PROTEOMICS, METABONOMICS OF EFFECTS OF NITRITE TREATMENT ON THE HEART

亚硝酸盐治疗对心脏影响的蛋白质组学、代谢组学

• 批准号: 8170918
• 负责人: MARTIN FEELISCH
• 金额: $ 1.74万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170918
• 关键词: Affect; Aliquot; Biological; Blood; Blood Circulation; Buffers; Cardiac; Cellular Stress Response; Cellular Structures; Computer Retrieval of Information on Scientific Projects Database; Computer software; Cytochrome P450; Data; Dietary intake; Digestion; Down-Regulation; Endocrine; Energy Metabolism; Equilibrium; Fingerprint; Funding; Gases; Gel; Glutathione; Grant; Health; Heart; Heat-Shock Proteins 70; Heme; High Pressure Liquid Chromatography; Human; Image; Institution; Intraperitoneal Injections; Manuscripts; Maps; Measures; Medicine; Molecular Chaperones; Nitric Oxide; Nitrites; Oxidation-Reduction; Peptides; Phase; Philadelphia; Physiological; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Publishing; Reperfusion Injury; Research; Research Personnel; Resistance; Resources; Signaling Molecule; Silver Staining; Sodium Nitrite; Soluble Guanylate Cyclase; Source; Spottings; Therapeutic Uses; Tissue Harvesting; Tissues; Trypsin; United States National Institutes of Health; Wistar Rats; Work; ascorbate; base; comparative; heme oxygenase-1; in vivo; insight; lectures; mRNA Differential Displays; male; meetings; oxidation; protein expression

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nitrite, stable and abundant in vivo, was long thought to be biologically inert at physiological concentrations. Formed endogenously through oxidation of nitric oxide (an ubiquitous signaling molecule known to elicit broad biological effects) and derived extensively from dietary sources, it has itself recently proven to be a signaling molecule, affecting soluble guanylyl cyclase and cytochrome P450 activities, heat shock protein 70 and heme oxygenase-1 expression, and protecting against cardiac ischemia-reperfusion injury. We are therefore engaged in characterizing the impact of changes in systemic nitrite availability on the cardiac proteome, using 2D gel-based differential display followed by LC-MS/MS or MALDI-TOF MS and peptide mass fingerprinting analyses, and correlate these data to metabonomic studies. Male Wistar rats administered a single intraperitoneal injection of sodium nitrite (0.1, 1 and 10 mg/kg) were anesthetized after 24 h, perfused free of blood and their cardiac tissue harvested. Immediately after homogenization the concentrations of NO-related metabolites were determined by gas phase chemiluminescence and HPLC. Tissue aliquots were denatured in IEF buffer and subjected to 2D-PAGE analysis, with Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest" software, excised and subjected to in-gel trypsin digestion. The resulting peptides were eluted, de-salted and analyzed by MALDI-TOF MS or by LC-MS and MS/MS. Spectra were analyzed with MoverZ" or MassLynx" and ProteinLynx" software, and PMF analysis was conducted. We have created preliminary 2D-PAGE reference maps of cardiac proteomes of control and nitrite-administered Wistar rats. We have identified more than 100 isolated protein spots by PMF and/or LC-MS/MS analyses. Quantitative comparative analyses have revealed significant changes to cardiac protein expression upon treatment with nitrite, including both up- and down-regulation of steady-state protein levels, and apparent alterations in post-translational protein modification. The proteins are involved in cell structure, energy metabolism, redox balance, and chaperone activity, etc. We also measured metabonomic changes and changes to the cellular redox status and correlated these to the proteomics data. We detected changes in the ratio of reduced/oxidized ascorbate and glutathione, and in the levels of S- and N-nitroso, and heme-nitroso species in the tissues. The results indicate specific changes in expression of cellular stress response proteins triggered by nitrite that may confer resistance to further oxidative insults. Our data suggest that nitrite may be able to act in a long-range endocrine fashion to establish this protective tone in the heart. Our approach is a step toward elucidating the scope and mechanism of cardioprotection and the potential activity of NO with newly discovered importance in human health and medicine. Our data may have direct and immediate implications for current experimental therapeutic uses of nitrite and may provoke a reassessment of the impact of daily dietary intake of nitrite. The work was included in lectures at the HUPO meeting in Totonto in September 2009 and the ASMS meeting in Philadelphia in May 2009. A manuscript describing these results was published in Circulation Research DH Perlman et al., Mechanistic Insights into Nitrite-Induced Cardioprotection Using an Integrated Metabonomic-Proteomic Approach. Circ. Res., 2009, 104, 796-804).

