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

 描述(申请人提供)：维生素D缺乏症在近一半的美国人口中普遍存在。缺乏与一系列疾病相关，从糖尿病、结直肠癌、慢性肾脏疾病，以及骨骼代谢疾病，如软骨病和骨质疏松症。维生素D补充剂有效性的一个重要障碍是，维生素D通过上调维生素D代谢酶细胞色素P450 24-羟基酶(CyP24A1)的表达，发出加速自身降解的信号。在人类中，这种酶通过两条不同的途径使维生素D失活；在主要位置(碳24)的羟化最终导致废物产品骨钙酸，而在次要位置(碳23)的羟化导致含有内酯的代谢物的产生，起到抑制骨吸收的作用。目前，维生素D类类似物(两者 拮抗剂和CYP24A1抑制剂)在设计时几乎没有考虑到这种代谢特征。这项建议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。我建议使用X射线结晶学、蛋白质核磁共振和功能分析相结合的方法来测试这一假设，即CYP24A1通过与有助于调节这些偏好的活性部位的动态相互作用而与底物结合。我的中期职业目标是将这一技术组合应用到我自己的研究实验室，专注于维生素D新陈代谢。由于到目前为止我的培训一直是蛋白质核磁共振光谱仪，我将在指导K99阶段期间专注于学习X射线结晶学。在这样做的同时，我建议确定CYP24A1与其底物的络合物的结构。我目前在国内膜P450酶结晶学方面的一家卓越实验室工作，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，并在我的职业生涯发展到独立助理教授阶段时继续应用这项技术。除技术培训外，还将制定广泛的职业发展计划，其中包括定期咨询由处于不同职业生涯阶段的教师组成的职业咨询委员会，进行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阶段下的技术和专业培训将转化为R00阶段下计划的工作，在R00阶段，我将结合结晶学和CYP24A1的核磁共振来评估蛋白质动力学在蛋白质功能中的作用。实现这项提案中描述的目标将极大地提高我们对维生素D如何分解的理解，这是与数百万患有维生素D缺乏症的美国人的健康相关的关键一步，并为我在这一领域建立一个全国公认的研究计划的长期职业目标提供宝贵的基础。

 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.