Regulation and Transport Mechanism of Choline Transporter-Like Protein 1 (CTL1/SLC44A1)

作为一种必需的营养素，胆碱主要用于膜磷脂的生物合成。一些胆碱在线粒体中被氧化成甜菜碱，只有少量的胆碱用于乙酰胆碱的合成。胆碱是带正电荷的季胺，需要蛋白质介导的转运才能通过膜。我们最近发现，胆碱转运蛋白样蛋白1（CTL 1）作为胆碱转运蛋白在质膜和线粒体的功能。CTL 1的功能取决于细胞分化状态和胆碱含量。胆碱缺乏会降低CTL 1水平，导致膜合成受损并增加脂滴的积累。胆碱添加刺激CTL 1基因表达并恢复其转运功能。关于CTL 1的调控和转运机制还有许多未知数。我们计划阐明：*1）基于细胞对胆碱的需求和特定细胞（肌肉和心脏）分化过程中控制CTL 1转录的核机制。为了建立这种广泛存在的和重要的调节反应的机制，我们将研究细胞核和表观遗传调节剂对CTL 1基因的调节。我们已经确定了哺乳动物CTL 1基因的特征，并产生了CTL 1的启动子-荧光素酶报告基因构建体。 我们将研究CTL 1启动子甲基化和核因子Sp1，NF 1和E2 F的特异性转录调控，我们知道DNA结合元件和功能。其他相关调节因子包括肌肉和心脏分化因子MyoD和GATA 4。它们的特殊作用也将在胆碱可用性的背景下使用全基因组和基因特异性方法进行仔细研究。*2）CTL 1的膜转运机制。我们已经制备了两种特异性抗体，并确定CTL 1转运蛋白具有9个跨膜结构域，细胞内N-末端和细胞外C-末端。然而，目前对负责CTL 1介导的转运的特定结构域和氨基酸残基的功能知之甚少。特别重要的是要确定哪些转运蛋白区域是关键的胆碱运输和膜靶向细胞表面和线粒体。将通过半胱氨酸扫描确认CTL 1膜位置。为了确定底物结合位点（带正电荷的胆碱），重点将放在主要位于外部区域的带负电荷的氨基酸残基和最后两个跨膜结构域上，所有这些都被发现是转运蛋白家族SLC 44 A中进化保守的。我们期望确定特定的结构组分如何支配CTL 1转运功能、寡聚化和对特异性抑制剂chemicholinium-3的抑制敏感性。

As an essential nutrient, choline is mostly consumed for the biosynthesis of membrane phospholipids. Some choline is oxidised in mitochondria to betaine and only small amount of choline is used for acetylcholine synthesis. Choline is positively charged quaternary amine and requires a protein-mediated transport to pass the membranes. We recently established that choline-transporter-like protein 1 (CTL1) function as a choline transporter at the plasma membrane and mitochondria. CTL1 function depends on cell differentiation state and choline content. Choline deficiency reduces CTL1 levels, causing impairments in the membrane synthesis and increasing accumulation of lipid droplets. Choline addition stimulates CTL1 gene expression and restores its transport function. There are multiple unknowns about the CTL1 regulation and transport mechanism. We plan to elucidate:***1) The nuclear mechanisms that control CTL1 transcription based on cellular demands for choline and during the process of specific cell (muscle and heart ) differentiation. To establish the mechanisms for this widely present and significant regulatory response we will examine the CTL1 gene regulation with both nuclear and epigenetic modulators. We have characterized mammalian CTL1 genes and produced promoter-luciferase reporter constructs for CTL1. We will investigate the CTL1 promoter methylation and specific transcriptional regulation with nuclear factors Sp1, NF1 and E2F for which we know DNA binding elements and function. Other relevant regulators will include muscle and heart differentiation factors MyoD and GATA4. They particular roles will be also carefully examined in the context of choline availability using genome-wide and gene-specific approaches. ***2) The membrane transport mechanism of CTL1. We have produced two specific antibodies and established that CTL1 transporter has nine transmembrane domains, intracellular N-terminus and extracellular C-terminus. Yet, little is currently known about the function of specific domains and amino acid residues responsible for CTL1-mediated transport. Of particular importance is to identify which transporter regions are critical for choline transport and for the membrane targeting at the cell surface and mitochondria. The CTL1 membrane position will be confirmed by cysteine scanning. For  determination of substrate binding site (positively charged choline),  focus will be on negatively charged amino acid residues mostly located in the outside area and on the last two transmembrane domains, all found to be the evolutionary conserved in the transporter family SLC44A. We expect to establish how specific structural components govern the CTL1 transport function, oligomerization and sensitivity to inhibition with  specific inhibitor chemicholinium-3.**