Nitric Oxide Synthases as Oxidative and Therapeutic Agents

一氧化氮(NO)参与多种生理和病理过程。人类体内的NO是由三种NO合成酶产生的。在分子水平上了解它们的催化和调节机制对于了解它们的功能和作为治疗剂的潜在用途至关重要。每种一氧化氮合酶在NO释放率、氧依赖性、超氧化物歧化释放量以及NO与过氧亚硝酸根形成的比率等方面存在显著差异。每一种一氧化氮合酶都可能进化为产生适合生物学中特定环境的产物和化学物质。我们假设一氧化氮合酶的变异体可以为特定的生物优势或劣势而设计(或自然创造)。我们将通过确定一氧化氮合酶变异的细胞后果来测试这一点，这些变异被设计成产生超生理量的一氧化氮，并通过表征自然出现在人类群体中的可能与心血管疾病的遗传易感性相关的一氧化氮合酶变异。 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SuperNOS突变基因导入哺乳动物细胞，以测试其作为SuperNO生成器的效果。(Ii)在我们的颈动脉损伤再狭窄模型中，利用病毒传递来测试它们产生治疗性NO的有效性。(Iii)确定一氧化氮合酶基因转移如何影响受损血管中的氧化/硝化生物标志物。(Iv)加入更多的突变，预计将进一步提高SuperNOS的疗效。目的2.研究内皮细胞和诱导型一氧化氮合酶特异性单核苷酸多态(SNPs)对功能的影响。人类诱导型一氧化氮合酶和内皮型一氧化氮合酶有五种天然变体，每一种都包含由其基因蛋白质编码区的SNP引起的氨基酸替换。我们将表达、纯化和广泛鉴定这些一氧化氮合酶变体，以确定每个点突变如何影响酶功能。目的3.检测内皮型一氧化氮合酶和诱导型一氧化氮合酶单核苷酸多态是否与冠状动脉疾病的发生有关。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)从一组特征良好的受试者中，确定导致iNOS和eNOS氨基酸替换的10个SNPs在患有和不患有冠心病的个体中的流行率。(Ii)测试改变酶功能的NOS SNP是否也可用于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指标之间的相关性。

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.