ID & CHARACTERIZE MEMBRANE TYPE SERINE PROTEASE: PROSTATE, EPITHELIAL CANCER

基本信息

批准号：
6220337
负责人：
TOSHIHIKO TAKEUCHI
金额：
$ 0.01万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-01 至 2000-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6220337
关键词：
anesthesiology bacteria biological products biomaterials biomedical resource human tissue model design /development nervous system proteins statistics /biometry technology /technique

项目摘要

Serine proteases of the chymotrypsin fold are of great interest due to detailed understanding of their enzymatic properties and their role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of ecotin and engineered ecotins has resulted in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, RT-PCR with PC-3 poly(A)+ mRNA was performed using degenerate oligonucleotide primers. These primers were designed using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in E. coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas. The Computer Graphics Laboratory facilities have been invaluable in the cloning and characterization of this novel serine protease.

胰凝乳蛋白酶折叠的丝氨酸蛋白酶引起了极大的兴趣由于详细了解了它们的酶学特性及其在许多生理和病理过程中发挥作用。我们有正在开发大分子抑制剂 Ecotin “折叠特异性”抑制剂，对成员具有选择性胰凝乳蛋白酶折叠类蛋白酶。抑制蛋白酶活性通过使用生态素和工程生态素已导致抑制大鼠前列腺分化并延缓人类 PC-3 前列腺癌肿瘤的生长。努力鉴定可能参与这些过程的蛋白酶，RT-PCR 使用简并寡核苷酸进行 PC-3 Poly(A)+ mRNA 分析引物。这些引物是使用保守蛋白设计的胰凝乳蛋白酶折叠丝氨酸蛋白酶特有的序列。五鉴定出蛋白酶：尿激酶型纤溶酶原激活剂，因子 XII、蛋白 C、胰蛋白酶原 IV 和我们提到的蛋白酶作为膜型丝氨酸蛋白酶 1 (MT-SP1)。克隆和 MT-SP1 cDNA 的表征表明它编码嵌合体含有跨膜信号锚、两个 CUB 结构域的蛋白质，四个 LDLR 重复序列和一个丝氨酸蛋白酶结构域。北方印迹显示 MT-SP1 在多种上皮组织中广泛表达在人体胃肠道中高水平表达前列腺。 MT-SP1 蛋白酶结构域的 His 标记融合体是在大肠杆菌中表达、纯化并自激活。生态素及其变体 Ecotins 是 MT-SP1 激活蛋白酶的亚纳摩尔抑制剂结构域，表明 MT-SP1 在前列腺分化和前列腺癌的生长。计算机图形学实验室设施对于克隆和鉴定具有无价的价值这种新型丝氨酸蛋白酶。