Substrates and functions of the Sideroflexin mitochondrial transporter family

7. 项目概要/摘要 代谢流产生能量和构建模块，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，其功能 为此，它们需要与细胞其他部分进行有效的代谢物交换。线粒体转运蛋白 促进线粒体内膜上的代谢物交换，从而在新陈代谢中发挥关键作用 和生理学。线粒体转运蛋白的改变与癌症、 然而，这些转运蛋白的身份和功能尚不清楚。一种代谢物，其 转运到线粒体对于生物合成和增殖至关重要的是氨基酸丝氨酸。我最近 确定 Sideroflexin1 (SFXN1) 是长期寻找的负责丝氨酸摄取到线粒体的转运蛋白 存在于一碳代谢途径中，该途径在癌症中通常上调。 SFXN1 是 哺乳动物中铁弹性蛋白的保守载体家族，由五个同源物组成。所有 Sideroflexins 均定位 线粒体，但其组织表达模式各不相同，并且其体内功能尚不清楚。我的 初步数据表明 Sideroflexins（包括 SFXN1）具有其他生理相关底物 除丝氨酸外，还有丙氨酸和半胱氨酸，在单碳以外的代谢中发挥着重要作用 途径。虽然 SFXN1 及其最接近的同源物 SFXN3 在功能上是冗余的，但其他 Sideroflexins 可以 不同程度地补偿 SFXN1 的损失，表明底物特异性和/或 亲和力。最遥远的同源物 SFXN4 具有一碳代谢之外的功能，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 呼吸作用，它负责摄取另一种在线粒体代谢中起关键作用的代谢物， 例如谷氨酰胺。为了广泛识别 SFXN1 和 SFXN4 候选底物，我将使用非靶向 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内。我会 使用 Seahorse 细胞外通量分析来确定哪个呼吸链复合体以及哪个电子 供体受到 SFXN4 丢失的影响，并进行阴性选择 CRISPR 筛选以探测 SFXN4 功能 以公正的方式。最后，我将使用基因敲除小鼠来确定 Sideroflexins 的生理功能。 通过阐明 Sideroflexins 的功能，Sideroflexins 是一个重要但基本上未经研究的潜在潜在家族 化疗相关的线粒体转运蛋白，我的研究将揭示人们知之甚少的问题 线粒体运输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.