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

• 批准号: 10546459
• 负责人: Nora Kory
• 金额: $ 24.9万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 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/10546459
• 关键词: Address; Affect; Affinity; Alanine; Amino Acids; Anabolism; Animals; Biological Assay; CRISPR screen; Carbon; Catabolism; Cells; Citric Acid Cycle; Compensation; 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; 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; Proliferating; Protein Biosynthesis; Proteins; Research; Resistance; Respiration; Respiratory Chain; Role; Serine; Substrate Specificity; Testing; Tissues; Tumor Tissue; Work; candidate identification; chemotherapy; differential expression; electron donor; experimental study; extracellular; human disease; in vivo; metabolomics; mitochondrial metabolism; uptake

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.项目总结/摘要 代谢通量产生能量和构建块，为细胞中发生的许多过程提供燃料， 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， 为此，它们需要与细胞的其余部分有效地交换代谢物。线粒体转运蛋白 因此，促进代谢物穿过线粒体内膜的交换在代谢中起关键作用 和生理学。线粒体转运蛋白的改变与包括癌症在内的疾病有关， 然而，这些转运蛋白的特性和功能还不十分清楚。一种代谢物， 转运到线粒体中对于生物合成和增殖至关重要的是氨基酸丝氨酸。我最近 发现Sideroflexin 1（SFXN 1）是长期寻找的负责丝氨酸摄取到线粒体的转运蛋白 在一碳代谢途径中，这通常在癌症中上调。SFXN 1是 在哺乳动物中由五种同源物组成的铁弯曲素的保守载体家族。所有Sideroflexins定位于 但它们的组织表达模式不同，它们在体内的功能是未知的。我 初步数据表明，包括SFXN 1在内的铁弯曲素具有其他生理学相关底物 除了丝氨酸，如丙氨酸和半胱氨酸，并发挥重要作用，在代谢以外的一个碳 通路虽然SFXN 1及其最接近的同源物SFXN 3在功能上是冗余的，但其他Sideroflexins可以 补偿SFXN 1的损失至不同程度，表明底物特异性的差异和/或 亲和力最远的同源物SFXN 4具有单碳代谢之外的功能， 不运输丝氨酸。我推测，由于SFXN 4是TCA循环通量和线粒体 呼吸，它负责摄取另一种代谢物，在线粒体代谢中起关键作用， 例如谷氨酰胺。为了广泛地鉴定SFXN 1和SFXN 4候选底物，我将使用非靶向的 代谢组学方法，并确定这些底物的转运是否发生在体外和细胞中。我会 使用Seahorse细胞外通量分析来确定哪种呼吸链复合物，从而确定哪种电子 供体受SFXN 4损失的影响，并进行阴性选择CRISPR筛选以探测SFXN 4功能 不带偏见地最后，我将使用基因敲除小鼠来确定Sideroflexins的生理功能。 通过阐明Sideroflexins的功能，一个重要但基本上未研究的潜在的 化疗相关的线粒体转运蛋白，我的研究将阐明鲜为人知的 线粒体运输在细胞代谢和生理学中的作用。