Mechanism and inhibition of collagen prolyl-4-hydroxylases

点击翻译按钮获取中文摘要

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.