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）参与了许多生理和病理过程。没有生成 人类通过三个无合酶。了解他们的催化和调节机制 分子水平对于理解其功能和作为治疗剂的潜在用途至关重要。 每个NOS的无释放速率，氧依赖性概况，能力，能力明显不同 未偶联的超氧化物释放，而NO与过氧亚硝酸盐的比率形成。每个NOS可能 演变为生成适合生物学特定情况的产品和化学物质。我们 假设可以为特定的生物学设计（或自然而然地创建）NOS变体 优势或劣势。我们将通过确定NOS变体的细胞后果来测试这一点 设计为生成超生理数量的NO，并通过表征NOS变体 自然出现在人口中，可能与遗传倾向有关 心血管疾病。 目标1。研究两个“超级无合酶”的功效以抑制新内膜 血管损伤后的增生。我们创建了两个NOS变体，最多生成25 与野生型酶相比，毫无疑问。我们将：（i）转染超新星突变基因 哺乳动物细胞以将功效作为超中期发电机测试。 （ii）利用病毒输送来测试其功效 用于在我们的颈动脉伤害再狭窄模型中产生治疗NO。 （iii）确定NOS基因如何 转移会影响受伤血管中的氧化/硝酸生物标志物。 （iv）合并其他 预计将进一步提高超其他事物的疗效的突变。 目标2。研究特定单核苷酸多态性（SNP）的功能影响 这发生在内皮和诱导的NOS中。每个人iNOS都有五个自然变体 和包含由SNP在蛋白质编码区域中的氨基酸取代的eNOS 他们的基因。我们将表达，纯化和广泛表征这些NOS变体，以确定如何 每个点突变都会影响酶功能。 AIM 3。测试ENOS和INOS SNP是否与冠状动脉的发展有关 疾病。 NOS SNP的体内意义在很大程度上是未知的。我们将：（i）定义 在有和没有CAD的个体中引起iNOS和ENOS的十个SNP 来自一系列特征良好的受试者。 （ii）测试是否也改变了酶功能的NOS SNP 有助于预测心血管疾病的风险增加。 （iii）测试NOS SNP与 氧化或亚硝化应激的临床标记，