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

线粒体维生素 B12 途径和 II 型脂肪酸合成研究的综合方法

基本信息

批准号：
10343768
负责人：
Hongying Shen
金额：
$ 24.9万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10343768
关键词：
Adipocytes Binding Biochemical Genetics Biochemistry Bioinformatics Biology Branched-Chain Amino Acids Brown Fat Candidate Disease Gene Cell physiology Cells Cellular biology Citric Acid Cycle Clustered Regularly Interspaced Short Palindromic Repeats Coenzyme A Computing Methodologies Cultured Cells Cytosol Databases Development Plans Diabetes Mellitus Diagnosis Diet Modification Disease Drug Metabolic Detoxication Environment Enzymatic Biochemistry Enzymes Exhibits Fatty Acids Funding Genes Genetic study Goals Health Homeostasis Human Human Genetics Human Genome Inborn Errors of Metabolism Institutes Intervention Knock-out Laboratories Lesion Light Link Lipids Lyase Malignant Neoplasms Membrane Lipids Mentors Metabolic Metabolic Diseases Metabolic Pathway Metabolism Methionine Methods Methylmalonyl-CoA Mutase Mitochondria Molecular Molecular Diagnosis Mutation National Research Service Awards Octanoic Acids Orphan Oxidoreductase Pathway interactions Phase Physiological Physiology Process Proteome Proteomics Recycling Regulation Research Research Personnel Resolution Role Sequence Homology Testing Thioctic Acid Tracer Training United States National Institutes of Health Vitamin B 12 X-Ray Crystallography base career development cofactor computerized tools genome editing genome-wide human disease in vitro activity inhibitor insight interest lipidomics loss of function metabolic abnormality assessment metabolomics methylmalonic aciduria methylmalonyl-coenzyme A novel programs propionyl-coenzyme A public database stable isotope succinyl-coenzyme A

项目摘要

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.

抽象的代谢途径的失调是代谢的先天错误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潜在的代谢途径的特征不佳。这些研究的结果将产生新的假设，用于未知原因的诊断代谢疾病，并提供疾病干预措施的替代方向。适当的以前已经获得了严格的毕业生包括膜脂质生物学在内的生物化学和细胞生物学培训。在正在进行的NRSA期间 F32博士后资金期间，适用的已开发了一种综合方法来研究 clybl的线粒体酶，并揭示其在确定线粒体方面的功能维生素B12（B12）依赖性过程。这一发现反映了以前的人类遗传研究 CLYBL的功能丧失与低循环B12水平的功能丧失。对于K99/R00应用程序，申请人的提议专注于人类中的两个线粒体定位的基本代谢途径：（1）至确定线粒体B12途径的缺失调节剂；（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，两种主要策略将应用：（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节器；（b）在培养细胞和高分辨率LC-中结合CRISPR编辑基于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）以证明代谢。申请人的房东实验室和研究所为拟议的研究。她的博士后导师Vamsi Mootha博士以前已经开发了全基因组的计算方法预测线粒体蛋白质组 - 与可以预测新型的代谢调节剂。该实验室也是代谢组学的早期采用者广泛研究所代谢计划的一部分。该应用程序已在LC-MS中获得了初步培训剖析极地代谢产物的方法。在K99/R00的资金期间，她将获得额外研究脂肪酸的培训和先进的天然蛋白质组学方法，以研究脂肪酸酰基链延伸在mtfasii期间。成功完成该项目将提供基本的见解线粒体辅因子代谢和脂质稳态调节，并可能引入新诊断代谢疾病的方向。意思是，职业发展计划将准备申请人过渡到代谢领域的独立研究者。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Systems biochemistry to "deorphanize" human mitochondrial proteome.

系统生物化学“去孤儿化”人类线粒体蛋白质组。

DOI：
10.1016/j.molcel.2022.07.005
发表时间：
2022
期刊：
Molecular cell
影响因子：
16
作者：
Miros,Francois;Liu,Ran;Shen,Hongying
通讯作者：
Shen,Hongying

Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.

DOI：
10.1038/s41467-022-30126-9
发表时间：
2022-05-05
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
Shi, Xiaojian;Reinstadler, Bryn;Shah, Hardik;To, Tsz-Leung;Byrne, Katie;Summer, Luanna;Calvo, Sarah E.;Goldberger, Olga;Doench, John G.;Mootha, Vamsi K.;Shen, Hongying
通讯作者：
Shen, Hongying

An IRON-clad Connection between Aging Organelles.

衰老细胞器之间的铁一般连接。