An integrated approach to the study of mitochondrial vitamin B12 pathway and type II fatty acid synthesis

摘要 代谢途径的失调是先天性代谢缺陷的直接原因， 导致癌症和糖尿病等常见疾病。申请人的长期目标是发展 一种结合计算、CRISPR基因组编辑和代谢组学的综合策略， 人类疾病潜在的代谢途径特征不明确。这些研究的结果将 产生用于诊断未知原因的代谢疾病的新假设，并提供 疾病干预的替代方向。申请人以前获得过严格的毕业证书 生物化学和细胞生物学培训，包括膜脂质生物学。在进行中的NRSA F32博士后资助期内，申请人已制定了综合研究方法， 线粒体酶的未知功能，CLYBL，并揭示其功能，在调节线粒体 维生素B12（B12）依赖性过程。这一发现反映了以前的人类遗传研究， CLYBL功能丧失与低循环B12水平有关。对于K99/R 00应用程序， 申请人提出集中于人中两种位于大肠杆菌中的必需代谢途径：（1） 确定线粒体B12途径缺失的调节因子;（2）进行功能丧失研究， 线粒体II型脂肪酸合成（mtFASII）。为了实现这些目标，两大战略将 （a）利用全基因组计算方法和公开数据库， 预测新的途径调节剂;（B）将培养细胞中的联合收割机CRISPR编辑和高分辨率LC- 基于MS的代谢组学（包括脂质组学）和蛋白质组学（包括自上而下的蛋白质组学） 新陈代谢.申请人的实验室和研究所为以下人员提供理想的培训环境： 提议的研究。她的博士后导师Vamsi Mootha博士的实验室此前开发了 全基因组计算方法来预测线粒体蛋白质组-相同的工具集， 可以预测新的代谢调节剂。该实验室也是代谢组学的早期采用者， 布罗德研究所代谢计划的一部分。申请人已获得LC-MS的初步培训 分析极性代谢物的方法。在K99/R 00资助期间，她将获得额外的 脂质组学和先进的天然蛋白质组学方法的培训，以研究脂肪酸酰基链延伸 在MTFASII期间。该项目的成功完成将提供基本的见解， 线粒体辅因子代谢和调节脂质稳态，并可能引入新的 代谢性疾病的诊断指南。同时，职业发展计划将编制 申请人转变为代谢领域的独立研究者。

Abstract The dysregulation of the metabolic pathways is the direct cause of inborn errors of metabolism and also leads to common diseases like cancers and diabetes. The applicant's long-term objective is to develop an integrated strategy combining computation, CRISPR genome editing and metabolomics to study the poorly characterized metabolic pathways underlying human diseases. The results of these studies will generate novel hypotheses for diagnosing metabolic diseases of unknown causes and provide alternative directions for disease interventions. The applicant has previously obtained rigorous graduate training in biochemistry and cell biology, including membrane lipid biology. During the ongoing NRSA F32 postdoctoral funding period, the applicant has developed an integrated approach to study a mitochondrial enzyme of unknown function, CLYBL, and revealed its function in regulating mitochondrial vitamin B12 (B12)-dependent processes. This finding mirrors previous human genetic studies that associate loss-of-function of CLYBL with low circulating B12 levels. For the K99/R00 application, the applicant proposes to focus on two mitochondria-localized, essential metabolic pathways in human: (1) to identify missing regulators of the mitochondrial B12 pathway; (2) to perform loss-of-function studies of the mitochondrial type II fatty acid synthesis (mtFASII). To achieve these goals, two major strategies will be applied: (a) to leverage genome-wide computational approaches and publicly available databases to predict new pathway regulators; (b) to combine CRISPR editing in cultured cells and high-resolution LC- MS based metabolomics (including lipidomics) and proteomics (including top-down proteomics) to probe metabolism. The applicant's host laboratory and institute provide an ideal training environment for the proposed research. Her postdoctoral mentor Dr. Vamsi Mootha's laboratory has previously developed genome-wide computational methods to predict the mitochondrial proteome – the same toolset that could predict novel metabolic regulators. The laboratory is also an early adopter of metabolomics and part of the Broad Institute Metabolism Program. The applicant has obtained initial training in LC-MS methods to profile polar metabolites. And during the K99/R00 funding period, she will receive additional training in lipidomics and advanced native proteomics method to study the fatty acid acyl-chain extension during the mtFASII. Successful completion of this project will provide fundamental insights into mitochondrial cofactor metabolism and regulation of lipid homeostasis, and might introduce new directions for diagnosing metabolic diseases. Meanwhile, the career development plan will prepare the applicant to transition into an independent investigator in the field of metabolism.