ANGIOTENSIN CONVERTING ENZYME GENE EXPRESSION

血管紧张素转换酶基因表达

• 批准号: 2224323
• 负责人: GANES C. SEN
• 金额: $ 21.83万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224323
• 关键词: cyclic AMP; enzyme structure; gene expression; genetic regulation; genetic transcription; genetically modified animals; glucocorticoids; hypertension; laboratory mouse; messenger RNA; methylation; peptidyl dipeptidase A; protein structure function; tissue /cell culture; transfection

Angiotensin-converting enzyme (ACE) is a glycosylated ectoprotein which is found in both cell-bound .and secreted forms. This enzyme is responsible for the synthesis of angiotensin II which has diverse physiological effects including maintenance of fluid and electrolyte balance and blood pressure. Elevated circulating levels of ACE are associated with the pathogenesis of 'essential' hypertension, heart failure, and renal failure and inhibitors of this enzyme are widely used for clinical management of these diseases. It is therefore important to understand the regulation of biosynthesis of ACE, its structure and its functions. In this application, we propose to use the powerful tools of molecular biology for this purpose. Previous investigations have shown that there are two isozymic forms of ACE, ACEp and ACET, which are expressed in a tissue specific manner. ACEp is synthesized in vascular endothelial cells and in the epithelial cells of kidney and intestine whereas ACET is synthesized only in sperm cells. Both isozymes originate from the same gene by alternative choice of two transcriptional start sites. The two resultant mRNAs, and the corresponding proteins, have common regions as well as isozyme-specific regions. We propose to investigate the molecular mechanisms responsible for tissue-specific expression of the two ACE mRNAs. For this purpose, we will study the cis-acting elements and trans-acting factors which regulate their transcription. The cis-acting elements will be identified by the expression of suitable chimeric reporter genes ,in transgenic mice and in transfected cells in culture. The trans-acting factors which bind to these cis-acting elements will be identified by electrophoretic mobility shift and footprinting assays. We also propose to ,study the routes of biosynthesis and secretion of the ACE proteins. Both permanent and transient transfectants will be used for this purpose. We will examine the effects of inhibiting glycosylation and cell-surface proteolytic activity on the synthesis, processing, and secretion of ACE. Using site-specific mutagenesis, we will identify the specific amino acid residues necessary for enzyme activity, membrane anchoring, and cleavage-processing of the protein. Finally, we will determine the enzymatic properties of cell-bound ACE by expressing a non-secretable form of ACE. The information obtained from the last aim ,will be valuable for understanding localized action of ACE in the body.

血管紧张素转换酶（ACE）是一种糖基化的外蛋白，是 在两个细胞结合的。和分泌形式中发现。 该酶是负责的 对于具有多种生理作用的血管紧张素II的合成 包括维持流体和电解质平衡和血压。 ACE的循环水平升高与 “必不可少的”高血压，心力衰竭和肾衰竭和抑制剂 该酶的其中广泛用于这些疾病的临床管理。 因此，重要的是要了解对生物合成的调节 王牌，其结构及其功能。 在此应用程序中，我们建议 为此，使用强大的分子生物学工具。 以前的 调查表明，ACE有两种同学形式ACEP 和以组织特定方式表达的acet。 Acep是 在血管内皮细胞中合成以及在 肾脏和肠道，而乙酸仅在精子细胞中合成。 两个都 同工酶源自同一基因。 转录启动站点。 由此产生的mRNA和 相应的蛋白质具有共同区域以及同工酶特异性 地区。 我们建议研究负责的分子机制 用于两个ACE mRNA的组织特异性表达。 为此，我们 将研究调节的顺式作用元素和跨作用因子 他们的转录。 顺式作用元素将由 在转基因小鼠中和在 在培养中转染的细胞。 与这些结合的跨性别因素 从电泳迁移率转移将确定顺式作用元素 和足迹测定法。 我们还建议，研究 ACE蛋白的生物合成和分泌。 永久和 瞬态转染物将用于此目的。 我们将检查 抑制糖基化和细胞表面蛋白水解活性的影响 关于ACE的合成，加工和分泌。 使用特定于网站的 诱变，我们将确定必要的特定氨基酸残基 用于酶活性，膜锚定和切割 蛋白质。 最后，我们将确定细胞结合的酶促特性 ACE通过表达不可销的ACE形式。 获得的信息 从最后一个目标来看，对于理解的本地行动是有价值 体内的王牌。