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

 描述（由申请人提供）：维生素D缺乏症在近一半的美国人口中普遍存在。缺乏与糖尿病、结肠直肠癌、慢性肾病和骨代谢紊乱如佝偻病和骨质疏松症等一系列疾病有关。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-羟化酶（CYP 24 A1）的表达而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;在初级位点（碳24）的羟基化最终导致废物骨化酸，而在次级位点（碳23）的羟基化导致产生含有内酯的代谢物，其在抑制骨吸收中起作用。目前，维生素D类型类似物（两者都是 拮抗剂和CYP 24 A1抑制剂）的设计很少考虑这种代谢特征。 这项工作的研究目标是确定人类CYP 24 A1酶的特征，该酶决定了维生素D对碳23或碳24的偏好。我建议使用X射线晶体学，蛋白质NMR和功能测定的组合来测试CYP 24 A1通过与活性位点的动态相互作用来结合底物，从而帮助调节这些偏好的假设。我的中期职业目标是将这种技术组合带入我自己的研究实验室，重点是维生素D代谢。由于我迄今为止的培训一直是作为一个蛋白质NMR光谱学家，我将专注于学习X射线晶体学在辅导K99阶段。在这样做的同时，我建议确定与其底物复合的CYP 24 A1的结构。我目前在该国一个杰出的实验室工作，从事膜P450酶的晶体学研究，因此，我具有独特的优势，能够获得在拟议项目中取得成功所需的技术专业知识，并随着我的职业生涯发展到独立助理教授阶段，继续应用这项技术。这种技术培训将由一个广泛的职业发展计划，需要，除其他项目外，定期咨询由教师组成的职业咨询委员会在他们的职业生涯的不同阶段，在实验室预算管理培训，接触研究生的选拔过程中，并初步接触到教学理念和实践。K99阶段的技术和专业培训将转化为R 00阶段计划的工作，其中我将联合收割机晶体学与CYP 24 A1的NMR相结合，以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如何分解的理解，这是与数百万维生素D缺乏症美国人健康相关的关键一步，并为我的长期职业目标提供了宝贵的基础。

 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.