Heteropolymer Ferritins Structure-Function Studies

杂聚物铁蛋白结构-功能研究

• 批准号: 8877681
• 负责人: Fadi Bou-Abdallah
• 金额: $ 26.06万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT POTSDAM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8877681
• 关键词: Affect; Bacteria; Biochemical Process; Biochemistry; Biological; Cells; Ceruloplasmin; Chemistry; Complex; Data; Defect; Deposition; Development; Differential Scanning Calorimetry; Disease; Drug Metabolic Detoxication; Electronics; Equation; Ferritin; Fluorescence Spectroscopy; Goals; Health; Heme; Heme Iron; Homeostasis; Human; Hydrogen Peroxide; Hydrolysis; Inherited; Invertebrates; Iron; Iron Overload; Kinetics; L-ferritin; Lead; Light; Magnetism; Mammals; Measurement; Metabolic; Minerals; Molecular; Mossbauer Spectroscopy; Mutation; Organism; Oxygen; Oxygen saturation measurement; Play; Process; Property; Proteins; Reaction; Recombinants; Research; Research Proposals; Role; Site-Directed Mutagenesis; Staging; Structure; Structure-Activity Relationship; Techniques; Tissues; Toxic effect; Variant; Vertebrates; crystallinity; design; in vivo; insight; iron metabolism; iron oxide; mineralization; nanoparticle; oxidation; plant fungi; programs; public health relevance; research study; thermostability; uptake

 DESCRIPTION (provided by applicant): Ferritins are multi-subunit iron storage and detoxification proteins that play a central role in the biological management of iron. In vertebrates, ferritins consist of 24 subunits of two types, H and L that co-assemble in various ratios with a tissue specific distribution. The homopolymer H- chain contains ferroxidase centers where the rapid oxidation of Fe(II) to Fe(III) occurs by either molecular oxygen or hydrogen peroxide. While ferritins from different organisms share many common structural features, the chemistries of iron uptake, oxidation, deposition and mobilization differ markedly. Extensive studies have been performed, separately, with either recombinant homopolymer H-chain or recombinant L-chain ferritin, but not with the heteropolymer H/L ferritin. [Surprisingly, and despite the widespread occurrence of heteropolymer ferritins of different H to L subunit ratio (isoferritins) in tissues of vertebrates], very little is known about these proteins and the complementary roles that H and L subunits play during iron uptake and mineralization. The goals of this research proposal are to investigate the structure-function relationships of iron uptake and deposition in recombinant heteropolymer ferritins, [which mimic naturally occurring ferritins in-vivo], and to characterize the stability and functionality of two pathogenic L-ferriti variants responsible for a hereditary ferritinopathy disorder. Specifically, we plan to study (a) te complementary roles of H and L subunits in iron oxidation and mineralization and identify iron-protein intermediates during this process, (b) the effect of L-chain mutations and iron content on the protein thermostability and (c) the magnetism and crystallinity of the iron core formed inside the ferritin cavity. To achieve this, a combination of site-directed mutagenesis, pH stat/oximetry, stopped-flow rapid kinetics techniques, UV- visible and fluorescence spectroscopy, differential scanning calorimetry and Mössbauer spectroscopy will be employed. The experiments proposed here should lead to a detailed understanding of the chemistry of iron deposition in heteropolymer ferritins and how different proportions of H and L subunits affect the biochemistry and functional properties of isoferritins. It will also provide insights into the biochemical processes responsible for the hereditary neuroferritinopathy disorder. Additionally, the proposed Mössbauer measurements of ferritin iron core should provide important insights into the structure, design, and development of controlled size nanoparticles within the ferritin shell for a broad range of applications ranging from electronics to biomedicine.

 描述（由申请人提供）：铁蛋白是多亚基铁储存和解毒蛋白，在铁的生物管理中发挥核心作用。在脊椎动物中，铁蛋白由两种类型的24个亚基组成，H和L以不同的比例与组织特异性分布共组装。均聚物H-链含有铁氧化酶中心，其中Fe（II）通过分子氧或过氧化氢快速氧化为Fe（III）。虽然来自不同生物体的铁蛋白具有许多共同的结构特征，但铁的吸收、氧化、沉积和动员的化学性质明显不同。已经分别用重组均聚物H-链或重组L-链铁蛋白进行了广泛的研究，但没有用杂聚物H/L铁蛋白。[令人惊讶的是，尽管在脊椎动物组织中广泛存在不同H与L亚基比例的杂聚物铁蛋白（异铁蛋白）]，但对这些蛋白质以及H和L亚基在铁吸收和矿化过程中发挥的互补作用知之甚少。本研究的目标是研究重组杂聚物铁蛋白中铁吸收和沉积的结构-功能关系，[其模拟体内天然存在的铁蛋白]，并表征两种致病性L-铁蛋白变体的稳定性和功能性，这两种变体负责遗传性铁蛋白病。具体而言，我们计划研究（a）在铁氧化和矿化的H和L亚基的互补作用，并确定在此过程中的铁蛋白中间体，（B）的L-链突变和铁含量对蛋白质的热稳定性的影响和（c）的磁性和结晶度的铁蛋白空腔内形成的铁核心。为了实现这一点，结合定点诱变，pH统计/血氧测定， 将采用停流快速动力学技术、紫外可见和荧光光谱法、差示扫描量热法和穆斯堡尔光谱法。这里提出的实验应该导致一个详细的了解铁沉积在杂聚铁蛋白的化学和不同比例的H和L亚基如何影响异铁蛋白的生物化学和功能特性。它也将提供深入了解生化过程负责遗传性神经铁蛋白病紊乱。此外，提议的铁蛋白铁核的穆斯堡尔测量应该为铁蛋白壳内可控尺寸纳米颗粒的结构、设计和发展提供重要的见解， 从电子到生物医学的广泛应用。