METALLOPROTEASES IN CONNECTIVE TISSUE MATRIX BREAKDOWN

结缔组织基质分解中的金属蛋白酶

• 批准号: 3159157
• 负责人: HIDEAKI NAGASE
• 金额: $ 11.79万
• 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/3159157
• 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 胶原酶和至少另外两种基质金属蛋白酶MNP- 2(“明胶酶”)和基质金属蛋白酶-3(“蛋白聚糖酶”)) 各种细胞外基质大分子，但这些酶 以非活性酵素的形式从细胞中分泌出来。长期的 拟议研究的目标是调查 这些酵母菌的激活机制及鉴定 活性酶的原位研究，以了解它们在 病理破坏以及在正常的周转中 结缔组织基质。 三种酶原(前胶原酶、前基质金属蛋白酶-2和原基质金属蛋白酶-3)将 从体外培养的人类风湿滑膜细胞中提纯 各种层析技术。它的主要结构 ProMMP2将从DNA序列中推导出来，即 与原基质金属蛋白酶-2的mRNA互补。序列信息 被用来研究细胞的激活机制 序列水平上的酶原。各种组织和血浆 将对蛋白酶和4-氨基苯基汞醋酸酯进行检测 它们激活proMMP2和proMMP3的能力，以及它们的 对酶原的作用将通过NH2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激活也将是 通过酵母菌的重组检测和活化 酵素。MMP2和MMP3的酶性质将是 通过它们对基底膜的作用进一步研究 IV型胶原、层粘连蛋白和真肌动蛋白等成分。这个 这些酶的底物特异性的特征是 它们对还原的、羧甲基化的转铁蛋白的作用。 将尝试在组织中定位活性的基质金属蛋白酶-3 发生快速基质再吸收的地方。特异性抗体 基质金属蛋白酶原-3的前肽区与单抗 将制备针对活性的基质金属蛋白酶-3，并将其用于 基质金属蛋白酶-3活性形式和酶原形式在乳腺癌中的双重定位 人类风湿滑膜和关节软骨及兔 产后子宫和白细胞介素1治疗关节软骨。 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位置 组织，也许可以提出具体的方法来限制 细胞外基质中发生的不必要的蛋白质分解 各种结缔组织病。