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: 7186419
• 负责人: Julian Limburg
• 金额: $ 25.81万
• 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 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7186419
• 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; Disruption; Endoplasmic Reticulum; Environment; Enzyme Kinetics; Enzymes; Family; Fibrosis; Goals; Heme Iron; 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; Rate; Reaction; Reporting; Research; Research Personnel; Roentgen Rays; Role; Scurvy; Sequence Homology; Spectrum Analysis; Structure; Symptoms; Therapeutic Agents; Universities; Work; X ray spectroscopy; X-Ray Crystallography; alpha ketoglutarate; base; design; enzyme mechanism; expectation; experience; in vivo; inhibitor/antagonist; innovation; interstitial; oxidation; prevent; programs; size; 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(申请人提供)：胶原蛋白-4-羟基酶(P4H)是胶原蛋白生物合成过程中的一种重要酶，P4H功能障碍可导致间质性肺纤维化和肝纤维化等纤维化疾病。脯氨酸的羟化是胶原生物合成的限速步骤，因此P4H的抑制剂是潜在的针对纤维化疾病的治疗药物。在没有抗坏血酸的情况下，人的P4H迅速失活。在体内，这种失活会导致羟化不足的胶原蛋白的形成，而这种胶原蛋白是不稳定的，从而导致许多典型的坏疽症状。P4H属于依赖于AKG的单核非血红素铁-酮戊二酸(AKG)双加氧酶家族。在哺乳动物中，P4H是一个220 kDa的同源四聚体，其中α亚基包含一个肽结合域和一个Fe(11)/AKG结合催化域，而β亚基是一种蛋白质二硫键异构酶，它既可以阻止α亚基聚集，又可以将P4H保留在内质网中。由于缺乏结构和光谱信息，设计针对人-P4H的抑制剂的尝试一直受到阻碍。光谱学研究的一个主要限制是需要毫摩尔浓度的蛋白质，而考虑到人-P4H的大小，这是很难达到的。在这项研究中，炭疽芽孢杆菌的一种细菌形式的Pro-4-羟基酶(anthrax-P4H)将作为研究人-P4H对肽-4-羟基羟化反应机理的模型酶。细菌酶是一种同源二聚体，与人P4Hα亚基的C末端催化结构域具有显著的序列同源性。炭疽-P4H具有很高的溶解性，因此适合于所提出的光谱和X射线结晶学研究。在专注于炭疽P4H的研究的同时，针对活性部位Fe(11)的已知人P4H抑制剂的机制将在人类和细菌P4H上进行研究。这项比较研究的结果将被用来评估人类P4H的独特特性，这些特性可以用来设计特定的抑制剂。这一全新的知识将对理解AKG依赖的单核非血红素铁氧合酶超家族的机制产生积极的影响。这些预期的发现具有潜在的重要意义，因为它们将为设计可能针对纤维化的治疗剂提供新的策略。