Substrates and functions of the Sideroflexin mitochondrial transporter family

7.项目摘要/摘要 代谢流产生能量和积木，为发生在细胞和 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，是一种功能 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，促进跨内线粒体膜的代谢物交换在新陈代谢中起关键作用 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，这些转运蛋白的身份和功能还没有被很好地理解。一种代谢物 转运到线粒体是生物合成和增殖的关键，氨基酸丝氨酸也是如此。我最近 发现铁弹性蛋白1(SFXN1)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，这在癌症中通常上调。SFXN1是 哺乳动物中保守的铁弹性蛋白载体家族，由五个同源物组成。所有的铁弹性蛋白都本地化 但它们的组织表达模式和在体内的功能尚不清楚。我的 初步数据表明，铁杆菌素，包括SFXN1，还有其他生理相关的底物 除了丝氨酸，如丙氨酸和半胱氨酸，并在一碳以外的代谢中发挥重要作用 路径。虽然SFXN1及其最接近的同系物SFXN3在功能上是多余的，但其他铁杆菌素可以 补偿SFXN1的损失，在不同程度上表明底物特异性和/或 亲和力。最远的同系物SFXN4具有一碳代谢以外的功能，很可能 不运输丝氨酸。我推测由于SFXN4是TCA循环通量和线粒体所必需的 呼吸作用负责摄取另一种在线粒体新陈代谢中起关键作用的代谢物， 比如谷氨酰胺。为了广泛识别SFXN1和SFXN4候选底物，我将使用 代谢组学方法，并确定这些底物是否在体外和细胞内发生转运。这就做 使用海马细胞外流量分析来确定哪个呼吸链复合体，从而确定哪个电子 供体受到SFXN4缺失的影响，并进行负选择CRISPR筛查以探测SFXN4功能 以一种公正的方式。最后，我将利用基因敲除小鼠来确定铁弹性蛋白的生理功能。 通过阐明铁弹性蛋白的功能，一个重要但在很大程度上未被研究的潜在的 与化疗相关的线粒体转运蛋白，我的研究将阐明人们对 线粒体转运在细胞代谢和生理学中的作用。

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.