REGULATION OF GENE EXPRESSION BY NO/CGMP

NO/CGMP 对基因表达的调节

• 批准号: 6019266
• 负责人: RENATE B PILZ
• 金额: $ 22.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019266
• 关键词: affinity chromatography; biological signal transduction; cGMP dependent protein kinase; chimeric proteins; cyclic GMP; enzyme activity; enzyme mechanism; enzyme substrate; genetic enhancer element; genetic promoter element; genetic regulatory element; immunofluorescence technique; nitric oxide; nucleic acid sequence; oligonucleotides; site directed mutagenesis; transcription factor; tumor necrosis factor alpha; yeast two hybrid system

DESCRIPTION (Investigator's Abstract): The nitric oxide (NO)cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction pathway is important for the regulation of many physiological and pathophysiological processes, e.g., in the cardiovascular system it is a major determinant of smooth muscle cell, endothelial cell and platelet functions and is implicated in the development of hypertension and atherosclerosis. However, compared to cAMP-dependent protein kinase, little is known about the downstream effects of G-kinase activation. The investigators found that G-kinase regulates gene expression: in response to NO or cGMP, the kinase translocates to the nucleus, induces phosphorylation of CREB-related proteins and activates the fos, junB and TNF-alpha promoters. Preliminary data suggest that G-kinase transactivates these promoters through similar cis-acting elements recognized by transcription factors of the AP-1 (Fos-Jun), CRE- and CCAAT-enhancer binding protein families and that subcellular localization and biological activity of the kinase is regulated by specific anchoring proteins. The speciic aims of this proposal are: (1) to define DNA sequences of NO/cGMP-response elements (NGREs): (II) to identify transcription factors targeted by NO/cGMP/G-kinase; and (III) to identify key substrates and cytoplasmic or nuclear anchoring proteins which bind to G-kinase. NGREs will be defined by deletion and site-directed mutagenesis of putative enhancer elements in the fos and TNF-alpha promoters. Transcription factors targeted by NO/cGMP will be identified in transactivation studies using Ga14-fusion products and dominant negative mutants of candidate transcription factors as well as in DNA binding studies and DNA affinity chromatography using oligodeoxynucleotides encoding NGREs. Protein interaction cloning will be employed to identify proteins interacting with G-kinase and the effect of these proteins on subcellular localization and function of G-kinase will be tested. The widespread importance of NO as a signaling molecule has been recognized recently; the studies proposed in this grant application should provide new insights into the mechanism of action of NO and cGMP in mammalian cells. Since pharmacological manipulation of the NO/cGMP signal transduction pathway offers therapeutic potential for a wide variety of human diseases, a better understanding of the long-term downstream effects of NO and cGMP is urgently needed.

描述（研究者摘要）：一氧化氮 (NO)cGMP/cGMP 依赖性蛋白激酶（G-激酶）信号转导 途径对许多生理和功能的调节非常重要 病理生理过程，例如，在心血管系统中，它是 平滑肌细胞、内皮细胞和血小板的主要决定因素 功能并与高血压的发展有关 动脉粥样硬化。 然而，与 cAMP 依赖性蛋白激酶相比，很少 已知 G 激酶激活的下游效应。 这 研究人员发现 G-激酶调节基因表达：响应 NO 或 cGMP，激酶易位至细胞核，诱导磷酸化 CREB ​​相关蛋白并激活 fos、junB 和 TNF-α 发起人。 初步数据表明 G-激酶反式激活这些 通过转录识别的相似顺式作用元件启动子 AP-1 (Fos-Jun)、CRE 和 CCAAT 增强子结合蛋白的因子 家族以及亚细胞定位和生物活性 激酶受特定锚定蛋白调节。 具体目标 该提案是： (1) 定义 NO/cGMP 响应元件的 DNA 序列 (NGRE)：(II) 识别目标转录因子 NO/cGMP/G-激酶； (III) 识别关键底物和细胞质或 与 G 激酶结合的核锚定蛋白。 NGRE 的定义为 假定的增强子元件的缺失和定点突变 fos 和 TNF-α 启动子。 NO/cGMP 靶向的转录因子将 在使用 Ga14 融合产物的反式激活研究中被识别，并且 候选转录因子的显性失活突变体以及 DNA 结合研究和 DNA 亲和层析使用 编码 NGRE 的寡脱氧核苷酸。 蛋白质相互作用克隆将 用于鉴定与 G 激酶相互作用的蛋白质以及 这些蛋白质对 G-激酶的亚细胞定位和功能的影响将是 已测试。 NO 作为信号分子的广泛重要性已被 最近被认可；本拨款申请中提出的研究应 为 NO 和 cGMP 的作用机制提供新的见解 哺乳动物细胞。 由于 NO/cGMP 信号的药理学操纵 转导途径为多种疾病提供了治疗潜力 人类疾病，更好地了解长期下游影响 迫切需要 NO 和 cGMP。