METALLOPROTEASES IN CONNECTIVE TISSUE MATRIX BREAKDOWN

结缔组织基质分解中的金属蛋白酶

• 批准号: 3159154
• 负责人: HIDEAKI NAGASE
• 金额: $ 13.63万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3159154
• 关键词: antibody formation; basement membrane; cartilage; collagenase; enzyme induction /repression; enzyme mechanism; enzyme structure; enzyme substrate; extracellular matrix; fibroblasts; guinea pigs; human tissue; laboratory mouse; laboratory rabbit; mercury; metalloenzyme; monoclonal antibody; pepsin; peptidases; protease inhibitor; rheumatism; synovial membrane; tissue /cell culture; zymogens

Connective tissue cells have the ability to synthesize and secrete collagenase and at least two other matrix metalloproteases MNP- 2 ("gelatinase") and MMP-3 ("proteoglycanase")) that digest various extracellular matrix macromolecules, but these enzymes are secreted from the cells as inactive zymogens. The long-term objectives of the proposed research are to investigate the mechanisms of activation of these zymogens and to identify active enzymes in situ in order to understand their roles in pathological destruction as well as in normal turnover of the connective tissue matrix. Three zymogens (procollagenase, proMMP-2 and proMMP-3) will be purified from human rheumatoid synovial cells in culture by various chromatographic techniques. The primary structure of proMMP-2 will be deduced from the DNA sequence which is complementary to proMMP-2 mRNA. The sequence information is used to investigate the mechanisms of activation of the proenzymes at the sequence level. Various tissue and plasma proteases and 4-aminophenylmercuric acetate will be tested for their abilities to activate proMMP-2 and proMMP-3, and their actions on proenzymes will be characterized by the NH2-terminal sequence analyses of active enzymes. The "cascade" hypothesis for the accelerated activation of these proenzymes will be also examined by the recombination of zymogens and activated enzymes. Enzymic properties of MMP-2 and MMP-3 will be further investigated by their action on the basement membrane components such as type IV collagen, laminin and eutactin. The substrate specificities of these enzymes are characterized by their action on reduced, carboxymethylated transferrin. Attempts will be made to localize active MMP-3 in the tissues where rapid matrix resorption is occurring. Antibodies specific to the propeptide region of proMMP-3 and monoclonal antibodies specific to the active MMP-3 will be prepared, and used for the dual localization of both active and zymogen forms of MMP-3 in human rheumatoid synovium and articular cartilage, and rabbit postpartum uterus and interleukin 1-treated articular cartilage. Once the detailed knowledge is obtained about the zymogen activation, enzyme specificities, and the sites of activation in the tissue, it may be possible to suggest specific ways to limit the unwanted proteolysis of the extracellular matrix that occurs in a variety of connective tissue diseases.

结缔组织细胞具有合成和分泌的能力 胶原酶和至少两种其他基质金属蛋白酶 MNP- 2（“明胶酶”）和 MMP-3（“蛋白聚糖酶”））消化 各种细胞外基质大分子，但这些酶 作为无活性的酶原从细胞中分泌。 长期来看 拟议研究的目标是调查 这些酶原的激活机制并确定 原位活性酶，以了解它们的作用 病理性破坏以及正常周转 结缔组织基质。 三种酶原（原胶原酶、proMMP-2 和 proMMP-3）将 从培养的人类风湿滑膜细胞中纯化 各种色谱技术。 的主要结构 proMMP-2 将从以下 DNA 序列中推导出来： 与 proMMP-2 mRNA 互补。 序列信息 用于研究激活机制 序列水平的酶原。 各种组织和血浆 将测试蛋白酶和 4-氨基苯汞乙酸盐 他们激活 proMMP-2 和 proMMP-3 的能力，以及他们 对酶原的作用以 NH2 末端为特征 活性酶的序列分析。 “级联”假说 为了加速这些酶原的激活也将 通过酶原的重组进行检查并激活 酶。 MMP-2 和 MMP-3 的酶特性为 通过它们对基底膜的作用进一步研究 IV 型胶原蛋白、层粘连蛋白和 eutactin 等成分。 这 这些酶的底物特异性的特点是 它们对还原的羧甲基化转铁蛋白的作用。 将尝试在组织中定位活性 MMP-3 发生快速基质吸收的地方。 特异性抗体 proMMP-3 和单克隆抗体的前肽区域 特定于活性 MMP-3 将被制备，并用于 MMP-3 的活性形式和酶原形式的双重定位 人类风湿滑膜和关节软骨，以及兔 产后子宫和白介素 1 处理的关节软骨。 一旦获得有关酶原的详细知识 激活、酶特异性和激活位点 组织，也许可以建议限制的具体方法 发生在细胞外基质中的不需要的蛋白水解 各种结缔组织疾病。