STRUCTURE AND FUNCTION OF BACTERIAL PROLYL-4-HYDROXYLASE

细菌脯氨酰-4-羟化酶的结构和功能

• 批准号: 7381959
• 负责人: Julian Limburg
• 金额: $ 13.62万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381959
• 关键词: STRUCTURE; FUNCTION; BACTERIAL; PROLYL; HYDROXYLASE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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(II)/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 for the 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(II) 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 the mechanism of action of the 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.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。胶原脯氨酸-4-羟化酶（P4H）是胶原生物合成中必需的酶，P4H功能的破坏可导致纤维化疾病，如间质性肺纤维化和肝纤维化。脯氨酸羟基化是胶原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。在缺乏抗坏血酸的情况下，人P4H迅速失活。在体内，这种失活导致低羟基胶原蛋白的形成，这是不稳定的，导致许多坏血病的经典症状。P4H属于单核非血红素铁α酮戊二酸（aKG）依赖双加氧酶家族。在哺乳动物中，P4H是一种220 kDa的同型四聚体，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亚基是一种蛋白质二硫异构酶，既可以防止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，设计人类p4h特异性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，考虑到人类p4h的大小，这很难实现。在这个提议中，将研究一种来自炭疽芽孢杆菌（炭疽- p4h）的细菌形式的脯氨酸-4-羟化酶作为人类p4h的模型。细菌酶是一种同型二聚体，与人类p4h α亚基的c端催化结构域具有显著的序列同源性。炭疽- p4h是高度可溶的，因此适合于提出的光谱和x射线晶体学研究。在对炭疽- p4h进行研究的同时，将研究已知的针对活性位点Fe（II）的人- p4h抑制剂对人和细菌p4h的作用机制。这项比较研究的结果将用于评估人类p4h的独特特征，这些特征可用于设计特定的抑制剂。这些基本的新知识将对理解依赖akg的单核非血红素铁加氧酶的作用机制产生积极的影响。预期的发现具有潜在的重要性，因为它们将为设计靶向纤维化的治疗药物提供新的策略。