Substrates and functions of the Sideroflexin mitochondrial transporter family

Sideroflexin 线粒体转运蛋白家族的底物和功能

基本信息

批准号：
10338199
负责人：
Nora Kory
金额：
$ 17.11万
依托单位：
HARVARD SCHOOL OF PUBLIC HEALTH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10338199
关键词：
Address Affect Affinity Alanine Amino Acids Anabolism Animals Biological Assay CRISPR screen Carbon Catabolism Cells Citric Acid Cycle Complex Culture Media Cysteine Cystine Cytosol Data Defect Diabetes Mellitus Disease Distant Family Genes Genus Hippocampus Glutamine Glycine Hematopoietic System Homeostasis Homologous Gene In Vitro Inner mitochondrial membrane Interphase Cell Knockout Mice Lead Light Link Lipids Liver Malignant Neoplasms Mammals Measures Metabolic Metabolic Pathway Metabolism Methods Mitochondria Mitochondrial Proteins Molecular Mus Normal tissue morphology Nucleotide Biosynthesis Null Lymphocytes Organelles Organism Pathway interactions Pattern Phenotype Physiological Physiology Play Process Protein Biosynthesis Proteins Research Resistance Respiration Respiratory Chain Role Serine Substrate Specificity Testing Tissues Tumor Tissue Work chemotherapy differential expression electron donor experimental study extracellular human disease in vivo metabolomics mitochondrial metabolism uptake

Address Affect Affinity Alanine Amino Acids Anabolism Animals Biological Assay CRISPR screen Carbon Catabolism Cells Citric Acid Cycle Complex Culture Media Cysteine Cystine Cytosol Data Defect Diabetes Mellitus Disease Distant Family Genes Genus Hippocampus Glutamine Glycine Hematopoietic System Homeostasis Homologous Gene In Vitro Inner mitochondrial membrane Interphase Cell Knockout Mice Lead Light Link Lipids Liver Malignant Neoplasms Mammals Measures Metabolic Metabolic Pathway Metabolism Methods Mitochondria Mitochondrial Proteins Molecular Mus Normal tissue morphology Nucleotide Biosynthesis Null Lymphocytes Organelles Organism Pathway interactions Pattern Phenotype Physiological Physiology Play Process Protein Biosynthesis Proteins Research Resistance Respiration Respiratory Chain Role Serine Substrate Specificity Testing Tissues Tumor Tissue Work chemotherapy differential expression electron donor experimental study extracellular human disease in vivo metabolomics mitochondrial metabolism uptake

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项目摘要

7. PROJECT SUMMARY/ABSTRACT Metabolic flux generates energy and building blocks to fuel the many processes occurring in cells and organisms. Mitochondria are central organelles in both catabolic and anabolic cellular metabolism, a function for which they require efficient exchange of metabolites with the rest of the cell. Mitochondrial transporters facilitating metabolite exchange across the inner mitochondrial membrane thus play a key role in metabolism and physiology. Alterations in mitochondrial transporters have been linked to diseases including cancer, however, the identities and functions of these transporters are not well understood. One metabolite whose transport into mitochondria is critical for biosynthesis and proliferation is the amino acid serine. I recently identified Sideroflexin1 (SFXN1) as the long-sought transporter responsible for serine uptake into mitochondria in the one-carbon metabolic pathway, which is commonly upregulated in cancer. SFXN1 is part of the conserved carrier family of Sideroflexins consisting of five homologues in mammals. All Sideroflexins localize to mitochondria but vary in their tissue expression patterns and their functions in vivo are unknown. My preliminary data suggest that Sideroflexins, including SFXN1, have other physiologically relevant substrates beside serine, such as alanine and cysteine, and play important roles in metabolism outside of the one-carbon pathway. While SFXN1 and its closest homologue, SFXN3, are functionally redundant, other Sideroflexins can compensate for loss of SFXN1 to varying degrees suggesting a difference in substrate specificities and/or affinities. The most distant homologue, SFXN4, has a function outside of one-carbon metabolism and likely does not transport serine. I hypothesize that since SFXN4 is required for TCA cycle flux and mitochondrial respiration, it is responsible for the uptake of another metabolite with a key role in mitochondrial metabolism, such as glutamine. To broadly identify SFXN1 and SFXN4 candidate substrates, I will use untargeted metabolomics methods and determine whether transport of these substrates occurs in vitro and in cells. I will use Seahorse extracellular flux analysis to determine which respiratory chain complex and thus which electron donor is affected by loss of SFXN4 and perform a negative selection CRISPR screen to probe SFXN4 function in an unbiased way. Finally, I will determine the physiological functions of Sideroflexins using knockout mice. By elucidating the functions of Sideroflexins, an important but largely unstudied family of potentially chemotherapeutically relevant mitochondrial transporters, my research will shed light on the poorly understood role of mitochondrial transport in cellular metabolism and in physiology.

7。项目摘要/摘要代谢通量会产生能量和构建块，以助长细胞中发生的许多过程有机体。线粒体是分解代谢和合成代谢细胞代谢的中心细胞器，一个功能他们需要有效地将代谢物与细胞的其余部分交换。线粒体转运蛋白促进内部线粒体膜的代谢物交换因此在代谢中起关键作用和生理学。线粒体转运蛋白的改变已与包括癌症在内的疾病有关但是，这些转运蛋白的身份和功能尚不清楚。一个代谢物进入线粒体对于生物合成至关重要，增殖是氨基酸丝氨酸。我最近确定的Sideroflexin1（SFXN1）是负责丝氨酸摄取线粒体的长期转运蛋白在单碳代谢途径中，通常在癌症中上调。 SFXN1是保守的Sideroflex蛋白家族由哺乳动物的五种同源物组成。所有Sideroflexins都本地化对于线粒体而言，其组织表达模式及其在体内的功能各不相同。我的初步数据表明，包括SFXN1在内的Sideroflexin具有其他与生理相关的底物除丝氨酸（例如丙氨酸和半胱氨酸）以外路径。虽然SFXN1及其最接近的同源物SFXN3在功能上是多余的，但其他Sideroflexins可以在不同程度上补偿SFXN1的损失，这表明底物特异性和/或亲和力。最遥远的同源物SFXN4具有一碳代谢以外的功能，并且可能不运输丝氨酸。我假设由于TCA循环通量和线粒体需要SFXN4 呼吸，它负责摄取另一种代谢物在线粒体代谢中具有关键作用的代谢产物，例如谷氨酰胺。要广泛识别sfxn1和sfxn4候选底物，我将使用不靶向的代谢组学方法并确定这些底物的转运是否在体外和细胞中发生。我会使用海马细胞外通量分析来确定哪种呼吸链复合物，从而确定哪个电子捐赠者受SFXN4的损失影响，并执行负面选择CRISPR屏幕以探测SFXN4功能以公正的方式。最后，我将使用基因敲除小鼠来确定副反合蛋白的生理功能。通过阐明Sideroflexin的功能，Sideroflexin的功能，这是一个重要但很大程度上未研究的家族化学治疗相关的线粒体转运蛋白，我的研究将揭示出鲜为人知的线粒体转运在细胞代谢和生理学中的作用。