ROLE OF CYS RESIDUES AS A THIOL/DISULFIDE SWITCH IN HEME OXYGENASE 2 PROTEIN

半胱氨酸残基作为血红素加氧酶 2 蛋白中硫醇/二硫键开关的作用

• 批准号: 8363844
• 负责人: Paul R Ortiz De Montellano
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363844
• 关键词: Acids; Affinity; Anti-Inflammatory Agents; Anti-inflammatory; Antioxidants; Apoptotic; Bilirubin; Biliverdin reductase; Biliverdine; Binding; Biological Process; Carbon Monoxide; Catalysis; Cysteine; Data; Diet; Disulfides; Enzymes; Exhibits; Funding; Grant; Heme; Heme Iron; Homeostasis; Iron; Label; Ligand Binding; Mammals; Mass Spectrum Analysis; Measurement; Monitor; NADPH-Ferrihemoprotein Reductase; NMR Spectroscopy; National Center for Research Resources; Oxidation-Reduction; Oxygenases; Principal Investigator; Property; Proteins; Recycling; Reporting; Research; Research Infrastructure; Resources; Role; Signaling Molecule; Source; Spectrum Analysis; Structure; Sulfhydryl Compounds; United States National Institutes of Health; cost; disulfide bond; heme oxygenase-1; heme oxygenase-2; protoporphyrin IX; two-dimensional

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Heme oxygenases (HO) are enzymes involved in degradation of Fe(III) protoporphyrin IX (heme) to biliverdin IX, Fe(II) and carbon monoxide (CO) in the presence of O2 and NADPH cytochrome P450 reductase. The HOs are the only enzymes that can degrade heme indicating their essential role in maintaining heme and iron homeostasis. In addition, the three products generated from HO catalysis have important biological functions. The iron is recycled since only ~3% of the amount of iron required daily is obtained by the diet. CO serves as a signaling molecule exhibiting anti-apoptotic, anti-inflammatory, and anti-proliferation properties. Whereas, biliverdin is reduced by biliverdin reductase (in mammals) to the potent antioxidant bilirubin, which is subsequently conjugated with glucoronic acid and later secreted. The HO2 protein consists of three Cys residues which are proposed to regulate the binding affinity of HO2 for heme. The presence or absence of the disulfide bond formed between residues Cys265 and Cys282 resulted in an ~10-fold difference in the HO2 affinity for heme suggesting an important role of Cys in regulating the cellular levels of the free heme, CO, Fe, and biliverdin. There is no structural information of the region comprising C265 and C282 residues since they are missing in the HO2 crystal structure and furthermore, no NMR structural data has been reported up to date. We are using (1H, 13C) 2 dimensional NMR spectroscopy to monitor the redox state of Cys residues and protein conformational changes upon heme ligand binding. The HO2 proteins used for NMR measurements are L-[3-13C]-cysteine labeled and the incorporation efficiency of isotopically labeled Cys will be determined by Mass Spectrometry analysis. Furthermore, we will determine the redox state of the Cys residues by absorbance spectroscopy and Mass Spectrometry analysis.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 血红素氧酶（HO）是参与Fe（III）原核IX（Heme）降解的酶，在O2和NADPH Cytothrome P450还原酶的情况下，在biliverdin IX，Fe（II）和一氧化碳（CO）中均降解。 HOS是唯一可以降解血红素的酶，表明其在保持血红素和铁稳态中的重要作用。此外，由HO催化产生的三种产品具有重要的生物学功能。铁被回收，因为每天只能通过饮食获得所需的铁量的〜3％。 CO用作具有抗凋亡，抗炎和抗增殖特性的信号分子。而双脂蛋白通过双脂蛋白还原酶（在哺乳动物中）还原为有效的抗氧化剂胆红素，随后将其与葡萄糖酸结合并随后分泌。 HO2蛋白由三个CYS残基组成，这些残基提出了调节HO2对血红素的结合亲和力。残基Cys265和Cys282之间形成的二硫键的存在或不存在导致HO2亲和力的差异约10倍，这表明CYS在调节自由Heme，CO，Fe，Fe和Biliverdin的细胞水平中具有重要作用。由于HO2晶体结构缺少C265和C282残基，该区域没有包含C265和C282残基的结构信息，此外，尚无NMR结构数据的最新信息。我们正在使用（1H，13C）2维NMR光谱法来监测血红素配体结合时Cys残基的氧化还原状态和蛋白质构象变化。用于NMR测量的HO2蛋白是标记的L- [3-13C] - 半胱氨酸，同位素标记的CYS的掺入效率将通过质谱分析确定。此外，我们将通过吸光度光谱和质谱分析来确定CYS残基的氧化还原状态。