Investigating the Role of SLC45A4 in GABA Metabolism

摘要 溶质载体(SLC)蛋白是控制葡萄糖跨膜交换的膜转运蛋白， 氨基酸、无机离子和其他小分子代谢物。许多SLC基因已经被证明是 人类孟德尔病的病因和一些SLC转运蛋白是重要的药物靶标。 然而，由于无数的技术困难，很大一部分SLC家庭成员仍然是孤儿运输者 没有已知的底物，这既代表着巨大的知识差距，也代表着新的 药物开发。为了系统地研究孤儿SLC转运蛋白的生化功能， 计算工作流是我们实验室开发的，它结合了公共转录和代谢组学 数据集，以揭示SLC转运蛋白的代谢功能。使用此关联分析，一个 未知基因SLC45A4被鉴定为γ-氨基丁酸的单一最大决定因素 人体癌细胞中的(GABA)水平。GABA最为人所知的是一种抑制性神经递质 哺乳动物中枢神经系统，在外周组织中发挥调节细胞增殖、分化、 和迁徙。鉴于这些功能在癌细胞和正常细胞中的重要性，有必要 了解SLC45A4如何在非神经细胞中GABA代谢的背景下发挥作用。使用稳定 同位素示踪发现，SLC45A4提高细胞内GABA水平不是通过促进GABA摄取，而是通过促进GABA摄取 通过促进GABA从头合成，暗示了一种全新的GABA调节机制 综合。因此，推测SLC45A4编码一种亚细胞鸟氨酸转运蛋白，支持 GABA生产。该计划将朝着三个方向前进：目标1，测定生化 SLC45A4促进细胞内GABA产生的机制；目标2，确定亚细胞 SLC45A4转运蛋白和GABA合成的区划；以及目标3，底物的确定 SLC45A4的选择性和酶学性质。总体目标是确定SLC45A4如何调节De Novo GABA合成，有助于人类生物化学的基础知识，并可能提供 针对GABA代谢的新疗法。

ABSTRACT Solute carrier (SLC) proteins are membrane transporters that govern the cross-membrane exchanges of glucose, amino acids, inorganic ions, and other small molecule metabolites. Many SLC genes have been shown to be causes of Mendelian diseases in humans, and a number of SLC transporters are important drug targets. However, due to myriad technical difficulties, a large fraction of SLC family members are still orphan transporters without known substrates, which represents both a significant knowledge gap and a huge opportunity for new drug development. In order to systematically study the biochemical functions of the orphan SLC transporters, a computational workflow was developed in our lab, which combines public transcriptomic and metabolomic datasets to uncover the metabolic function of SLC transporters. Using this association analysis, an uncharacterized gene, SLC45A4, was identified to be the single greatest determinant of γ-Aminobutyric acid (GABA) levels in human cancer cells. GABA, which is mostly known as an inhibitory neurotransmitter in the mammalian central nervous system, functions in peripheral tissues to regulate cell proliferation, differentiation, and migration. Given the importance of these functions in both cancer and normal cells, it is essential to understand how SLC45A4 functions in the context of GABA metabolism in non-neuronal cells. Using stable isotope tracing, it was found that SLC45A4 increases cellular GABA levels not by promoting GABA uptake but by facilitating GABA de novo synthesis, suggesting an entirely new mechanism of the regulation of GABA synthesis. Therefore, it is hypothesized that SLC45A4 encodes a subcellular ornithine transporter that supports GABA production. This program will move forward in three directions: Aim 1, determining the biochemical mechanism by which SLC45A4 promotes cellular GABA production; Aim 2, determining the subcellular compartmentalization of SLC45A4 transporter and GABA synthesis; and Aim 3, determining substrate preference and enzymatic properties of SLC45A4. The overall goal is to determine how SLC45A4 regulates de novo GABA synthesis, which contributes to the fundamental knowledge of human biochemistry and may provide new therapeutics targeting GABA metabolism.