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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.

描述（由申请人提供）：近一半的美国人口普遍缺乏维生素 D。缺乏维生素D与一系列疾病有关，包括糖尿病、结直肠癌、慢性肾病以及佝偻病和骨质疏松症等骨代谢紊乱。维生素 D 补充剂有效性的一个重大障碍是，维生素 D 通过上调维生素 D 代谢酶细胞色素 P450 24-羟化酶 (CYP24A1) 的表达来发出其自身降解增加的信号。在人类中，这种酶通过两种不同的途径使维生素 D 失活：初级位点（碳 24）的羟基化最终产生废产物骨柠檬酸，而次级位点（碳 23）的羟基化则导致产生含内酯的代谢物，该代谢物在抑制骨吸收中发挥作用。目前，维生素 D 类型类似物（均拮抗剂和 CYP24A1 抑制剂）的设计很少考虑这种代谢特征。本提案中工作的研究目标是确定人类 CYP24A1 酶的特征，该特征决定了对维生素 D 的碳 23 或碳 24 的偏好。我建议结合使用 X 射线晶体学、蛋白质 NMR 和功能测定来测试 CYP24A1 通过与有助于调节这些偏好的活性位点的动态相互作用来结合底物的假设。我的中期职业目标是将这种技术组合推进到我自己的研究实验室，重点关注维生素 D 代谢。由于迄今为止我的培训内容是蛋白质 NMR 光谱学家，因此我将在 K99 指导阶段重点学习 X 射线晶体学。在此过程中，我建议确定 CYP24A1 及其底物复合物的结构。我目前在该国膜 P450 酶晶体学方面最杰出的实验室之一工作，因此，我处于独特的地位，可以获得在拟议项目中取得成功所需的技术专业知识，并在我的职业发展到独立助理教授阶段时继续应用这项技术。这种技术培训将得到广泛的职业发展计划的补充，其中包括定期咨询由处于职业生涯不同阶段的教师组成的职业咨询委员会、实验室预算管理培训、接触研究生选拔过程以及初步接触教学理念和实践。 K99阶段的技术和专业培训将转化为R00阶段计划的工作，其中我将结合晶体学和CYP24A1的NMR来评估蛋白质动力学在蛋白质功能中的作用。实现本提案中描述的目标将极大地提高我们对维生素 D 分解方式的理解，这是与数百万维生素 D 缺乏症美国人的健康相关的关键一步，并为我在该领域建立全国认可的研究计划的长期职业目标奠定了宝贵的基础。