TYPE V COLLAGENASE IN RHEUMATOID ARTHRITIS

细胞外基质的降解需要协调的 构成金属蛋白酶家族的酶的作用。 这种降解能力在类风湿性关节炎中最为明显， 关节炎在这种疾病中，金属蛋白酶的过度产生 介导关节软骨和软骨下骨的降解， 导致严重畸形。 V型胶原酶/明胶酶B是一种 这种多基因酶家族的一员，表现出高度特异性 对于变性胶原蛋白，降解天然的V型和Xi型胶原蛋白， 软骨的结构成分。 建议的主要目标 研究的目的是了解这种酶的调节，并确定其 在类风湿关节炎中的确切作用和意义。 朝向 实现这一目标，我们建议1。调查的作用 血红素样羧基端结构域在钙依赖性激活 明胶酶B。 这将通过生成一系列COOH-末端 (CTD)截短突变体和评估截短对酶的影响 潜伏期和构象。 将鉴定CTD“调节肽” 通过产生一系列GST-CDT肽， 将检查CTD截短酶的活性，2.证实 假设明胶酶B催化结构域中存在盐键， 负责酶的Ca 2+非依赖性活性。 NH 2-和 催化结构域的COOH末端区域将交联， 交联对酶的Ca 2+依赖性活性的影响将 评估，3。鉴定活性多肽中的TIMP-1结合残基 明胶酶B位点丙氨酸扫描定点突变，4. 研究纤连蛋白样结合域在底物中的作用 通过产生缺失突变体来提高酶的特异性。 特定氨基 负责酶的底物特异性的酸残基将是 通过丙氨酸扫描定点突变鉴定，最后5. 明胶酶B的区域负责蛋白质-蛋白质和蛋白质- 基质膜相互作用将使用截短的突变体来鉴定 上面生成的。 这些研究将进一步加深我们对这种酶的作用的理解 并可能导致新的治疗方法， 类风湿性关节炎中关节组织的退化。

The degradation of the extracellular matrix requires the coordinated action of enzymes which constitute a family of metalloproteinases. Nowhere is this degradative ability more apparent than in rheumatoid arthritis. In this disease, excessive production of metalloproteinases mediates the degradation of articular cartilage and subchondral bone, resulting in severe deformity. Type V collagenase/gelatinase B is a member of this multigene family of enzymes which exhibits high specificity for denatured collagen, degrades native types V and XI collagen which is a structural component of cartilage. The main objective of the proposed research is to understand the regulation of this enzyme and to define its precise role and significance in rheumatoid arthritis. Towards the achievement of this goal we propose to 1. Investigate the role of hemopexin-like carboxy terminal domain in Ca2+-dependent activation of gelatinase B. This will be done by generating a series of COOH-terminal (CTD) truncated mutant and assessing the effect of truncation on enzyme latency and conformation. The CTD "regulatory peptide" will be identified by generating a series of GST-CDT peptide and the effect of each peptide on activity of CTD-truncated enzymes will be examined, 2. Substantiate the hypothesis that a salt linkage present in gelatinase B catalytic domain is responsible for Ca2+-independent activity of the enzyme. The NH2- and COOH-terminal regions of catalytic domain will be cross linked and the effect of cross linking on the Ca2+-dependent activity of the enzyme will be assessed, 3. Identify the TIMP-1 binding residues within the active site of gelatinase B by alanine-scanning site-directed mutagenesis, 4. Investigate the role of the fibronectin-like binding domain in substrate specificity of the enzyme by generating deletion mutants. Specific amino acid residues responsible for substrate specificity of the enzyme will be identified by alanine-scanning site directed mutagenesis and finally 5. Region(s) of gelatinase B responsible for protein-protein and protein- matrix membrane interaction will be identified using truncated mutants generated above. These studies will further our understanding on the action of this enzyme and may lead to new therapeutic approaches which would focus on protection of articular tissues from degradation in rheumatoid arthritis.