ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF LUNG DISEASE

一氧化氮在肺部疾病发病机制中的作用

• 批准号: 6290428
• 负责人: Joel Moss
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290428
• 关键词: RNA splicing; alveolar macrophages; clinical research; cytokine; enzyme induction /repression; gene deletion mutation; human subject; interview; lipopolysaccharides; neoplastic cell culture for noncancer research; nitric oxide; nitric oxide synthase; pathologic process; protein sequence; respiratory epithelium; southern blotting

Superoxide and nitric oxide are important free radical mediators of diverse biological processes. NO is an important regulator of bronchodilation and vasomotor tone. Superoxide is a mediator in host defense. The effects of these agents, in part, result from post- translational modification of proteins. NO is lipophilic and diffuses readily through cellular membranes, interacting not only with plasma and extracellular proteins but with cytoplasmic proteins as well. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an abundant glycolytic enzyme with a highly reactive active site thiol, may represent an intracellular target of NO. It is inactivated by NO through nitrosylation of its active-site cysteine and post- translationally modified by NAD or NADH in the presence of nitric oxide species and thiols. Replacement of NAD by NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased the degree of modification from ~1% to 25%. It had been concluded, therefore, that NADH was the preferred substrate and thiols stimulated covalent attachment of NADH via a transnitrosation reaction. In contrast to these reports, observations in the laboratory suggested that the covalent attachment of NADH to GAPDH proceeded in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species. Inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that the linkage occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of- flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at cysteine-149, the active site thiol. This thiol linkage was stable to mercuric chloride. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO and is mediated by superoxide. - nitric oxide, cytokines, lipopolysaccharide - Human Subjects

超氧化物和一氧化氮是多种生物过程中重要的自由基介质。NO是一种重要的支气管扩张和血管舒张性调节因子。超氧化物歧化酶是机体防御的中介物。这些药物的作用部分是由蛋白质的翻译后修饰造成的。NO是亲脂性的，很容易通过细胞膜扩散，不仅与血浆和细胞外蛋白相互作用，还与细胞质蛋白相互作用。甘油醛-3-磷酸脱氢酶(GAPDH)是一种丰富的糖酵解酶，具有高活性的硫醇，可能是NO的胞内靶标。它被NO通过其活性部位半胱氨酸的亚硝化而失活，并在存在一氧化氮和硫醇的情况下被NAD或NADH翻译后修饰。在SIN-1和二硫苏糖醇存在下，NADH取代NAD的修饰度从~1%提高到25%。因此，我们得出结论，NADH是最好的底物，硫醇通过转亚硝化反应促进NADH的共价连接。与这些报道相反，实验室的观察表明，NADH与GAPDH的共价结合是在低分子量硫醇存在下进行的，与NO无关。氧和过渡金属离子的去除抑制了修饰，这与活性氧物种的作用一致。超氧化物歧化酶的抑制，黄嘌呤氧化酶/次黄嘌呤的刺激，以及过氧化氢酶的缺乏，都支持这样的假设，即由硫醇氧化产生的超氧化物参与了这一假设。电喷雾质谱仪显示NADH分子与GAPDH发生共价连接。磷酸二酯酶裂解产物的表征表明，这种连接是通过NADH的烟酰胺进行的。经Lys-C消化、高效液相色谱分离、基质辅助激光解吸电离飞行时间分析和Edman测序，证明NADH结合在活性部位半胱氨酸-149上。该硫醇键对氯化汞很稳定。因此，与NAD不同的是，GAPDH与NADH的连接是在硫醇存在的情况下发生的，不依赖于NO，是由超氧化物介导的。-一氧化氮、细胞因子、脂多糖-人类受试者