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: 3774337
• 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/3774337
• 关键词: 3T3 cells; RNA splicing; enzyme activity; enzyme biosynthesis; enzyme mechanism; gene deletion mutation; human genetic material tag; messenger RNA; metastasis; molecular cloning; neoplastic cell; secretion; species difference; structural genes; tissue /cell culture; zymogens

Malignantly transformed cells secrete a variety of proteases and protease precursors which have been hypothesized to be involved in the invasiveness, metastasis, immune suppression, and paraneoplastic syndromes associated with cancer. Our work has concentrated on the function of a secreted lysosomal cysteine proprotease called procathepsin L or MEP (for major excreted protein of transformed cells). Cathepsin L is a broad spectrum acid protease found both in lysosomes and secreted as the proenzyme into the extracellular space. We have cloned the genes for mouse and human procathepsin L and compared their structures and modes of regulation. Each has 8 exons and 7 introns located at the same positions within the coding region, but the structural gene for procathepsin L in the mouse spans 7400 bp, while the human procathepsin L gene is 5100 bp, with the size difference accounted for almost entirely by intron size. Unlike the mouse gene which has a unique start site of transcription and a single major mRNA species, at least two major mRNAs are transcribed from the human procathepsin L gene. These two mRNAs differ in their 5' untranslated sequences, and represent alternative splicing or alternative promoters. To determine why procathepsin L is secreted in large amounts by the activity of malignantly transformed cells and other cells which overproduce it, we have initiated a deletion analysis of the human procathepsin L cDNA. Expression of deleted forms of human procathepsin L in mouse NIH 3T3 cells results in either failure to form a stable structure (deletions of the "pro" piece) or failure to be secreted (carboxy-terminal deletions). To evaluate the function of secreted procathepsins in metastasis we have compared the synthesis, secretion, and activity of cathepsins L and B in a series of mouse B 16 melanoma cells of varying metastatic potential, and find no clear correlation with ability to metastasize.

转化细胞分泌多种蛋白酶和蛋白酶 前体已经被假设参与了 侵袭、转移、免疫抑制和副肿瘤综合征 与癌症有关。我们的工作集中在一个 分泌的溶酶体半胱氨酸前蛋白酶，称为前组织蛋白酶L或MEP（用于 转化细胞的主要分泌蛋白）。组织蛋白酶L是一种广泛的 一种酸性蛋白酶，存在于溶酶体中， 进入细胞外空间。我们克隆了老鼠的基因 和人组织蛋白酶原L，并比较了它们的结构和表达模式。 调控每个基因有8个外显子和7个内含子，位于相同的位置 在编码区内，但在编码区中的组织蛋白酶原L的结构基因， 小鼠全长7400 bp，而人的组织蛋白酶原L基因为5100 bp， 大小差异几乎完全由内含子大小决定。 与小鼠基因不同，小鼠基因具有独特的转录起始位点， 一个主要的mRNA种类，至少有两个主要的mRNA转录自 人类组织蛋白酶原L基因这两种mRNA的5'端不同， 非翻译序列，并代表选择性剪接或选择性剪接。 发起人。为了确定为什么前组织蛋白酶L大量分泌， 恶性转化细胞和其他细胞的活性， 过度生产，我们已经开始了人类的缺失分析， 前组织蛋白酶L cDNA缺失型人组织蛋白原L的表达 在小鼠NIH 3T3细胞中，要么无法形成稳定的 结构（“原”片段缺失）或不能分泌 （羧基末端缺失）。为了评价分泌的功能， 在转移中，我们比较了前组织蛋白酶的合成、分泌和 组织蛋白酶L和B在一系列小鼠B 16黑素瘤细胞中的活性 不同的转移潜力，并没有发现明确的相关性， 转移