Characteristics and novel functions of SLC44A transporters

SLC44A转运蛋白的特点和新功能

• 批准号: RGPIN-2020-04573
• 负责人: Bakovic, Marica
• 金额: $ 2.4万
• 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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740049
• 关键词: Characteristics; novel; functions; SLC44A; transporters

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are membrane phospholipids involved in the fundamental processes of cell division, survival and signaling. PC and PE are synthesized de novo by the CDP-choline and CDP-ethanolamine (Kennedy) pathway, in which the extracellular substrates choline and ethanolamine are transported into the cell, phosphorylated, and coupled with diacylglycerol to form the final phospholipid product. While multiple transport systems have been established for choline, ethanolamine transport is poorly characterized, and there is no single protein assigned a transport function for ethanolamine. We investigate transporters from the family of Solute Carriers 44A (SLC44A) known as Choline Transporter-Like proteins-1 and -2 (CTL1 and CTL2). CTL1/SLC44A1 is a choline/proton antiporter and delivers choline for the CDP-choline pathway and choline oxidation to betaine. There are indications that CTL2/SLC44A2 is also a choline transporter at the plasma membrane and mitochondria, but its function is not firmly established. We propose that i. CTL1 and CTL2 are authentic plasma membrane and mitochondria ethanolamine transporters -We will characterize cellular and mitochondrial transport of CTL1 and CTl2 in cells that maintain CTL1 but lack CTL2 and vice versa. We will employ CTL1/2 mutants, siRNA and specific antibodies to demonstrate for the first time that CTL1 and CTL2 are choline and also ethanolamine transporters in mammalian systems. ii. CTL1 and CTL2 regulate mitochondrial function-Our preliminary data suggest that the luck of CTL1 induces mitophagy and fragmentation of mitochondrial networks. We propose that CTL1 regulate mitochondrial fusion-fission processes. We will investigate mechanisms of mitochondrial fusion and metabolism (electron transport complexes, ATP synthesis, ROS production, membrane potential, etc.) in CTL1 and CTL2 deficient and over-expressing cells. The initial findings will be corroborated in mitochondria and primary cells isolated from CTL1 and/or CTL2 knockout mice. We expect to demonstrate for the first time that CTL1 and perhaps CTL2 are critical regulators of mitochondrial turnover and mitochondrial energy homeostasis. iii. CTL1 and CTL2 couple phospholipid synthesis with the whole-body energy metabolism-CTL1 and CTL2 knockout mice were produced and characterized by the International Mouse Phenotyping Consortium. Reduced body weight and increased muscle/fat ratio are common features of CTL1 and CTL2 knockout mice. We will characterize CTL1 mice in full detail and compare to better known CTL2 mice, including the regulation of the  Kennedy pathway, lipid synthesis and energy metabolism in skeletal muscle, liver and adipose tissue, applying protocols extensively used in previous studies of our Pcyt2 knockout mice (Pcyt2 is the main regulatory enzyme in the CDP-ethanolamine Kennedy pathway).

磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE）是参与细胞分裂、存活和信号传导的基本过程的膜磷脂。PC和PE通过CDP-胆碱和CDP-乙醇胺（Kennedy）途径从头合成，其中细胞外底物胆碱和乙醇胺被转运到细胞中，磷酸化，并与二酰基甘油偶联以形成最终的磷脂产物。虽然已经建立了多个运输系统的胆碱，乙醇胺运输的特点很差，有没有一个单一的蛋白质分配的运输功能乙醇胺。 我们研究溶质载体44 A（SLC 44 A）家族的转运蛋白，称为胆碱转运蛋白样蛋白-1和-2（CTL 1和CTL 2）。CTL 1/SLC 44 A1是胆碱/质子反向转运蛋白，并为CDP-胆碱途径和胆碱氧化为甜菜碱递送胆碱。有迹象表明，CTL 2/SLC 44 A2也是质膜和线粒体上的胆碱转运蛋白，但其功能尚未确定。我们建议我。CTL 1和CTL 2是真正的质膜和线粒体乙醇胺转运蛋白-我们将表征维持CTL 1但缺乏CTL 2的细胞中CTL 1和CT 12的细胞和线粒体转运，反之亦然。我们将采用CTL 1/2突变体，siRNA和特异性抗体首次证明CTL 1和CTL 2是哺乳动物系统中的胆碱和乙醇胺转运蛋白。 二. CTL 1和CTL 2调节线粒体功能-我们的初步数据表明，CTL 1的运气诱导线粒体自噬和线粒体网络的碎片化。我们建议，CTL 1调节线粒体融合-分裂过程。我们将研究线粒体融合和代谢的机制（电子传递复合物，ATP合成，ROS产生，膜电位等）。在CTL 1和CTL 2缺陷和过表达的细胞中。最初的发现将在从CTL 1和/或CTL 2敲除小鼠分离的线粒体和原代细胞中得到证实。我们希望首次证明CTL 1和CTL 2是线粒体周转和线粒体能量稳态的关键调节因子。三. CTL 1和CTL 2偶联磷脂合成与全身能量代谢-CTL 1和CTL 2敲除小鼠由国际小鼠表型鉴定协会生产和表征。体重减轻和肌肉/脂肪比增加是CTL 1和CTL 2基因敲除小鼠的共同特征。我们将全面详细描述CTL 1小鼠的特征，并与更知名的CTL 2小鼠进行比较，包括肯尼迪通路的调节，骨骼肌，肝脏和脂肪组织中的脂质合成和能量代谢，应用在我们的Pcyt 2敲除小鼠的先前研究中广泛使用的协议（Pcyt 2是CDP-乙醇胺肯尼迪通路中的主要调节酶）。