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

摘要 代谢途径的失调是新陈代谢先天错误的直接原因，也 会导致癌症和糖尿病等常见疾病。申请者的长期目标是发展 一种结合计算、CRISPR基因组编辑和代谢组学的综合策略来研究 人类疾病背后的代谢途径特征不佳。这些研究的结果将 为诊断未知原因的代谢性疾病产生新的假设，并提供 疾病干预的替代方向。申请人以前曾获得过严格的毕业生资格。 生物化学和细胞生物学方面的培训，包括膜脂生物学。在正在进行的NRSA期间 F32博士后资助期，申请人已经发展了一种综合的方法来学习 功能未知的线粒体酶CLYBL，并揭示了其调节线粒体的功能 维生素B12(B12)依赖的过程。这一发现反映了之前的人类基因研究 CLYBL功能丧失与循环B12水平降低有关。对于K99/R00应用程序， 申请人建议重点研究人类线粒体定位的两条基本代谢途径：(1)至 识别线粒体B12途径缺失的调节子；(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 应用：(A)利用全基因组计算方法和公开可用的数据库 预测新的途径调控因子；(B)将培养细胞中的CRISPR编辑与高分辨率LC- 以MS为基础的代谢组学(包括脂质组学)和蛋白质组学(包括自上而下的蛋白质组学)来探测 新陈代谢。申请者所在的实验室和研究所为 拟开展的研究。她的博士后导师Vamsi Mootha博士的实验室之前已经开发出 预测线粒体蛋白质组的全基因组计算方法--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， 博德研究所新陈代谢计划的一部分。申请人已接受LC-MS的初步培训 方法对极性代谢物进行分析。在K99/R00资助期间，她将获得额外的 脂组学培训和先进的天然蛋白质组学方法研究脂肪酸酰链延伸 在MtFASII期间。该项目的成功完成将为以下方面提供基本见解 线粒体辅因子代谢和调节脂质稳态，并可能引入新的 代谢性疾病诊断指南。与此同时，职业发展计划将为 申请转变为新陈代谢领域的独立研究员。

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.