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

基本信息

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
项目类别：
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

项目摘要

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）参与许多生理和病理过程。 NO 产生于人类通过三种NO合酶。了解它们的催化和调节机制分子水平对于理解它们的功能和作为治疗剂的潜在用途至关重要。每种 NOS 在 NO 释放速率、氧依赖性、未耦合的超氧化物释放，以及 NO 与形成的过氧亚硝酸盐的比率。每个 NOS 可能进化以产生适合生物学特定情况的产品和化学物质。我们假设 NOS 变体可以针对特定的生物制剂进行工程改造（或自然产生）优点或缺点。我们将通过确定 NOS 变异的细胞后果来测试这一点旨在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出现在人群中，可能与遗传倾向有关心血管疾病。目标1.研究两种“超级NO合酶”抑制新生内膜的功效血管损伤后增生。我们创建了两个 NOS 变体，最多可生成 25 个与野生型酶相比，NO 多出一倍。我们将： (i) 将 superNOS 突变基因转染至哺乳动物细胞来测试作为超级一氧化氮发生器的功效。 (ii) 利用病毒传递来测试其功效用于在我们的颈动脉损伤再狭窄模型中产生治疗性 NO。 (iii) 确定NOS基因如何转移影响受损血管中的氧化/硝化生物标志物。 (iv) 纳入额外的预计突变会进一步提高 superNOS 的功效。目标 2. 研究特定单核苷酸多态性 (SNP) 的功能影响发生在内皮细胞和诱导型 NOS 中。人类 iNOS 有五种自然变体和 eNOS，其含有由蛋白质编码区中的 SNP 产生的氨基酸取代他们的基因。我们将表达、纯化并广泛表征这些 NOS 变体，以确定如何每个点突变都会影响酶的功能。目标 3. 测试 eNOS 和 iNOS SNP 是否与冠状动脉的发育相关疾病。 NOS SNP 的体内意义很大程度上未知。我们将： (i) 定义导致患有和不患有 CAD 个体的 iNOS 和 eNOS 氨基酸替换的 10 个 SNP 来自一群特征良好的受试者。 (ii) 测试 NOS SNP 是否也会改变酶功能用于预测心血管疾病风险增加。 (iii) 测试 NOS SNP 如何与氧化或亚硝化应激的临床标志物，