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Crystallographic and NMR studies of the Human Cytochrome P450 enzyme responsible for inactivating vitamin D

负责使维生素 D 失活的人类细胞色素 P450 酶的晶体学和核磁共振研究

基本信息

批准号：
9343024
负责人：
David Fernando Estrada
金额：
$ 24.9万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9343024
关键词：
Acids Active Sites Advisory Committees Affect Age American Binding Biochemical Biological Assay Bone Diseases Bone Resorption Budgets Calcitriol Carbon Catabolism Characteristics Cholecalciferol Chronic Kidney Failure Colorectal Cancer Complex Consultations Country Crystallization Crystallography Cytochrome P450 Data Development Plans Diabetes Mellitus Didelphidae Diet Disease Drug Targeting Educational process of instructing Effectiveness Enzymes Exposure to Faculty Foundations Fracture Future Goals Health Homeostasis Human Hydroxyl Radical Hydroxylation Hypercalcemia Knowledge Laboratories Lactones Learning Maintenance Manuscripts Mediating Membrane Mentors Metabolic Bone Diseases Metabolism Mitochondria Mixed Function Oxygenases Molecular Conformation Mutagenesis Nuclear Magnetic Resonance Nuclear Protein Osteoporosis Pathway interactions Phase Philosophy Physiologic calcification Play Population Positioning Attribute Process Production Protein Dynamics Proteins Rattus Reporting Research Rickets Risk Roentgen Rays Role Series Side Signal Transduction Site Structure Student Selections Supplementation Technical Expertise Techniques Testing Training Translating Up-Regulation Vitamin D Vitamin D Analog Vitamin D Deficiency Vitamin D supplementation Vitamin D3 Receptor Waste Products Work X-Ray Crystallography analog base bone health career career development common treatment design graduate student hydroxyl group improved inhibitor/antagonist metabolic profile novel preference professor programs prohormone protein function protein structure public health relevance response stem structural biology tool vitamin metabolism

项目摘要

DESCRIPTION (provided by applicant): 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.