Nitric Oxide Synthases As Oxidative And Therapeutic Agents

作为氧化剂和治疗剂的一氧化氮合成酶

• 批准号: 7664775
• 负责人: DENNIS J STUEHR
• 金额: $ 10.35万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7664775
• 关键词: Amino Acid Substitution; Biological Markers; Biology; Blood Vessels; Cardiovascular Diseases; Cardiovascular Physiology; Cells; Chemistry; Clinical; Clinical Markers; Consumption; Coronary Arteriosclerosis; Data; Dependence; Development; Disadvantaged; Dissociation; Engineering; Environment; Enzymes; Equilibrium; Feedback; Free Radicals; Gene Transfer; Genes; Genetic Polymorphism; Genetic Predisposition to Disease; Heme; Human; Hyperplasia; Individual; Inflammation; Injury; Kinetics; Link; Mammalian Cell; Measures; Modeling; Molecular; Mutation; Nitric Oxide; Nitric Oxide Synthase; Open Reading Frames; Oxygen; Pathologic Processes; Peroxonitrite; Physiological; Point Mutation; Population; Prevalence; Process; Production; Rate; Recombinants; Relative (related person); Role; Single Nucleotide Polymorphism; Site; Stimulus; Stress; Structure; Superoxides; Testing; Therapeutic; Therapeutic Agents; Time; Variant; Viral; Work; cardiovascular disorder risk; cohort; dimer; follow-up; in vivo; injured; mutant; oxidation; protein protein interaction; research study; response; restenosis; vector

Nitric oxide (NO) participates in many physiological and pathological processes. NO is generated in humans by three NO synthases. Understanding their catalytic and regulatory mechanisms at the molecular level is critical for understanding their functions and potential use as therapeutic agents. Each NOS differs markedly in their rate of NO release, oxygen dependence profile, capacity for uncoupled superoxide release, and the ratio of NO versus peroxynitrite formed. Each NOS likely evolved to generate products and chemistries appropriate for specific circumstances in biology. We hypothesize that NOS variants can be engineered (or have been created naturally)for specific biologic advantage or disadvantage. We will test this by determining cellular consequences of NOS variants engineered to generate superphysiological amounts of NO, and by characterizing NOS variants that naturally appear in the human population and may be associated with genetic predisposition toward cardiovascular disease. Aim 1. Investigate efficacy of two "super NO synthases" for inhibiting neointimal hyperplasia following vascular injury. We have created two NOS variants that generate up to 25 times more NO compared to wild type enzyme. We will: (i) transfect the superNOS mutant genes into mammalian cells to test efficacy as superNO generators. (ii) Utilize viral delivery to test their efficacy for generating therapeutic NO in our carotid-injury restenosis model. (iii) Determine how NOS gene transfer impacts oxidative/nitrative biomarkers in the injured vessels. (iv) Incorporate additional mutations predicted to further increase efficacy of superNOS. Aim 2. Investigate the functional impact of specific single nucleotide polymorphisms (SNPs) that occur in endothelial and inducible NOS. There are five natural variants each of Human iNOS and eNOS that contain amino acid substitutions resulting from SNP's in the protein-coding region of their genes. We will express, purify, and extensively characterize these NOS variants to determine how each point mutation impacts enzyme function. Aim 3. Test if the eNOS and iNOS SNP's are linked to the development of coronary artery disease. The in vivo significance of NOS SNP's is largely unknown. We will: (i) Define the prevalence of ten SNPs that cause amino acid substitutions in iNOS and eNOS in individuals with and without CAD from a cohort of well-characterized subjects. (ii) Test if NOS SNP's that alter enzyme function also serve to predict increased risk for cardiovascular disease. (iii) Test how NOS SNPs correlate with clinical markers of oxidative or nitrosative stress,

一氧化氮(NO)参与多种生理和病理过程。否生成于 人类有三个没有合成酶。了解它们的催化和调节机制 分子水平对于了解它们的功能和作为治疗剂的潜在用途是至关重要的。 每种一氧化氮合酶在NO释放率、氧依赖谱、 解偶联的超氧化物释放，形成NO与过氧亚硝酸根的比值。每个NOS可能 进化以产生适合于生物学中特定环境的产品和化学物质。我们 假设一氧化氮合酶变异体可以针对特定的生物进行工程设计(或自然创造) 优势或劣势。我们将通过确定一氧化氮合酶变体的细胞后果来测试这一点 被设计成产生超生理量的一氧化氮，并通过表征一氧化氮合酶变体 自然地出现在人类群体中，可能与遗传易感性有关 心血管疾病。 目的1.研究两种“超一氧化氮合酶”抑制新生内膜的效果 血管损伤后的增生。我们已经创建了两个NOS变体，可以生成多达25个 比野生型酶多2倍。我们将：(I)将超一氧化氮合酶突变基因导入 哺乳动物细胞测试作为SuperNO生成器的效果。(2)利用病毒传递来测试其疗效 在我们的颈动脉损伤再狭窄模型中产生治疗性一氧化氮。(三)确定一氧化氮合酶基因如何 转移影响受损血管中的氧化/硝化生物标志物。(Iv)加入额外的 突变预测将进一步提高SuperNOS的疗效。 目的2.研究特异性单核苷酸多态(SNPs)对功能的影响 发生在内皮细胞和诱导型一氧化氮合酶中。人类诱导型一氧化氮合酶有五种天然变体 和内皮型一氧化氮合酶，它们包含由SNP引起的氨基酸替换 他们的基因。我们将表达、纯化并广泛地表征这些NOS变体，以确定如何 每一个点突变都会影响酶的功能。 目的3.检测eNOS和iNOS SNP是否与冠状动脉的发育有关 疾病。一氧化氮合酶SNP在体内的意义在很大程度上是未知的。我们将：(I)界定 10个导致iNOS和eNOS氨基酸替换的SNPs在有无冠心病患者中的作用 从一群特征良好的受试者中挑选出来的。(Ii)测试一氧化氮合酶SNP是否也能改变酶功能 用来预测心血管疾病风险的增加。(Iii)测试一氧化氮合酶SNPs与 氧化或亚硝化应激的临床标志物，