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 亚硝酸盐在体内稳定而丰富，长期以来被认为在生理浓度下是生物惰性的。通过氧化一氧化氮（一种普遍存在的信号分子，已知可引起广泛的生物学效应）内源性形成，并广泛来源于饮食来源，它本身最近被证明是一种信号分子，影响可溶性鸟苷酸环化酶和细胞色素P450活性，热休克蛋白70和血红素加氧酶-1表达，并保护心脏缺血-再灌注损伤。 因此，我们正在研究全身亚硝酸盐利用率变化对心脏蛋白质组的影响，使用二维凝胶差异显示，然后进行LC-MS/MS或MALDI-TOF MS和肽质量指纹分析，并将这些数据与代谢组学研究相关联。 单次腹腔注射亚硝酸钠（0.1、1和10 mg/kg）给药的雄性Wistar大鼠在24 h后麻醉，无血灌注并收获其心脏组织。匀浆后立即通过气相化学发光和HPLC测定NO相关代谢物的浓度。将组织等分试样在IEF缓冲液中变性，并进行2D-PAGE分析，用考马斯或银染色。 使用PDQuest软件对蛋白质点进行成像和定量，切除并进行凝胶内胰蛋白酶消化。将所得肽洗脱、脱盐并通过MALDI-TOF MS或LC-MS和MS/MS进行分析。使用MoverZ”或MassLynx”和ProteinLynx”软件分析光谱，并进行PMF分析。 我们已经创建了对照组和亚硝酸盐给药的Wistar大鼠心脏蛋白质组的初步2D-PAGE参考图谱。 我们已经通过PMF和/或LC-MS/MS分析鉴定了100多个分离的蛋白质点。定量比较分析显示，与亚硝酸盐治疗后，心脏蛋白质表达的显着变化，包括上调和下调的稳态蛋白质水平，并在翻译后蛋白质修饰的明显改变。这些蛋白质参与细胞结构、能量代谢、氧化还原平衡和伴侣活性等。我们还测量了代谢组学变化和细胞氧化还原状态的变化，并将这些与蛋白质组学数据相关联。我们检测到的比例的变化，还原/氧化的抗坏血酸和谷胱甘肽，并在S-和N-亚硝基的水平，和血红素亚硝基物种在组织中。 结果表明亚硝酸盐引发的细胞应激反应蛋白表达的特定变化，可能赋予进一步的氧化损伤的抵抗力。我们的数据表明，亚硝酸盐可能能够以长距离内分泌方式发挥作用，在心脏中建立这种保护性基调。我们的方法是朝着阐明心脏保护的范围和机制以及NO的潜在活性迈出的一步，这些NO在人类健康和医学中具有新发现的重要性。我们的数据可能对目前亚硝酸盐的实验性治疗用途有直接和直接的影响，并可能引起对每日膳食摄入亚硝酸盐的影响的重新评估。这项工作被列入2009年9月在托托托举行的住房和城市规划组织会议和2009年5月在费城举行的ASMS会议的讲座。描述这些结果的手稿发表在Circulation Research DH Perlman et al.，使用综合代谢组学-蛋白质组学方法对亚硝酸盐诱导的细菌保护的机制见解。 Circ.结果：2009，104，796-804）。