CLEAVAGE-SECRETION OF ANGIOTENSIN-CONVERTING ENZYME

血管紧张素转换酶的裂解-分泌

• 批准号: 6604258
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6604258
• 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(改编自应用)：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组成部分 压力。对血管紧张素转换酶基因敲除小鼠的研究揭示了血管紧张素转换酶的其他作用 在肾脏生理和男性生育能力方面。尽管ACE主要以 细胞表面蛋白，在正常情况下以可溶性形式存在于 血清和其他体液。因为文献中的信息表明 细胞结合血管紧张素转换酶的特定生理功能可能不同于 循环中的血管紧张素转换酶，从细胞结合的血管紧张素转换酶产生可溶性血管紧张素转换酶 可能在生物调节方面具有重要意义。关于ACE的分析 利用天然血管紧张素转换酶产生细胞和细胞的切割-分泌过程 用血管紧张素转换酶或其突变体的表达载体进行表达，结果显示 ACE的胞外结构域在质膜附近的特定位置被切割，但 切割的特异性不是通过位于或周围的序列来保持的 但由ACE蛋白的远端胞外结构域存在。 ACE-分泌酶是一种膜固定型金属蛋白酶，酵母菌含有 具有哺乳动物血管紧张素转换酶分泌酶特性的血管紧张素转换酶活性。这个 哺乳动物分泌酶的活性可通过细胞处理而上调 使用佛波酯、钙调蛋白抑制剂或蛋白酪氨酸磷酸酶 抑制剂。本申请的目的是：1)定义 激活分泌酶的ACE的胞外结构域，2)克隆和鉴定 ACE-分泌酶，以及3)确定是否磷酸化状态 血管紧张素转换酶的胞质结构域和分泌酶调节 血管紧张素转换酶的裂解-分泌。枯草杆菌缺失和替换突变体的分泌 将监测ACE-CD4嵌合体，以识别ACE中 触发它的卵裂分泌。克隆人血管紧张素转换酶基因 将进行酵母和人体细胞的互补。酵母菌突变体 缺乏血管紧张素转换酶-分泌酶活性将由系统地产生 突变其已知的金属蛋白水解酶。然后，突变体将被用于 表达人cdna文库，并对修复结果进行评分 血管紧张素分泌。同样，缺乏ACE分泌酶的哺乳动物细胞将被 通过化学诱变产生，然后FACS分选和这些突变体 细胞将被人类ACE-分泌酶的克隆补充。这个 PMA、钙调蛋白抑制剂和钙调素对血管紧张素转换酶分泌的调节机制 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细胞质结构域 血管紧张素转换酶和分泌酶或其相关蛋白质。涉及到特定的 酪氨酸和丝氨酸/苏氨酸蛋白激酶通路在这一调节中将 通过使用特殊的化学抑制剂，显性-负性突变体进行研究 蛋白激酶和缺乏特定蛋白激酶的细胞系。

项目成果

Unaltered cleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-alpha-converting enzyme-deficient mice.

肿瘤坏死因子-α转换酶缺陷小鼠中血管紧张素转换酶的裂解和分泌未改变。

• DOI: 10.1074/jbc.274.15.10511
• 发表时间: 1999
• 期刊: The Journal of biological chemistry
• 作者: Sadhukhan,R;Santhamma,KR;Reddy,P;Peschon,JJ;Black,RA;Sen,I
• 通讯作者: Sen,I

Role of tyrosine phosphorylation in the regulation of cleavage secretion of angiotensin-converting enzyme.

酪氨酸磷酸化在血管紧张素转换酶裂解分泌调节中的作用。

• DOI: 10.1074/jbc.m407176200
• 发表时间: 2004
• 期刊: The Journal of biological chemistry
• 作者: Santhamma,KizhakkekaraR;Sadhukhan,Ramkrishna;Kinter,Michael;Chattopadhyay,Saurabh;McCue,Brian;Sen,Indira
• 通讯作者: Sen,Indira