CLEAVAGE-SECRETION OF ANGIOTENSIN-CONVERTING ENZYME

血管紧张素转换酶的裂解-分泌

• 批准号: 6389472
• 负责人: INDIRA SEN
• 金额: $ 25.9万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6389472
• 关键词: calmodulin; cell sorting; endopeptidases; enzyme activity; enzyme mechanism; enzyme structure; genetic library; glycosylation; isozymes; molecular cloning; peptidyl dipeptidase A; phorbols; phosphatase inhibitor; phosphorylation; protease inhibitor; protein binding; protein protein interaction; protein purification; protein structure function; renin angiotensin system; site directed mutagenesis

DESCRIPTION (Adapted from the application): Angiotensin-converting enzyme (ACE) is a key component of the renin-angiotensin system that regulates blood pressure. Studies with ACE knockout mice have revealed additional roles of ACE in renal physiology and male fertility. Although ACE exists primarily as a cell-surface protein, a soluble form is present under normal conditions in serum and other body fluids. Because information in the literature suggests that the specific physiological function of cell-bound ACE may differ from those of ACE in circulation, production of soluble ACE from cell-bound ACE could be significant in biological regulation. Analysis of ACE cleavage-secretion processes using natural ACE-producing cells and cells transfected with expression vectors of ACE or its mutants, revealed that the ectodomain of ACE is cleaved at a specific site near the plasma membrane, but the cleavage specificity is maintained not by sequence at or around the cleavage site but by the presence of the distal ectodomain of the ACE protein. ACE-secretase is a membrane-anchored metalloprotease and yeasts contain an ACE-cleaving activity with the properties of the mammalian ACE-secretase. The activity of the mammalian secretase can be upregulated by treatment of cells with phorbol esters, calmodulin inhibitors or protein tyrosine phosphatase inhibitors. The aims of this application are: 1) to define the sequence in the ectodomain of ACE that activates the secretase, 2) to clone and characterize the ACE-secretase, and 3) to determine whether the phosphorylation states of the cytoplasmic domains of ACE and the secretase regulates the rate of cleavage-secretion of ACE. Secretion of deletion and substitution mutants of ACE-CD4 chimeras will be monitored for identifying the domain in ACE that triggers its cleavage-secretion. For cloning human ACE secretase, genetic complementation of yeast and human cells will be carried out. A yeast mutant that lacks the ACE-secretase activity will be generated by systematically mutating its known metalloproteases. The mutant will then be used for expressing a human cDNA library and scored for the restoration of ACE-secretion. Similarly, mammalian cells devoid of ACE-secretase will be generated by chemical mutagenesis followed by FACS sorting and these mutant cells will be complemented with the clone for the human ACE-secretase. The mechanism of regulation of ACE secretion by PMA, calmodulin inhibitors and tyrosine phosphatase inhibition will be explored by determining whether the relevant protein kinases bind to and phosphorylate the cytoplasmic domains of ACE and the secretase or its associated proteins. Involvement of specific tyrosine and serine/threonine protein kinase pathways in this regulation will be investigated by using specific chemical inhibitors, dominant-negative mutant kinases and cell lines devoid of specific kinases.

描述（改编自申请）：血管紧张素转换酶（ACE） 是调节血液的肾素-血管紧张素系统的关键成分 压力对ACE基因敲除小鼠的研究揭示了ACE的其他作用 肾脏生理学和男性生育能力。虽然ACE主要作为 细胞表面蛋白，在正常条件下以可溶形式存在于 血清和其他体液。因为文献资料显示 细胞结合ACE的特定生理功能可能不同于 循环中ACE的那些，从细胞结合的ACE产生可溶性ACE 在生物调节中可能很重要。ACE分析 使用天然ACE产生细胞的裂解-分泌过程和细胞 用ACE或其突变体的表达载体转染，发现 ACE的胞外域在质膜附近的特定位点被切割，但 切割特异性不是通过位于或周围的序列来维持的， 切割位点，但通过ACE蛋白的远端胞外域的存在。 ACE-分泌酶是一种膜锚定金属蛋白酶，酵母含有一种 具有哺乳动物ACE分泌酶特性的ACE切割活性。的 哺乳动物分泌酶的活性可以通过处理细胞 与佛波醇酯、钙调蛋白抑制剂或蛋白酪氨酸磷酸酶 抑制剂的本申请的目的是：1）定义序列， 激活分泌酶的ACE胞外域，2）克隆和表征 ACE-分泌酶，和3）确定是否磷酸化状态， ACE和分泌酶的胞质结构域调节 ACE的裂解-分泌。缺失和取代突变体的分泌 将监测ACE-CD 4嵌合体以鉴定ACE中的结构域， 触发了它的分裂分泌用于克隆人ACE分泌酶， 将进行酵母和人细胞的互补。一种酵母突变体 缺乏ACE分泌酶活性的细胞将通过系统性地 突变其已知的金属蛋白酶。突变体将被用于 表达人cDNA文库，并对 ACE分泌。类似地，缺乏ACE-分泌酶的哺乳动物细胞将被 通过化学诱变，然后通过FACS分选产生，并且这些突变体 细胞将与人ACE-分泌酶的克隆互补。的 PMA、钙调素抑制剂和 酪氨酸磷酸酶抑制将通过确定是否 相关蛋白激酶结合并磷酸化 ACE和分泌酶或其相关蛋白。具体参与 酪氨酸和丝氨酸/苏氨酸蛋白激酶途径在这种调节中将 通过使用特定的化学抑制剂，显性负突变体， 激酶和缺乏特异性激酶的细胞系。