP; member; migration; protein expression; protein folding; protein function; repaired; response; skeletal dysplasia; synthetic peptide; thrombospondin 3; thrombospondin 4

A detailed comprehension of the role of the thrombospondins (TSPs), including cartilage oligomeric matrix protein (COMP), in the regulation of extracellular matrix (ECM) structure and cellular behavior during tissue genesis and repair is the long-term goal of the proposed studies. Specific focus for the next period of support will be on the following areas. Specific Aim 1. To identify key amino acids for the interactions of COMP with proteins and proteoglycans (PGs). We have found that COMP binds to aggrecan, as well as to integrins in chondrocytes and fibroblasts. In addition, growth plate disorganization is observed in TSP-1-, TSP-3-, and COMP-null mice. We have recently solved the structure of the signature domain of COMP by X-ray crystallography. Based on these data, we plan to determine the molecular basis for the interaction of COMP with collagens, integrins, and glycosaminoglycans (GAGs) by site-directed mutagenesis within the context of the intact molecule and the recombinant signature domain. The aspartic acids that comprise the potential metal ion-dependent adhesion site (MIDAS) and the adjacent to MIDAS motif will be mutated to alanines to establish the importance of these sites in collagen and zinc binding, and matrix assembly. In addition, the specificity of the MIDAS will be explored using peptide libraries. The binding site for GAGs will be identified by X-ray crystallography. Existing structural data, naturally occurring mutations, and published synthetic peptide data will be used to identify amino acids for mutagenesis. This approach will enable us to probe the function of specific motifs within the context of the correctly folded protein. Specific Aim 2. To determine the effects of COMP on cellular phenotype and ECM structure. COMP orchestrates collagen fibril formation, ECM organization, and chondrocyte survival and differentiation during growth plate development. The importance of COMP is underscored by the fact that naturally occurring mutations in it result in skeletal dysplasias. In Aim 1, we will identify the integrin, collagen and GAG binding sites in the COMP molecule. In this aim, we will identify the integrin-mediated cellular responses to COMP in chondrocytes and fibroblasts. We will also explore the effect of the absence of COMP expression using primary cultures of chondrocytes from COMP-null mice. We hypothesize that wild-type COMP promotes cell adhesion, migration, proliferation and survival, while suppressing apoptosis. The specific signaling pathways that mediate the cellular responses to COMP in 2- and 3-D culture systems will be identified. In addition, proliferation, apoptosis and matrix structure in the growth plate of wild-type and COMP-null mice will be analyzed. We will also determine whether or not the integrin and GAG binding sites collaborate with the collagen-binding site to regulate ECM structure. Finally, we will explore the effect of some of the disease causing mutations on the ability of COMP to support cellular processes and matrix assembly. The data from these studies will provide key insights into the function of the signature domain of COMP in terms of its interactions with various proteins, PGs and cells.

详细了解血小板反应蛋白（TSP），包括软骨寡聚基质蛋白（COMP），在组织发生和修复过程中细胞外基质（ECM）结构和细胞行为的调节中的作用是拟议研究的长期目标。下一阶段支持的具体重点将在以下几个方面。具体目标 1. 确定 COMP 与蛋白质和蛋白聚糖 (PG) 相互作用的关键氨基酸。我们发现 COMP 与聚集蛋白聚糖以及软骨细胞和成纤维细胞中的整合素结合。此外，在 TSP-1-、TSP-3- 和 COMP-null 小鼠中观察到生长板瓦解。我们最近通过X射线晶体学解析了COMP的特征域结构。基于这些数据，我们计划通过在完整分子和重组特征域的背景下进行定点诱变来确定 COMP 与胶原蛋白、整合素和糖胺聚糖 (GAG) 相互作用的分子基础。构成潜在金属离子依赖性粘附位点 (MIDAS) 和邻近 MIDAS 基序的天冬氨酸将突变为丙氨酸，以确定这些位点在胶原蛋白和锌结合以及基质组装中的重要性。此外，将使用肽库探索 MIDAS 的特异性。 GAG 的结合位点将通过 X 射线晶体学来鉴定。现有的结构数据、自然发生的突变和已发表的合成肽数据将用于识别用于诱变的氨基酸。这种方法将使我们能够在正确折叠的蛋白质的背景下探测特定基序的功能。具体目标 2. 确定 COMP 对细胞表型和 ECM 结构的影响。 COMP 在生长板发育过程中协调胶原纤维的形成、ECM 组织以及软骨细胞的存活和分化。 COMP 的重要性通过以下事实得到强调：COMP 中自然发生的突变会导致骨骼发育不良。在目标 1 中，我们将识别 COMP 分子中的整合素、胶原蛋白和 GAG 结合位点。为此，我们将鉴定软骨细胞和成纤维细胞中整合素介导的对 COMP 的细胞反应。我们还将使用 COMP 缺失小鼠的软骨细胞原代培养物来探讨缺少 COMP 表达的影响。我们假设野生型 COMP 促进细胞粘附、迁移、增殖和存活，同时抑制细胞凋亡。将确定在 2-D 和 3-D 培养系统中介导细胞对 COMP 反应的特定信号传导途径。此外，还将分析野生型和COMP缺失小鼠生长板的增殖、凋亡和基质结构。我们还将确定整合素和 GAG 结合位点是否与胶原蛋白结合位点协作来调节 ECM 结构。最后，我们将探讨一些引起突变的疾病对 COMP 支持细胞过程和基质组装的能力的影响。这些研究的数据将为了解 COMP 特征域在与各种蛋白质、PG 和细胞的相互作用方面的功能提供重要见解。