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体。 NO是支气管扩张和血管舒张肌张力的重要调节剂。超氧化物是宿主防御的调解人。这些药物的作用部分是由蛋白质的翻译后修饰引起的。否是亲脂性的，并且很容易通过细胞膜扩散，不仅与血浆和细胞外蛋白质相互作用，而且与细胞质蛋白相互作用。甘油醛-3-磷酸脱氢酶（GAPDH）是一种具有高反应性活性位点硫醇的丰富糖溶解酶，可能代表NO的细胞内靶标。 NO通过硝基化的活性位点半胱氨酸而被NO灭活，并在存在一氧化氮物种和硫醇的情况下由NAD或NADH进行了翻译后修饰。在存在SIN-1（3-多酚辛二氨基胺）和二硫代醇的存在下，NADH替换NAD，将修饰程度从〜1％提高到25％。因此，已经得出结论，NADH是首选的底物，硫醇通过经硝化反应刺激了NADH的共价附着。与这些报告相反，实验室的观察结果表明，在存在低分子量硫醇的情况下，NADH与GAPDH的共价附着，与No无关。去除氧和过渡金属离子抑制了修饰，这与活性氧的作用一致。超氧化物歧化酶抑制，黄嘌呤氧化酶/低黄嘌呤的刺激以及缺乏过氧化氢酶的作用支持以下假说：涉及硫醇氧化产生的超氧化物涉及。电喷雾质谱法显示NADH分子与GAPDH的共价连接。磷酸二酯酶裂解产物的表征表明，连接发生通过NADH的烟酰胺发生。 LYS-C-C消化GAPDH，然后通过高性能液相色谱分离肽，基质辅助激光解吸电离电离时间 - 飞行时间分析和Edman测序，证明了NADH附着在Cysteine-149上发生了NADH附着，这是活性位点硫醇。这种硫醇连接与氯化汞稳定。因此，与NAD相比，GAPDH与NAD的连接发生在硫醇存在下，与NO无关，并由超氧化物介导。 - 一氧化氮，细胞因子，脂多糖 - 人类受试者