METALLOPROTEASES IN CONNECTIVE TISSUE MATRIX BREAKDOWN

结缔组织基质分解中的金属蛋白酶

• 批准号: 3159158
• 负责人: HIDEAKI NAGASE
• 金额: $ 12.49万
• 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/3159158
• 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它基质金属蛋白酶MNP-1， 明胶酶（“明胶酶”）和MMP-3（“蛋白聚糖酶”）） 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 拟议研究的目标是调查 这些酶原的激活机制，并确定 活性酶，以了解它们在 病理性破坏以及在正常营业额的 结缔组织基质 三种酶原（前胶原酶、proMMP-2和proMMP-3）将 从培养的人类风湿性滑膜细胞中纯化， 各种色谱技术。 的一级结构 proMMP-2将从DNA序列推导出， 与proMMP-2 mRNA互补。 的序列信息 是用来调查的激活机制， 在序列水平上的前酶。 各种组织和血浆 蛋白酶和4-氨基苯汞乙酸将被测试， 它们激活proMMP-2和proMMP-3的能力，以及它们的 对酶原的作用将通过NH 2-末端来表征 活性酶的序列分析。 “级联”假说 对于这些酶原的加速活化， 通过酶原的重组和活化 内切酶 MMP-2和MMP-3的酶性质将被确定。 进一步研究了它们对基底膜的作用 例如IV型胶原蛋白、层粘连蛋白和euclyn。 的 这些酶的底物特异性的特征在于 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 发生快速基质再吸收。 特异性抗体 MMP-3前肽区与单克隆抗体 将制备对活性MMP-3特异的蛋白质，并用于 MMP-3活性和酶原形式的双重定位， 人类风湿性滑膜和关节软骨以及兔 产后子宫和白细胞介素1治疗的关节软骨。 一旦获得了关于酶原的详细知识 激活，酶的特异性，和激活的网站， 组织，这可能是可能的，建议具体的方式来限制 细胞外基质的不需要的蛋白水解， 各种结缔组织疾病。