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）：胶原蛋白脯氨酰-4-羟化酶（P4 H）是胶原蛋白生物合成中的必需酶，已知P4 H功能的破坏会导致纤维化疾病，如间质性肺纤维化和肝纤维化。脯氨酸的羟化是胶原生物合成的限速步骤，因此P4 H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P4 H迅速失活。在体内，这种失活导致形成不稳定的羟基化不足的胶原蛋白，导致坏血病的许多典型症状。P4 H属于单核非血红素铁α酮戊二酸（aKG）依赖性双加氧酶家族。在哺乳动物中，P4 H是220 kDa同源四聚体，其中α亚基含有肽结合结构域和Fe（II）/aKG结合催化结构域，β亚基是蛋白质二硫键异构酶，其用于防止α亚基聚集并将P4 H保留在内质网中。由于缺乏结构和光谱信息，设计对人P4 H特异性的抑制剂的尝试受到阻碍。光谱研究的一个主要限制是需要蛋白质的毫摩尔浓度，这是难以实现的人P4 H的大小。在这个建议中，细菌形式的脯氨酰-4-羟化酶从炭疽杆菌（炭疽-P4 H）将被研究作为模式酶的肽酰脯氨酸羟基化的机制，由人类-P4 H。该细菌酶是一种同源二聚体，与人-P4 H α亚基的C-末端催化结构域具有显著的序列同源性。炭疽-P4 H是高度可溶的，因此适合于所提出的光谱学和X射线晶体学研究。与聚焦于炭疽-P4 H的研究平行，将在人和细菌P4 H两者上研究靶向活性位点Fe（II）的人-P4 H的已知抑制剂的机制。这项比较研究的结果将用于评估可用于设计特异性抑制剂的人P4 H的独特特征。基础新知识将积极影响aKG依赖性单核非血红素铁加氧酶超家族机制的理解。预期的发现可能很重要，因为它们将为设计靶向纤维化的治疗药物提出新的策略。

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