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

胆碱是一种必需的营养物质，主要用于膜磷脂的生物合成。部分胆碱在线粒体中氧化为甜菜碱，只有少量胆碱用于合成乙酰胆碱。胆碱是带正电的季胺，需要蛋白质介导的转运才能通过细胞膜。我们最近发现胆碱转运蛋白样蛋白1 （CTL1）在质膜和线粒体中作为胆碱转运蛋白起作用。CTL1的功能取决于细胞分化状态和胆碱含量。胆碱缺乏降低CTL1水平，导致膜合成受损，增加脂滴积累。添加胆碱刺激CTL1基因表达，恢复其转运功能。关于CTL1的调控和转运机制存在诸多未知因素。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CTL1转录的核机制。为了建立这种广泛存在和重要的调控反应的机制，我们将研究CTL1基因与核和表观遗传调节剂的调控。我们已经鉴定了哺乳动物CTL1基因，并产生了CTL1的启动子荧光素酶报告结构。我们将研究CTL1启动子甲基化和特定的转录调控与核因子Sp1， NF1和E2F，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GATA4。它们的特殊作用也将在使用全基因组和基因特异性方法的胆碱可用性的背景下仔细检查。***2) CTL1的膜转运机制。我们制备了两种特异性抗体，并确定了CTL1转运蛋白具有9个跨膜结构域、细胞内n端和细胞外c端。然而，目前对ctl1介导转运的特定结构域和氨基酸残基的功能知之甚少。特别重要的是确定哪些转运体区域对胆碱运输和针对细胞表面和线粒体的膜至关重要。通过半胱氨酸扫描确认CTL1膜的位置。为了确定底物结合位点（带正电的胆碱），重点将放在主要位于外部区域和最后两个跨膜结构域的带负电的氨基酸残基上，这些残基都是转运蛋白家族SLC44A中进化保守的。我们希望确定特定的结构成分如何控制CTL1的运输功能，寡聚化和对特定抑制剂化学-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.**