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的重要自由基介质。 NO 是支气管扩张和血管舒缩张力的重要调节剂。超氧化物是宿主防御的介质。这些药物的作用部分是由蛋白质的翻译后修饰引起的。 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。 3-磷酸​​甘油醛脱氢酶 (GAPDH) 是一种丰富的糖酵解酶，具有高活性活性位点硫醇，可能是 NO 的细胞内靶标。它通过其活性位点半胱氨酸的亚硝基化而被 NO 灭活，并在一氧化氮物质和硫醇存在的情况下被 NAD 或 NADH 进行翻译后修饰。在 SIN-1（3-吗啉代亚胺）和二硫苏糖醇存在的情况下，用 NADH 替换 NAD 可将修饰程度从约 1% 增加至 25%。因此，得出的结论是，NADH 是首选底物，硫醇通过转亚硝化反应刺激 NADH 的共价附着。与这些报道相反，实验室观察表明，NADH 与 GAPDH 的共价连接是在低分子量硫醇存在的情况下进行的，与 NO 无关。氧和过渡金属离子的去除抑制了改性，这与活性氧的作用一致。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作用的缺乏支持了硫醇氧化产生的超氧化物参与的假设。电喷雾质谱显示 NADH 分子与 GAPDH 共价连接。磷酸二酯酶裂解产物的表征表明，连接是通过 NADH 的烟酰胺发生的。 GAPDH 的 Lys-C 消化，然后通过高效液相色谱、基质辅助激光解吸电离飞行时间分析和 Edman 测序进行肽分离，证明 NADH 附着发生在半胱氨酸 149（活性位点硫醇）处。该硫醇键对氯化汞稳定。因此，与 NAD 不同，GAPDH 与 NADH 的连接是在硫醇存在的情况下发生的，不依赖于 NO，并且由超氧化物介导。 - 一氧化氮、细胞因子、脂多糖 - 人类受试者