CLEAVAGE/SECRETION OF ANGIOTENSIN CONVERTING ENZYME

血管紧张素转换酶的裂解/分泌

• 批准号: 2735258
• 负责人: INDIRA SEN
• 金额: $ 18.68万
• 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/2735258
• 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）是一种广泛分布的金属蛋白酶 它在控制血压和体液方面起着关键作用， 电解质稳态 虽然ACE主要以膜的形式存在- 结合酶，在正常条件下存在于血液中的可溶形式 血浆和其他体液。 来描述结构关系 在蛋白质的细胞结合形式和可溶形式之间， 为了确定它们的生物合成模式，我们使用了各种表达方法， 克隆的ACE cDNA的系统。 我们的研究表明，ACE是锚定在 细胞质膜通过其C- 末端和其胞外分泌是通过蛋白水解酶实现的。 切割该膜锚定结构域。 负责的蛋白酶是 血管紧张素转换酶：一种在细胞膜上或膜附近切割血管紧张素转换酶的细胞酶， 规范的时尚。 还确定了确切的切割位点。 我们 假设ACE特异性结构特征被一种 细胞膜相关的分泌酶活性负责 细胞外释放这种蛋白质。 为了验证这个假设，我们将 确定其细胞内，跨膜和细胞外的作用 结构域在裂解-分泌过程中，通过缺失、突变或 将每个结构域与不可裂解的 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，在切割位点处及其周围的残基 区域中的取代或缺失。 我们将决定 糖基化，设计并表达一个细胞结合的非- ACE的分泌形式，并确定其酶性质。 我们也 建议纯化和表征ACE-分泌酶活性， 负责这个分裂分泌过程。 为此，我们将 开发用于测量ACE-分泌酶活性的无细胞测定系统， 过程 为此，我们将开发一种无细胞测定系统， 测定ACE-分泌酶活性， 性质，纯化至均一，并开发抗体，因此， 我们将来可以分离它的cDNA克隆。 因此，拟议项目 将更好地了解细胞结合的ACE是如何转化为 不溶形式，两种形式的相对比例如何 生理调节，以及这种转换是否具有细胞- 特异性，从而在一些组织中引起局部ACE作用。 上方和 除了ACE系统，我们提出的研究，将是一个原型 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 是选择性地切割和释放许多重要的生物蛋白质。