The long-term aim of this research is to define the network of integrated metabolic events that underlie vitamin K anabolism and catabolism, in order to understand inter-subject variability in response to this essential dietary nutrient. In ths new application, we will establish the P450 enzymes, CYP4F2 and CYP4F11 as the key vitamin K hydroxylases that initiate catabolism of this essential dietary constituent. In addition, we will determine the role of the newly identified human UBDIA1 enzyme in the interconversion of vitamins K1 and K2. These efforts will establish the link between the core vitamin K cycle proteins and ancillary enzymes that modulate the availability of the hydroquinone cofactors that drive Gla protein production. Because there is a high degree of inter-individual variability in response to supplementation with vitamin K in highly vulnerable K-deficient populations (e.g. those suffering from chronic kidney disease, cancer patients), these studies are of high significance. The goals of this research proposal are three-fold, (i) to establish the relative roes of human CYP4F2 and CYP4F11 in the initiation of vitamin K catabolism, (ii) to establish the role and chemical mechanism of action of UBIAD1 in conversion of vitamin K 1 to K2 (MK-4), and (iii) to ascertain the influence of common genetic variation in each of these three genes on vitamin K homeostatis in human tissues and cellular models of increasing complexity, as a precursor to future in vivo studies. In preliminary studies, we have accomplished the important tasks of heterologous expression of each of the three target enzymes, and development of highly sensitive and specific tandem MS assays for analysis of vitamin K in a variety of biological matrices. With these tools in hand, we propose, in Aim1, to evaluate the roles of CYP4F2 and CYP4F11 in vitamin K catabolism.
In Aim 2, we will express and purify human UBIAD1 to enable an unambiguous assessment of the enzyme's catalytic capability and chemical mechanism for conversion of vitamin K1 to K3.
In Aim 3, we will use banked, genotyped human liver tissue and a novel engineered cell system for analysis of Gla protein production to determine the effect of common genetic variation in these newly described vitamin K cycle-associated genes on cellular vitamin K homeostasis.
We hypothesize that novel vitamin K response genes, CYP4F2 and CYP4F11- newly identified as vitamin K hydroxylases, and UBIAD1- a vitamin K2 biosynthetic gene, are intimately associated with vitamin K status in humans. It follows then that genetically determined variability in these enzyme activities will affect patient response to supplementation with exogenous vitamin K - an increasingly interesting therapeutic strategy to combat (i) variability in response to the vitamin K antagonist class of oral anticoagulants and (ii vascular calcification and bone fracture risk in susceptible populations that are prone to vitamin K deficiency. We propose a series of enzymological and mechanistic studies that will establish the role of these enzymes (and their common genetic variants) in an expanded picture of the canonical vitamin K cycle in both hepatic and extrahepatic tissues where vitamin K-dependent processes have diverse physiological roles in controlling hemostasis, bone metabolism and arterial calcification, respectively.
|Roth, J A; Boudreau, D; Fujii, M M et al. (2014) Genetic risk factors for major bleeding in patients treated with warfarin in a community setting. Clin Pharmacol Ther 95:636-43|
|Haque, Jamil A; McDonald, Matthew G; Kulman, John D et al. (2014) A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites. Blood 123:582-9|