Characterization of enzymes in the vitamin K cycle

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 DESCRIPTION (provided by applicant): Vitamin K-dependent (VKD) carboxylation, an essential post-translational modification catalyzed by gamma- glutamyl carboxylase (GGCX), is required for the biological function of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase (VKOR) and vitamin K reductase (VKR) in a pathway known as the vitamin K cycle. Warfarin, the most widely prescribed anticoagulant for thromboembolic disorders, works to impair the biosynthesis of functional clotting factors by inhibiting VKOR. Despite significant progress in the field, fundamental questions remain: 1) What are the mechanisms for VKOR active site regeneration and warfarin inhibition? - 2) What are the identities of the VKR enzymes? - 3) Why do some mutations of GGCX result in a bleeding disorder, named as combined vitamin K-dependent coagulation factors deficiency (VKCFD), while others are linked with Pseudoxanthoma elasticum (PXE)-like syndrome? The objectives of the current proposal are to identify and characterize the components of the vitamin K cycle, understand how these various components contribute to VKD carboxylation in their native milieu, and determine how naturally-occurring GGCX mutations contribute to different disease states. To accomplish these goals, we propose the following specific aims: Aim 1) To study the structure-function relationships of VKOR and its paralog enzyme VKORC1L1 using the recently established cell-based assay; Aim 2) To characterize and identify VKR using genome editing techniques TALENs (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 in our AV12 and HEK293 reporter cell lines; Aim 3) To establish a cell-based assay for the GGCX function study using the TALENs-meditated gene knockout of the endogenous GGCX gene in our HEK293 reporter cells - allowing us to study how GGCX mutations are related to VKCFD and PXE-like syndromes. Information derived from these studies will help us understand how the various vitamin K cycle components contribute to these complex mechanisms; thereby, gaining new therapeutic insights into the control of thrombosis and by improving warfarin therapy treatments.