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: 7407572
• 负责人: Minae Mure
• 金额: $ 26.62万
• 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/7407572
• 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（由申请人提供）：胶原脯氨酰-4-羟化酶（P4H）是胶原生物合成中的必需酶，已知 P4H 功能的破坏会导致纤维化疾病，例如间质性肺纤维化和肝纤维化。脯氨酸的羟基化是胶原蛋白生物合成的限速步骤，因此 P4H 抑制剂是针对纤维化疾病的潜在治疗剂。在没有抗坏血酸的情况下，人 P4H 会迅速失活。在体内，这种失活导致羟基化不足的胶原蛋白的形成，这种胶原蛋白不稳定，导致坏血病的许多典型症状。 P4H 属于单核非血红素α酮戊二酸铁 (aKG) 依赖性双加氧酶家族。在哺乳动物中，P4H是220kDa同源四聚体，其中α亚基包含肽结合结构域和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P4H保留在内质网中。由于缺乏结构和光谱信息，设计人 P4H 特异性抑制剂的尝试受到阻碍。光谱研究的一个主要限制是需要毫摩尔浓度的蛋白质，考虑到人类 P4H 的大小，这是很难达到的。在本提案中，将研究来自炭疽杆菌（anthrax-P4H）的细菌形式的脯氨酰-4-羟化酶，作为人-P4H 肽基脯氨酸羟化机制的模型酶。该细菌酶是同源二聚体，与人 P4H α 亚基的 C 端催化结构域具有显着的序列同源性。 Anthrax-P4H 具有高度可溶性，因此适用于所提出的光谱和 X 射线晶体学研究。与关注炭疽-P4H的研究并行，将在人类和细菌P4H上研究靶向活性位点Fe(II)的已知人类P4H抑制剂的机制。这项比较研究的结果将用于评估 human-P4H 的独特特征，这些特征可用于设计特定的抑制剂。基本的新知识将积极影响对 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，因为它们将为设计针对纤维化的治疗药物提出新策略。