Gamma-glutamyl carboxylation of vitamin K-dependent proteins is essential for their activation, and a long-term goal of my laboratory is to understand the mechanism and regulation of carboxylation and how dysfunction leads to disease. Carboxylation was originally thought to be important only to hemostasis, but is now known to have a much broader impact on human health, with vitamin K-dependent functions that include calcification, apoptosis, signal transduction and growth control. Carboxylation occurs in the endoplasmic reticulum during the secretion of vitamin K-dependent proteins, which are modified by the combined action of the gamma-glutamyl carboxylase and vitamin K oxidoreductase (VKORC1), as well as unknown components. One area of research is to define this complex process by determining how the secretory process impacts carboxylation, and by identifying other proteins required for carboxylation. Another area of interest is to determine why mutations in the carboxylase cause two distinct diseases, i.e., VKCFD1 in which bleeding is severe or pseudoxanthoma elasticum that is associated with excessive calcification and skin defects. Naturally occurring VKORC1 mutations have also been identified. These mutations alter the response of patients to drugs like warfarin (Coumadin), which is used by millions of people to suppress vitamin K-dependent clotting activity. The mechanism of warfarin inhibition is poorly understood, and a final research focus is to understand warfarin inhibition and the consequence of VKORC1 mutations. Approaches used in our research include mouse models, proteomics, mutagenesis, cellular systems, and biochemical studies.
- Rishavy MA, Hallgren KW, Wilson LA, Usubalieva A, Runge KW, Berkner KL. The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation. J Biol Chem. 2013 288(44):31556-66. doi: 10.1074/jbc.M113.497297. PMID: 23918929
- Hallgren KW, Zhang D, Kinter M, Willard B, Berkner KL. Methylation of γ-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the γ-glutamyl carboxylase. J Proteome Res. 2013 12(6):2365-74. doi: 10.1021/pr3003722. PMID: 22536908
- Rishavy MA, Berkner KL. Vitamin K oxygenation, glutamate carboxylation, and processivity: defining the three critical facets of catalysis by the vitamin K-dependent carboxylase. Adv Nutr. 2012 3(2):135-48. doi: 10.3945/an.111.001719. Review. PMID: 22516721
- Rishavy MA, Hallgren KW, Berkner KL. The vitamin K-dependent carboxylase generates γ-carboxylated glutamates by using CO2 to facilitate glutamate deprotonation in a concerted mechanism that drives catalysis. J Biol Chem. 2011 286(52):44821-32. doi: 10.1074/jbc.M111.249177. PMID: 21896484
- Rishavy MA, Usubalieva A, Hallgren KW, Berkner KL. Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation. J Biol Chem. 2011286(9):7267-78. doi: 10.1074/jbc.M110.172213. PMID: 20978134
- Li Q, Grange DK, Armstrong NL, Whelan AJ, Hurley MY, Rishavy MA, Hallgren KW, Berkner KL, Schurgers LJ, Jiang Q, Uitto J. Mutations in the GGCX and ABCC6 genes in a family with pseudoxanthoma elasticum-like phenotypes. J Invest Dermatol. 2009 129(3):553-63. doi: 10.1038/jid.2008.271. PMID: 18800149
- Rishavy MA, Berkner KL. Insight into the coupling mechanism of the vitamin K-dependent carboxylase: mutation of histidine 160 disrupts glutamic acid carbanion formation and efficient coupling of vitamin K epoxidation to glutamic acid carboxylation. Biochemistry. 2008 47(37):9836-46. doi: 10.1021/bi800296r. PMID: 18717596
- Rishavy MA, Hallgren KW, Yakubenko AV, Shtofman RL, Runge KW, Berkner KL. Brønsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation. Biochemistry. 2006 45(44):13239-48. PMID: 17073445
Tens of millions of patients worldwide take warfarin to prevent life-threatening blood clots. The drug is more effective in some patients than others, however, and determining dosage for individual patients can be problematic. Researchers are working to more clearly understand warfarin's biological effects in the body to overcome these limitations.