CLEAVAGE/SECRETION OF ANGIOTENSIN CONVERTING ENZYME

血管紧张素转换酶的裂解/分泌

• 批准号: 6030696
• 负责人: INDIRA SEN
• 金额: $ 19.43万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6030696
• 关键词: cell free system; endopeptidases; enzyme activity; enzyme mechanism; glycosylation; goats; immunofluorescence technique; immunoprecipitation; laboratory rabbit; membrane proteins; membrane structure; peptidyl dipeptidase A; protease inhibitor; protein purification; protein structure function; secretion; site directed mutagenesis

Angiotensin-converting enzyme (ACE) is a widely distributed metalloprotease which plays a key role in the control of blood pressure and fluid and electrolyte homeostasis. Although ACE exists primarily as a membrane - bound enzyme, a soluble form is present under normal conditions in blood plasma and other body fluids. To delineate the structural relationship between the cell-bound and the soluble forms of the protein and to determine the modes of their biosynthesis, we have used various expression systems of cloned ACE cDNA. Our studies indicate that ACE is anchored in the cellular plasma membrane through a hydrophobic domain near its C- terminal and its extracellular secretion is achieved by a proteolytic cleavage of this membrane anchoring domain. The responsible protease is a cellular enzyme that cleaves ACE at or near the plasma membrane in a regulated fashion. The exact cleavage site has also been determined. We hypothesize that recognition of specific structural features of ACE by a cell-membrane associated secretase activity is responsible for the extracellular release of this protein. To test this hypothesis, we will determine the role of its intracellular, transmembrane and extracellular domains in the process of cleavage-secretion, by deleting, mutating or exchanging each domain, with the corresponding domain of an uncleavable transmembrane protein. We will define the role of specific amino acid residues at and around the cleavage site by introducing suitable amino acid substitution or deletions in the region. We will determine the role of glycosylation in this process, design and express a cell-bound non- secretable form of ACE and determine its enzymatic properties. We also propose to purify and characterize the ACE-secretase activity which is responsible for this cleavage-secretion process. For this purpose we will develop a cell-free assay system for measuring the ACE-secretase activity, process. For this purpose we will develop a cell-free assay system for measuring the ACE-secretase activity, characterize its enzymatic properties, purify it to homogeneity and develop antibody to it, so t hat we can isolate its cDNA clone in the future. Thus, the proposed project will provide a better understanding of how cell-bound ACE is converted to athe soluble form, how the relative proportion of the two forms is physiologically regulated and whether this conversion has a cell- specificity thereby causing localized ACE action in some tissue. Above and beyond the ACE system, our proposed study, will be the prototype for an important and general mode of biological regulation and will identify the first member of a new class of membrane-bound proteases whose primary role is to selectively cleave and release many biologically important proteins.

血管紧张素转换酶(ACE)是一种分布广泛的金属蛋白酶 它在控制血压和液体方面起着关键作用 电解质动态平衡。尽管ACE主要以膜的形式存在- 结合酶，在正常情况下以可溶形式存在于血液中 血浆和其他体液。勾勒出结构关系 在细胞结合形式和可溶形式的蛋白质之间 确定了它们的生物合成方式，我们用了各种表达方式 血管紧张素转换酶基因克隆系统。我们的研究表明血管紧张素转换酶与 细胞质膜通过其C-端附近的疏水结构域 末端及其胞外分泌是通过蛋白水解物实现的 该膜锚定结构域的裂解。负责的蛋白酶是一种 在细胞膜上或质膜附近裂解ACE的细胞酶 受监管的时尚。准确的切割位置也已确定。我们 假设血管紧张素转换酶的特定结构特征由 细胞-膜相关的分泌酶活性是导致 这种蛋白质在细胞外的释放。为了检验这一假设，我们将 确定其在细胞内、跨膜和细胞外的作用 在切割-分泌过程中，通过删除、突变或 交换每个域，与不可拆分的相应域 跨膜蛋白。我们将定义特定氨基酸的作用 通过引入合适的氨基酸在裂解部位及其周围残基 区域内的替换或删除。我们将确定 在这个糖基化过程中，设计并表达了一种细胞结合的非 分泌形式的血管紧张素转换酶并测定其酶学性质。我们也 提出了血管紧张素转换酶分泌酶活性的纯化和鉴定 负责这一卵裂-分泌过程。为此，我们将 开发一种检测ACE分泌酶活性的无细胞检测系统 进程。为此，我们将开发一种无细胞检测系统，用于 ACE-分泌酶活性的测定及其酶学性质 性质，将其提纯为均一，并产生抗体，因此 我们可以在将来分离它的cdna克隆。因此，拟议的项目 将更好地理解细胞绑定ACE是如何转换为 对于可溶性形式，这两种形式的相对比例是多少 生理调节，以及这种转换是否有一个细胞- 特异性，从而导致局部的血管紧张素转换酶作用于某些组织。以上和 除了ACE系统，我们提议的研究将成为一种 生物调节的重要和一般模式，并将确定 一类新的膜结合蛋白酶的第一个成员，其主要作用是 选择性地裂解和释放许多具有生物重要性的蛋白质。