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: 7723068
• 负责人: MARTIN FEELISCH
• 金额: $ 0.52万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723068
• 关键词: Affect; Aliquot; Biological; Blood; Buffers; Cardiac; Cellular Stress Response; Cellular Structures; Computer Retrieval of Information on Scientific Projects Database; Computer software; Cytochrome P450; Daily; 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; Hour; Human; Image; Institution; Intraperitoneal Injections; Manuscripts; Maps; Measures; Medicine; Molecular Chaperones; Nitric Oxide; Nitrites; Oxidation-Reduction; Peptides; Phase; Physiological; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Range; Reperfusion Injury; Research; Research Personnel; Resistance; Resources; Signaling Molecule; Silver Staining; Sodium Nitrite; Soluble Guanylate Cyclase; Source; Spottings; Therapeutic; Therapeutic Uses; Thinking; Tissue Harvesting; Tissues; Trypsin; Two-Dimensional Gel Electrophoresis; United States National Institutes of Health; Wistar Rats; ascorbate; base; comparative; heme oxygenase-1; in vivo; mRNA Differential Displays; male; 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 thought for decades 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 very recently proven to be a signaling molecule in its own right, affecting soluble guanylyl cyclase and cytochrome P450 activities, heat shock protein 70 and heme oxygenase-1 expression, and protecting against cardiac ischemia-reperfusion injury. Based on this recent evidence, we are seeking to characterize 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, then 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 24 hours later, 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, followed by Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest" software, excised and subjected to in-gel trypsin digestion. 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 peptide mass fingerprinting analysis was conducted by submission of peak lists to MASCOT. Using 2D gel electrophoresis, we have created preliminary 2D reference maps of cardiac proteomes of control Wistar rats and those systemically administered physiologic and therapeutic levels of nitrite. We have confirmed the identity of over a hundred isolated protein spots through in-gel digestion followed by MALDI-TOF MS and peptide mass fingerprinting or by LC-MS/MS analyses. Quantitative comparative analyses have revealed significant changes to cardiac protein expression upon treatment with nitrite. These changes consisted of both up- and down-regulation of steady-state protein levels, as well as apparent alterations in post-translational protein modification and have included proteins involved in cell structure, energy metabolism, redox balance, and chaperone activity, among others. Additionally, we have measured metabonomic changes and changes to the cellular redox status that we have correlated to these proteomics data. Specifically, we have detected changes in the ratio of reduced to oxidized ascorbate and glutathione, as well as changes in the levels of S-nitroso, N-nitroso, and heme-nitroso species in the tissues. The results obtained using this combined proteomics/metabonomics approach 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, due to its stability in vivo, may be able to act in a long-range endocrine fashion to establish this protective tone in the heart. Our integrated proteomic and metabonomic approach is a step toward elucidating the scope and mechanism of cardioprotection and the potential activity of this biological compound 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. A manuscript describing our results is being prepared.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 亚硝酸盐在体内稳定而丰富，几十年来一直被认为在生理浓度下是生物惰性的。它由一氧化氮(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，广泛来自饮食来源，最近被证明是一种独立的信号分子，影响可溶性鸟苷酸环化酶和细胞色素P450的活性，热休克蛋白70和血红素加氧酶-1的表达，并对心肌缺血再灌注损伤具有保护作用。基于这些最新的证据，我们试图利用2D凝胶差异显示、LC-MS/MS或MALDI-TOF MS和多肽质量指纹图谱分析来表征全身亚硝酸盐可获得性变化对心肌蛋白质组的影响，然后将这些数据与代谢组学研究相关联。雄性Wistar大鼠一次性腹腔注射亚硝酸钠(0.1、1和10 mg/kg)，24小时后麻醉，无血灌流，取心脏组织。匀浆后即刻用气相化学发光法和高效液相色谱法测定NO相关代谢物的浓度。组织匀浆在IEF缓冲液中变性，2D-PAGE分析，考马斯亮蓝或银染。用PDQuest软件对蛋白质斑点进行成像和定量，切下蛋白质，用凝胶内胰酶消化，用MALDI-TOF MS或LC-MS和MS/MS洗脱、脱盐和分析多肽，用MoverZ“or MassLynx”和ProteinLynx软件分析Spectra，并将峰列表提交给吉祥物进行多肽质量指纹图谱分析。使用2D凝胶电泳法，我们已经创建了对照Wistar大鼠以及系统地给予生理和治疗水平的亚硝酸盐的心脏蛋白质组的初步2D参考图。我们已经通过凝胶内消化、MALDI-TOF MS和多肽质量指纹图谱或LC-MS/MS分析确认了100多个分离的蛋白质点的身份。定量比较分析显示，在亚硝酸盐治疗后，心脏蛋白表达发生了显著变化。这些变化包括稳定状态蛋白质水平的上调和下调，以及翻译后蛋白质修饰的明显变化，包括参与细胞结构、能量代谢、氧化还原平衡和伴侣活性的蛋白质等。此外，我们还测量了与这些蛋白质组学数据相关的代谢组学变化和细胞氧化还原状态的变化。具体地说，我们检测到了组织中还原型与氧化型抗坏血酸和谷胱甘肽比率的变化，以及S亚硝基、N-亚硝基和血红素亚硝基的水平变化。利用这种蛋白质组学/代谢组学相结合的方法获得的结果表明，亚硝酸盐触发的细胞应激反应蛋白的表达发生了特定的变化，这可能使人对进一步的氧化损伤产生抵抗。我们的数据表明，由于亚硝酸盐在体内的稳定性，它可能能够以一种长期的内分泌方式在心脏中建立这种保护性基调。我们集成的蛋白质组学和代谢组学方法是朝着阐明心脏保护的范围和机制以及这种新发现的对人类健康和医学具有重要意义的生物化合物的潜在活性迈出的一步。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，并可能引发对每天饮食摄入亚硝酸盐的影响的重新评估。一份描述我们结果的手稿正在准备中。