PROTEASES AND GROWTH FACTOR REGULATION

蛋白酶和生长因子调节

• 批准号: 3330293
• 负责人: RON G ROSENFELD
• 金额: $ 20.44万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3330293
• 关键词: blood proteins; chemical binding; circadian rhythms; decidua; endopeptidases; enzyme activity; female; high performance liquid chromatography; hormone binding protein; hormone regulation /control mechanism; human tissue; immunocytochemistry; in situ hybridization; insulinlike growth factor; laboratory rabbit; laboratory rat; male; menstrual cycle; molecular cloning; northern blottings; pregnancy; prostate; protease inhibitor; protein purification; proteolysis; receptor binding; tissue /cell culture

The mitogenic and anabolic actions of the insulin-like growth factors (IGFs) are modulated by a minimum of six serum binding proteins (BPs) . The major carrier of IGF peptides in plasma is IGFBP-3, which normally complexes with IGF-I (or -II) and an acid-labile subunit, to form a 150K complex. We have recently shown, by western ligand blotting techniques, that there is a dramatic reduction of intact IGFBP-3 in the sera of pregnant women, mice and rats, and that this decrease can be attributed to the presence of a pregnancy-associated protease (P-A-P) which cleaves IGFBP-3 into smaller fragments. We have demonstrated a similar mechanism in seminal plasma, and in several malignancy-related proteases. We propose that proteolysis of IGFBP-3 is a regulatory process, whereby alterations in the affinity of IGFBP-3 for IGF peptides modulates access of these IGFs to cell membrane receptors. We plan to identify and characterize IGFBP-3 proteases, with special attention to the P-A-P and the protease activity of seminal plasma, which we have tentatively ldentified as prostate-specific antigen (PSA). P-A-P will be purified and its cDNA cloned. Employing an IGFBP protease assay developed in our, laboratories, together with PSA and purified P-A-P, we will perform enzyme kinetic studies, identify specific protease inhibitors, determine cleavage site specificity, and quantitate protease activity. The source of P-A-P will be determined by decidual, trophoblast and hepatic explant and cell cultures, and hormonal regulation of protease activity will be studied. Immunohistochemical, in situ hybridization, and Northern blot studies will be performed to identify cells which produce IGFBP-3 and/or IGFBP-3 proteases. Finally, the IGFBP-3 fragments will be characterized in terms of: 1) their size; 2) their affinity for IGF-I and -II; 3) their ability to complex with the acid-labile subunit to form the normal ternary complex; and 4) their effect upon the ability of IGF-I and -II to bind to membrane receptors and exert biological action.

胰岛素样生长因子的促有丝分裂和合成代谢作用 胰岛素样生长因子（IGFs）由至少六种血清结合蛋白（BP）调节。 血浆中IGF肽的主要载体是IGFBP-3，其通常 与IGF-I（或-II）和酸不稳定亚基形成150 K复合物， 复杂. 我们最近通过蛋白质配体印迹技术， 血清中完整的IGFBP-3显著减少， 孕妇，小鼠和大鼠，这种减少可以归因于 妊娠相关蛋白酶（P-A-P）的存在， IGFBP-3分解成更小的片段。 我们已经证明了一个类似的机制 在精浆和几种恶性肿瘤相关蛋白酶中。 我们认为IGFBP-3的蛋白水解是一个调节过程， IGFBP-3对IGF肽亲和力的改变调节了 胰岛素样生长因子对细胞膜受体的作用。 我们计划识别和 表征IGFBP-3蛋白酶，特别注意P-A-P和 精浆蛋白酶活性，我们已经初步 前列腺特异性抗原（PSA）。 P-A-P将被净化 并克隆了其cDNA。 采用我们开发的IGFBP蛋白酶测定法， 实验室，连同PSA和纯化的P-A-P，我们将执行 酶动力学研究，鉴定特异性蛋白酶抑制剂，确定 切割位点特异性，并定量蛋白酶活性。 源 P-A-P的含量将通过蜕膜、滋养层和肝外植体来确定 和细胞培养，蛋白酶活性的激素调节将是 研究了 免疫组化、原位杂交和北方印迹 将进行研究以鉴定产生IGFBP-3和/或IGFBP-4的细胞。 IGFBP-3蛋白酶。 最后，将对IGFBP-3片段进行表征 在以下方面：1）它们的大小; 2）它们对IGF-I和IGF-II的亲和力; 3）它们的 与酸不稳定亚基复合以形成正常 三元复合物;和4）它们对IGF-I和IGF-II的能力的影响， 与膜受体结合并发挥生物学作用。