title: Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback.
abstract: Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.
authors.affiliation: Drug Discovery Core, University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, UK.
authors.forename: K
authors.initials: K
authors.lastname: Wilson
authors.affiliation: School of Biological Sciences and School of Biomedical Sciences, The University of Edinburgh, C.H. Waddington Building, King's Buildings, Max Born Crescent, Edinburgh, UK.
authors.forename: M
authors.initials: M
authors.lastname: Auer
authors.affiliation: Drug Discovery Core, University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, UK.
authors.forename: M
authors.initials: M
authors.lastname: Binnie
authors.affiliation: MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, UK.
authors.forename: X
authors.initials: X
authors.lastname: Zheng
authors.affiliation: School of Biological Sciences and School of Biomedical Sciences, The University of Edinburgh, C.H. Waddington Building, King's Buildings, Max Born Crescent, Edinburgh, UK.
authors.forename: N T
authors.initials: NT
authors.lastname: Pham
authors.affiliation: MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, UK.
authors.forename: J P
authors.initials: JP
authors.lastname: Iredale
authors.affiliation: Drug Discovery Core, University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, UK.
authors.forename: S P
authors.initials: SP
authors.lastname: Webster
authors.affiliation: MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Mole
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007737:Kynurenine / Q000031:analogs & derivatives* / Q000633:toxicity; D050601:Kynurenine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D059008:Time-Lapse Imaging; D014162:Transfection; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C005045:3-hydroxykynurenine; D007737:Kynurenine; D052247:Nitric Oxide Synthase Type II; D050601:Kynurenine 3-Monooxygenase; D010430:Pentosyltransferases; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)