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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 血红素加氧酶（HO）是在O2和NADPH细胞色素P450还原酶存在下参与Fe（III）原卟啉IX（血红素）降解为胆绿素IX、Fe（II）和一氧化碳（CO）的酶。血红素氧合酶是唯一能降解血红素的酶，这表明它们在维持血红素和铁稳态中起着重要作用。此外，HO催化产生的三种产物具有重要的生物学功能。铁被回收，因为每天所需铁量的约3%是通过饮食获得的。CO作为一种信号分子，具有抗凋亡、抗炎和抗增殖的特性。然而，胆绿素被胆绿素还原酶（在哺乳动物中）还原为有效的抗氧化剂胆红素，随后与葡萄糖醛酸结合并随后分泌。HO 2蛋白由三个Cys残基组成，其被提议调节HO 2对血红素的结合亲和力。残基Cys 265和Cys 282之间形成的二硫键的存在或不存在导致HO 2对血红素的亲和力的~10倍差异，表明Cys在调节游离血红素、CO、Fe和胆绿素的细胞水平中的重要作用。没有包含C265和C282残基的区域的结构信息，因为它们在HO 2晶体结构中缺失，此外，迄今为止还没有报道NMR结构数据。我们正在使用（1H，13 C）二维NMR光谱监测氧化还原状态的半胱氨酸残基和血红素配体结合后的蛋白质构象变化。用于NMR测量的HO 2蛋白是L-[3- 13 C]-半胱氨酸标记的，同位素标记的Cys的掺入效率将通过质谱分析测定。此外，我们将通过吸收光谱和质谱分析来确定Cys残基的氧化还原状态。