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.
Warfarin is the most widely used oral anticoagulant for the prevention and treatment of thrombosis with over 2 million new prescriptions written in the United States annually. Warfarin disrupts the vitamin K cycle, which is important for the biosynthesis of functional clotting factors. Knowledge gained from our study of the enzymes in the vitamin K-cycle could have major implications in current warfarin therapies and new alternative approaches to controlling coagulation.
Showing the most recent 10 out of 13 publications
