TYPE V COLLAGENASE IN RHEUMATOID ARTHRITIS

细胞外基质的降解需要协调 构成金属蛋白酶家族的酶的作用。这个 组成这个家族的酶是相关的，因为它们被释放出来 从细胞中以潜伏的形式，它们含有锌，需要二价 阳离子具有生物活性。总而言之，这些酶具有 能够降解细胞外基质的几乎所有成分。 这种降解能力在类风湿中表现得最为明显。 关节炎。在这种疾病中，金属蛋白酶的过度产生 调节关节软骨和软骨下骨的降解， 导致严重的畸形。V型胶原酶是这一类的成员 对变性表现出高度特异性的多基因酶家族 胶原蛋白，降解天然的V型胶原蛋白和裂解XI型胶原蛋白 这是软骨的一种结构成分。该计划的主要目标是 拟议的研究是为了了解这种酶的调节，并 明确其在类风湿性关节炎中的确切作用和意义。 为了实现这一目标，我们建议：1.研究机制 通过鉴定V型胶原酶的序列 利用定点定位技术研究负责这一过程的酶的结构 诱变和核磁共振波谱。这将通过生成一个 保守氨基酸序列的系列突变(PRCGVPD)和 评估取代对酶潜伏期和 构象。氨基酸侧链部分与催化剂相互作用 锌离子将通过~(113)Cd-核磁共振波谱进行鉴定和确认 Cd取代的野生型和突变型酶，2.功能检测和 推测的活性中心残基在催化和反应中的意义 配基结合。将在活动站点中创建一系列突变 潜伏酶和重组突变酶的活性将被检测 对于它们降解和/或结合V型和XI型胶原的能力 底物。突变的蛋白质将被评估它们的能力。 结合锌和钙离子与二价阳离子结合将与 酶活性和底物结合。钙离子的数量 结合酶将通过核磁共振波谱测定，3。 确定起到活性碱催化剂作用的酸性氨基酸 该酶的定点诱变和X射线结晶学研究 以及，4.研究纤维连接蛋白结合域和 V型胶原酶底物特异性中的胶原样结构域 缺失突变体的产生及嵌合蛋白的构建 中性粒细胞胶原酶/V型胶原酶。这些研究将进一步 我们对这种酶的作用的理解可能会导致新的 注重关节保护的治疗方法 类风湿性关节炎降解产生的组织。

The degradation of the extracellular matrix requires the coordinated action of enzymes which constitute a family of metalloproteinases. The enzymes which make up this family are related in that they are released from the cell in a latent form, they contain zinc and require divalent cations for biological activity. Together, these enzymes have the ability to degrade virtually all components of the extracellular matrix. 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 is a member of this multigene family of enzymes which exhibits high specificity for denatured collagen, degrades native type V collagen and cleaves type 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. study the mechanism of activation of type V collagenase by identifying the sequences in the structure of the enzyme responsible for this process using site-directed mutagenesis and NMR spectroscopy. This will be done by generating a series of mutations in the conserved amino acid sequence (PRCGVPD) and assessing the effect of the substitutions on enzyme latency and conformation. Amino acid side chain moieties interacting with catalytic zinc ion will be identified and confirmed by 113Cd-NMR spectroscopy of Cd-substituted wild type and mutant enzymes, 2. examine the function and significance of the presumptive active site residues in catalysis and ligand binding. A series of mutations will be created in the active site of the latent enzyme and the recombinant mutant enzymes will be assayed for their ability to degrade and/or bind to type V and XI collagen substrates. The mutant proteins will be evaluated for their ability to bind Zn2+ and Ca2+ and divalent cation binding will be correlated with enzymatic activity and substrate binding. The number of Ca ions associated with the enzyme will be determined by NMR spectroscopy, 3. identify the acidic amino acids that function as the active base catalyst in this enzyme by site-directed mutagenesis and X-ray crystallography and, 4. investigate the role of the fibronectin binding domain and collagen-like domain in substrate specificity of type V collagenase by generating deletion mutants and constructing chimeric proteins of neutrophil collagenase/type V collagenase. 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.