Mechanism and inhibition of collagen prolyl-4-hydroxylases

胶原蛋白脯氨酰-4-羟化酶的作用机制及抑制作用

• 批准号: 8072106
• 负责人: Minae Mure
• 金额: $ 24.89万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072106
• 关键词: Active Sites; Anabolism; Anthrax disease; Ascorbic Acid; Bacillus anthracis; Binding; Biological Models; C-terminal; Catalytic Domain; Collagen; Comparative Study; Core Facility; Cytochromes; Data; Dioxygenases; Disease; Endoplasmic Reticulum; Environment; Enzyme Kinetics; Enzymes; Family; Fibrosis; Goals; Human; Hydroxylation; Iron; Kansas; Knowledge; Liver Fibrosis; Mammals; Membrane; Modeling; Mononuclear; Oxidases; Oxygen; Oxygenases; Peptides; Procollagen-Proline Dioxygenase; Proline; Protein Disulfide Isomerase; Proteins; Pulmonary Fibrosis; Reaction; Reporting; Research; Research Personnel; Roentgen Rays; Role; Scurvy; Sequence Homology; Spectrum Analysis; Structure; Symptoms; Therapeutic Agents; Universities; Work; X-Ray Crystallography; alpha ketoglutarate; base; design; enzyme mechanism; expectation; experience; in vivo; inhibitor/antagonist; innovation; interstitial; oxidation; prevent; programs; therapeutic target; three dimensional structure

DESCRIPTION (provided by applicant): Collagen prolyl-4-hydroxylase (P4H) is an essential enzyme in collagen biosynthesis and disruption of P4H function is/known to contribute to fibrotic diseases such as interstitial pulmonary fibrosis and liver fibrosis. Hydroxylation of proline is the rate limiting step in collagen biosynthesis, so inhibitors of P4H are potential therapeutic agents that could target fibrotic diseases. In the absence of ascorbic acid, human P4H rapidly inactivates. In vivo, this inactivation leads to the formation of underhydroxylated collagen which is unstable leading to many of the classical symptoms of scurvy. P4H belongs to the family of mononuclear non-heme iron alpha ketoglutarate (aKG) dependent dioxygenases. In mammals, P4H is a 220 kDa homotetramer where the alpha subunit contains both a peptide binding domain and an Fe(ll)/aKG-binding catalytic domain, and the beta subunit is a protein disulfide isomerase that serves to both prevent the alpha subunit from aggregating and to retain P4H in the endoplasmic reticulum. Attempts to design inhibitors that are specific for human-P4H have been hampered by a lack of structural and spectroscopic information. A major limitation to spectroscopic studies is the need for millimolar concentrations of protein that are difficult to achieve given the size of human-P4H. In this proposal, a bacterial form of prolyl-4-hydroxylase from Bacilllus anthracis (anthrax-P4H) will be studied as model enzyme for the mechanism of peptidyl proline hydroxylation by human-P4H. The bacterial enzyme is a homodimer with significant sequence homology to the C-terminal catalytic domain of the alpha subunit of human-P4H. Anthrax-P4H is highly soluble and so is suitable for the proposed spectroscopic and X-ray crystallographic studies. In parallel to the studies focusing on anthrax-P4H, the mechanism of known inhibitors of human-P4H that target the active site Fe(ll) will be studied on both the human and bacterial P4Hs. The results of this comparative study will be used to assess the unique features of human-P4H that could be used to design specific inhibitors. The fundamental new knowledge will positively impact understanding of the mechanism of the superfamily of aKG-dependent mononuclear non-heme iron oxygenases. The anticipated findings are potentially important as they will suggest new strategies for the design of therapeutic agents that could target fibrosis.

描述（由申请人提供）：胶原蛋白 - 4-羟化酶（P4H）是胶原蛋白生物合成的必不可少的酶，P4H功能的破坏是/已知的/已知有助于纤维化疾病，例如间质肺纤维化和肝纤维化。脯氨酸的羟基化是胶原生物合成的速率限制步骤，因此P4H的抑制剂是可能靶向纤维化疾病的潜在治疗剂。在没有抗坏血酸的情况下，人P4H迅速失活。在体内，这种灭活导致形成二羟基化的胶原蛋白，这是不稳定的，导致许多经典的坏血病症状。 P4H属于单核非血红素α酮谷氨酸酯（AKG）依赖性双氧酶的家族。 In mammals, P4H is a 220 kDa homotetramer where the alpha subunit contains both a peptide binding domain and an Fe(ll)/aKG-binding catalytic domain, and the beta subunit is a protein disulfide isomerase that serves to both prevent the alpha subunit from aggregating and to retain P4H in the endoplasmic reticulum.缺乏结构和光谱信息，试图设计针对人类P4H的抑制剂的尝试受到阻碍。光谱研究的主要局限性是，鉴于人类P4H的大小，难以实现毫米浓度的蛋白质。在此提案中，将研究一种来自炭疽芽孢杆菌（炭疽-P4H）的细菌形式的细菌形式，作为模型酶，用于通过人-P4H的肽基脯氨酸羟基羟基化机制。细菌酶是与人类P4Hα亚基的C末端催化结构域具有显着序列同源的同型二聚体。炭疽-P4H高度溶解，因此适用于建议的光谱和X射线晶体学研究。与关注炭疽-P4H的研究并行，将研究针对活性位点Fe（LL）的人类P4H抑制剂的机制（人类和细菌P4HS）。这项比较研究的结果将用于评估可用于设计特定抑制剂的人类P4H的独特特征。基本的新知识将积极影响对AKG依赖性单核非血红素铁氧酶超家族机制的理解。预期的发现可能很重要，因为它们将提出针对靶向纤维化的治疗剂设计的新策略。

项目成果

A general protease digestion procedure for optimal protein sequence coverage and post-translational modifications analysis of recombinant glycoproteins: application to the characterization of human lysyl oxidase-like 2 glycosylation.

• DOI: 10.1021/ac2017037
• 发表时间: 2011-11-15
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Rebecchi, Kathryn R.;Go, Eden P.;Xu, Li;Woodin, Carrie L.;Mure, Minae;Desaire, Heather
• 通讯作者: Desaire, Heather