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.

详细理解血小板蛋白（TSP）的作用，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（ECM）结构（ECM）结构和细胞行为的调节中，是拟议的研究的长期目标。下一个支持时期的特定重点将放在以下领域。具体目标1。鉴定与蛋白质和蛋白聚糖（PGS）相互作用的关键氨基酸。我们发现，COMP与脂肪蛋白的结合以及软骨细胞和成纤维细胞中的整合素结合。另外，在TSP-1-，TSP-3-和Comp-Null小鼠中观察到生长板混乱。我们最近通过X射线晶体学解决了COMP的签名结构域的结构。基于这些数据，我们计划通过位于完整的分子和重组签名域的背景下，通过位置定向的诱变来确定COMP与胶原蛋白，整联蛋白和糖胺聚糖（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MIDAS基序相邻的天冬氨酸将突变到丙氨酸中，以确定这些位点在胶原蛋白和锌结合中的重要性，以及基质组件。另外，将使用肽库探索MIDAS的特异性。 GAG的结合位点将通过X射线晶体学识别。现有的结构数据，天然发生的突变和已发表的合成肽数据将用于鉴定氨基酸以进行诱变。这种方法将使我们能够在正确折叠蛋白的上下文中探测特定基序的功能。具体目的2。确定COMP对细胞表型和ECM结构的影响。 Compsestrestrestresscons在生长板开发过程中，胶原原纤维形成，ECM组织和软骨细胞的生存和分化。 COMP的重要性强调了以下事实：其自然发生的突变导致骨骼发育不良。在AIM 1中，我们将确定COMP分子中的整联蛋白，胶原蛋白和GAG结合位点。在此目标中，我们将确定整联蛋白介导的细胞对软骨细胞和成纤维细胞中对COMP的反应。我们还将探索使用来自Comp-Null小鼠的软骨细胞的原代培养物缺乏COMP表达的效果。我们假设野生型COMP可以促进细胞粘附，迁移，增殖和存活，同时抑制凋亡。将确定介导2和3-D培养系统中介导细胞反应的特定信号通路。另外，将分析野生型和Comp-Null小鼠生长板中的增殖，凋亡和基质结构。我们还将确定整联蛋白和插科打结合位点是否与胶原结合位点合作以调节ECM结构。最后，我们将探讨某些疾病的影响，导致突变对COMP支持细胞过程和基质组装的能力。这些研究的数据将在与各种蛋白质，PG和细胞的相互作用方面提供对COMP的签名域功能的关键见解。