Vitamin D deficiency is prevalent in nearly half of the U.S. population. Deficiency has been correlated with a spectrum of diseases ranging from diabetes, colorectal cancer, chronic kidney disease, and bone metabolism disorders such as rickets and osteoporosis. A significant obstacle toward the effectiveness of vitamin D supplementation is the fact that vitamin D signals an increase in its own degradation by up-regulating expression of the vitamin D metabolizing enzyme cytochrome P450 24-hydroxylase (CYP24A1). In humans, this enzyme deactivates vitamin D via two distinct pathways; hydroxylation at a primary site (carbon 24) ultimately results in the waste product calcitroic acid while hydroxylation at a secondary site (carbon 23) results in production of a lactone-containing metabolite that plays a role in inhibiting bone resorption. Currently, vitamin D type analogs (both antagonists and CYP24A1 inhibitors) are being designed with little consideration of this metabolic profile. The research goal of the work in this proposal is to identify characteristics o the human CYP24A1 enzyme that determines a preference for either carbon 23 or carbon 24 of vitamin D. I propose to use a combination of X-ray crystallography, protein NMR, and functional assays to test the hypothesis that CYP24A1 binds substrate via dynamic interactions with the active site that help modulate these preferences. My intermediate career goal is to carry this combination of techniques forward into my own research lab with a focus on vitamin D metabolism. Since my training to date has been as a protein NMR spectroscopist, I will focus on learning X-ray crystallography during the mentored K99 phase. While doing so, I propose to determine the structure of CYP24A1 in complex with its substrate. I currently work in one of the preeminent labs in the country with respect to the crystallography of membrane P450 enzymes and as such I am uniquely positioned to gain the technical expertise necessary to be successful in the proposed project, as well as to continue to apply this technique as my career advances to the independent assistant professor stage. This technical training will be complimented by an extensive career development plan that entails, among other items, regular consultation with a career advisory committee composed of faculty at different stages of their careers, training in laboratory budget management, exposure to the graduate student selection process, and initial exposure to teaching philosophy and practicalities. The technical and professional training under the K99 phase will then translate into work planned under the R00 phase, in which I will combine crystallography with NMR of CYP24A1 to evaluate the role of protein dynamics in protein function. Accomplishing the aims described in this proposal will greatly improve our understanding of how vitamin D is broken down, a critical step with health relevance to the millions of Americans with vitamin D deficiency, as well as provide a valuable foundation for my long-term career goal of establishing a nationally recognized research program in this field.
Vitamin D deficiency has been correlated to a variety of diseases, including chronic kidney disease, colorectal cancer, and bone disorders such as osteoporosis and rickets. Supplementation with dietary vitamin D is a common treatment, but has limited benefits due to the body's natural response of increasing the breakdown and therefore inactivation of supplemented vitamin D. Therefore, the goal of this proposal is to use structural biology to examine the function of the human protein Cytochrome P450 24-hydroxylase; the key enzyme responsible for vitamin D inactivation and an important drug target in the treatment of vitamin D deficiency.
| Estrada, D Fernando (2018) The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species. J Biol Chem 293:4167-4179 |
