SYNTHESIS AND FUNCTION OF A TRANSFORMATION-DEPENDENT SECRETED LYSOSOMAL PROTEASE

转化依赖性分泌型溶酶体蛋白酶的合成和功能

• 批准号: 3939320
• 负责人: M M GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3939320
• 关键词: chemical carcinogen; chromatography; electrophoresis; fibroblasts; genetic manipulation; genetic mapping; genetic markers; genetic transcription; growth factor; immunoprecipitation; lysosomes; mannose; messenger RNA; molecular biology; molecular cloning; neoplasm /cancer genetics; neoplastic transformation; oncoproteins; phorbols; platelet derived growth factor; radiotracer; secretion; tissue /cell culture

Cultured mouse fibroblasts which are malignantly transformed or treated with TPA or growth factors such as PDGF synthesize and secrete a 39,000 Mr-phos-phoglycoprotein (major excreted protein, MEP) in large amounts. The purified protein contains mannose 6-phosphate, the lysosomal recognition marker. It is processed intracellularly in both transformed and nontransformed cells to give two specific lower molecular weight forms with a lysosomal localization. The secreted form of MEP is the precursor to a lower molecular weight novel thiol protease (cathepsin) with an acid pH optimum capable of hydrolyzing a wide variety of proteins including the extracellular matrix proteins collagen, fibronectin and laminin. The specificity of peptide bond cleavage and the profile of inhibition of MEP is the same as cathepsin L. Sequence analysis indicates that mouse MEP and its human homolog represent precursors to cathespin L. Overproduction of cloned mouse or human MEP/cathespin L in a nontransformed cell results in secretion of this lysosomal enzyme. Secreted MEP can bind to the mannose 6-phosphate receptor of many cells and be endocytosed and processed intracellularly. In antigen presenting cells, MEP uptake reduces the efficiency of antigen presentation, thereby interfering with immune response. Transformation, TPA and PDGF stimulate MEP synthesis by increasing levels of MEP specific mRNA transcription. We have cloned a functional MEP gene from the mouse and have identified the 5' flanking region presumed to contain the MEP promoter. We are studying this system as a model of regulation of lysosomal protease synthesis, processing and secretion and how malignantly transformed cells perturb normal host functions.

培养的小鼠成纤维细胞恶性转化或 用TPA或生长因子如PDGF处理合成和 分泌39,000个MR-磷酸糖蛋白(主要排泄 蛋白质，MEP)大量存在。纯化的蛋白质中含有 甘露糖6-磷酸，溶酶体识别标记。它是 在转化和非转化中都在细胞内处理 细胞产生两种特定的低分子形式，具有 溶酶体定位。MEP的分泌形式是 一种低相对分子质量新型硫醇蛋白酶的前体 (组织蛋白酶)，具有最适pH的酸性，能够水解a 包括细胞外基质在内的各种蛋白质 胶原蛋白、纤维连接蛋白和层粘连蛋白。它的特殊性 多肽键的断裂和对MEP的抑制是 序列分析表明，小鼠MEP 和它的人类同源物代表了天冬氨酸L的前体。 克隆的小鼠或人的MEP/天冬氨酸天冬氨酸L在一种 未转化的细胞导致这种溶酶体酶的分泌。 分泌的MEP可与甘露糖6-磷酸受体结合 许多细胞，并被内吞并在细胞内加工。在……里面 抗原提呈细胞，MEP的摄取降低了 抗原提呈，从而干扰免疫反应。 转化、TPA和PDGF通过促进MEP合成 MEP特异性mRNA转录水平升高。我们有 从小鼠中克隆了一个有功能的MEP基因，并鉴定了 推测含有MEP启动子的5‘侧翼区。我们 正在研究这个系统作为调节溶酶体的模型 蛋白水解酶的合成、加工和分泌及其恶性程度 转化的细胞扰乱了正常的宿主功能。