My laboratory studies the mechanism of vitamin K-dependent protein carboxylation, a process critical to human health. Carboxylation activates vitamin K-dependent proteins and was originally associated only with hemostatic vitamin K-dependent proteins synthesized in liver. However, the identification of nonhemostatic vitamin K-dependent proteins and demonstration that virtually all tissues contain at least one vitamin K-dependent protein indicates other roles for carboxylation, and this post-translational modification is now known to be important to diverse functions like apoptosis, signal transduction and arterial calcification. Vitamin K-dependent protein carboxylation therefore has broad physiological impact, yet there are many aspects of this complex process that are not understood.
A major effort in the laboratory focuses on the carboxylase that converts clusters of Glu’s in vitamin K-dependent proteins to carboxylated Glu’s to render them active. The carboxylase is a bifunctional enzyme that oxygenates vitamin K and then uses this energy of oxygenation to drive Glu carboxylation. The reaction is complicated and also unique, as the carboxylase has no homology to other known enzymes, and our studies have provided novel insights into the catalytic mechanism. Another interesting and poorly-understood aspect of the mechanism is how the carboxylase can activate multiple vitamin K-dependent proteins in a single tissue even though it has widely different affinities for the vitamin K-dependent proteins. Understanding this mechanism could be important in explaining why naturally-occurring human mutations in the carboxylase cause two distinctly different diseases, i.e. pseudoxanthoma elasticum that is associated with dermal defects and VKCFD1 (for combined deficiency of vitamin K-dependent coagulation factors) that is associated with serious bleeding. Our laboratory also studies how the carboxylase is regulated by self-carboxylation, which we discovered, and how the secretory machinery impacts carboxylation, which occurs in the endoplasmic reticulum during vitamin K-dependent protein secretion. Finally, our interesting finding that the carboxylase has been acquired in bacteria by horizontal gene transfer has opened up a new direction on the adaptation of the carboxylase for functions other than Glu carboxylation. General approaches used to pursue these areas of research include cell biology, molecular biology, biochemistry, proteomics and mouse models.
Another major focus is on the contribution of other proteins to vitamin K-dependent protein carboxylation. Continuous carboxylation requires recycling of the oxidized vitamin K back to the reduced form, which is accomplished by a vitamin K oxidoreductase (VKOR) that was recently identified. Understanding the function of VKOR is essential, as this enzyme is the target of the anticoagulant drug warfarin that is used by millions of people, for example in the treatment of deep vein thrombosis or atrial fibrillation. Our studies suggest that VKOR reduction of vitamin K may be the limiting step in carboxylation, which we are currently testing. One direction is to identify and analyze the redox protein that is required for VKOR activity, because VKOR becomes oxidized when it reduces vitamin K and therefore must be reduced to regenerate an active enzyme. Other directions address the questions of whether VKOR and the carboxylase, which are both integral membrane proteins, exist in a complex to cycle vitamin K and whether other components also exist in such a complex.