Thrombospondin 4 A1

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调节细胞外基质（ECM）结构和细胞行为的作用是拟议研究的长期目标。下一个支助期的具体重点将放在以下领域。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。我们已经发现COMP与软骨细胞和成纤维细胞中的聚集蛋白聚糖以及整合素结合。此外，在TSP-1-、TSP-3-和COMP-敲除小鼠中观察到生长板解体。我们最近解决了COMP的签名域的结构，通过X射线晶体学。基于这些数据，我们计划确定COMP与胶原蛋白，整合素和糖胺聚糖（GAG）的相互作用的分子基础，通过定点诱变的背景下的完整分子和重组签名域。将包含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。将通过X射线晶体学鉴定GAG的结合位点。现有的结构数据、天然存在的突变和已发表的合成肽数据将用于鉴定用于诱变的氨基酸。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。观察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原纤维形成、ECM组织和软骨细胞存活和分化。COMP的重要性是由其天然发生的突变导致骨骼发育不良的事实所强调的。在目的1中，我们将鉴定COMP分子中的整合素、胶原和GAG结合位点。在这个目标中，我们将确定整合素介导的细胞反应，COMP在软骨细胞和成纤维细胞。我们还将使用来自COMP缺失小鼠的软骨细胞的原代培养物来探索COMP表达缺失的影响。我们推测野生型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-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基因敲除小鼠生长板中的增殖、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结合位点协作来调节ECM结构。最后，我们将探讨一些致病突变对COMP支持细胞过程和基质组装能力的影响。这些研究的数据将为COMP的特征结构域在与各种蛋白质、PG和细胞相互作用方面的功能提供关键见解。

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.