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

胆碱转运蛋白样蛋白1（CTL1/SLC44A1）的调控及转运机制

• 批准号: RGPIN-2015-05580
• 负责人: Bakovic, Marica
• 金额: $ 2.91万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613635
• 关键词: Regulation; Transport; Mechanism; Choline; Transporter

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.

胆碱作为一种必需的营养物质，主要用于膜磷脂的生物合成。线粒体中的一些胆碱被氧化成甜菜碱，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1(CTL1)在细胞膜和线粒体上作为胆碱转运蛋白发挥作用。CTL1的功能依赖于细胞分化状态和胆碱含量。胆碱缺乏会降低CTL1水平，导致细胞膜合成受损，增加脂滴的积累。胆碱的加入可刺激CTL1基因的表达，恢复其转运功能。关于CTL1的调控和转运机制有多种未知之处。我们计划澄清： 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的和重要的调控反应的机制，我们将研究核和表观遗传调节因子对CTL1基因的调控。我们已经鉴定了哺乳动物的CTL1基因，并为CTL1生产了启动子-荧光素酶报告载体。我们将用已知DNA结合元件和功能的核因子Sp1、NF1和E2F来研究CTL1启动子的甲基化和特异性转录调控。其他相关监管机构将包括肌肉和心脏分化因子MyoD和GATA4。还将利用全基因组和基因特异性的方法，在胆碱可获得性的背景下仔细研究它们的特殊作用。 2)CTL1的膜转运机制。我们已经产生了两种特异性抗体，证实了CTL1转运蛋白有九个跨膜区，即细胞内的N端和细胞外的C端。然而，目前对负责CTL1介导的转运的特定结构域和氨基酸残基的功能知之甚少。尤其重要的是要确定哪些转运体区域对胆碱的运输以及针对细胞表面和线粒体的膜至关重要。半胱氨酸扫描将确定CTL1膜的位置。为了确定底物结合部位(带正电的胆碱)，重点将放在带负电的氨基酸残基上，主要位于外区和最后两个跨膜结构域，都是在转运蛋白家族SLC44A中发现的进化保守的。我们希望确定特定的结构成分如何控制CTL1的运输功能、寡聚和对特定抑制剂Chemicholdium-3抑制的敏感性。