Functional Characterization of the NPC Homologue NPC1L1

NPC 同源物 NPC1L1 的功能表征

• 批准号: 7008112
• 负责人: YIANNIS A IOANNOU
• 金额: $ 36.41万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7008112
• 关键词: HMG coA reductases; Niemann Pick disease; cell line; cholesterol; clinical research; disease /disorder model; enzyme activity; genetically modified animals; homeostasis; immunoelectron microscopy; immunofluorescence technique; lipid metabolism; lipid transport; low density lipoprotein receptor; model design /development; permease; posttranslational modifications; protein localization; protein sequence; protein structure function; western blottings

DESCRIPTION (provided by applicant): Recently, we have identified a large polytopic membrane protein, NPC1L1 that shares significant homology (42% amino acid identity) with the Niemann-Pick C1 (NPC1) disease protein. NPC1 disease is a severe lysosomal lipidosis in which the egress of cholesterol and other lipids from the endosomal/lysosomal (E/L) system is defective, leading to neurodegeneration and premature demise. The proteins responsible for the two forms of NPC disease, NPC1 and NPC2, were recently identified. Preliminary characterization of these proteins suggests that NPC1 may act as a lipid permease on the membranes of late endosomes, whereas NPC2 is a small, soluble, cholesterol-binding lysosomal protein. The role of our newly identified NPC1 homologue, NPC1L1, is currently unknown and no diseases have been ascribed to its loss of function. Based on the homology between NPC1 and NPC1L1 and our preliminary data, we hypothesize that NPC1L1 has a lipid permease function similar to that of NPC1 but resides in a different subcellular location. Therefore, the overall objectives of the proposed research are to characterize the function of NPC1L1 and determine its role in subcellular lipid and/or cholesterol transport. Efforts will first be directed towards the analysis and characterization of the topology and intracellular location of the NPC1L1 protein to determine the cellular location in which it functions. Solution of its membrane topology will establish the direction of its potential pump activity and also its relationship to the newly identified resistance-nodulation-division (RND) family of eukaryotic permeases. Next, analysis and characterization of the function(s) of the NPC1L1 protein and its potential regulation by subcellular cholesterol and or lipid levels will be carried out to determine whether NPC1L1 exhibits a fatty acid, or other lipid, permease activity. These studies will be accomplished by expression of NPC1L1 in prokaryotes engineered to contain mutations in their endogenous RND permease genes relevant to our studies. Expression in yeast and mammalian cells will also be utilized, as needed, to further characterize the function(s) of this protein. Finally, generation and characterization of an NPC1L1 knockout mouse model should provide us with the necessary data to completely characterize the physiological function of NPC1L1 and its involvement in lipid/cholesterol transport or homeostasis.

描述（由申请人提供）：最近，我们鉴定了一种大的多胞膜蛋白 NPC1L1，它与 Niemann-Pick C1 (NPC1) 疾病蛋白具有显着的同源性（42% 氨基酸同一性）。 NPC1 疾病是一种严重的溶酶体脂质沉积症，其中胆固醇和其他脂质从内体/溶酶体 (E/L) 系统的排出有缺陷，导致神经变性和过早死亡。最近确定了导致两种形式的鼻咽癌疾病（NPC1 和 NPC2）的蛋白质。这些蛋白质的初步表征表明，NPC1 可能充当晚期内涵体膜上的脂质渗透酶，而 NPC2 是一种小的、可溶性、胆固醇结合溶酶体蛋白。我们新发现的 NPC1 同源物 NPC1L1 的作用目前尚不清楚，也没有任何疾病归因于其功能丧失。基于 NPC1 和 NPC1L1 之间的同源性以及我们的初步数据，我们假设 NPC1L1 具有与 NPC1 相似的脂质渗透功能，但位于不同的亚细胞位置。因此，本研究的总体目标是表征 NPC1L1 的功能并确定其在亚细胞脂质和/或胆固醇转运中的作用。首先将致力于分析和表征 NPC1L1 蛋白的拓扑结构和细胞内位置，以确定其发挥功能的细胞位置。其膜拓扑结构的解决方案将确定其潜在泵活性的方向以及其与新鉴定的真核渗透酶抗性结瘤分裂（RND）家族的关系。接下来，将对 NPC1L1 蛋白的功能及其通过亚细胞胆固醇和/或脂质水平的潜在调节进行分析和表征，以确定 NPC1L1 是否表现出脂肪酸或其他脂质通透酶活性。这些研究将通过在原核生物中表达 NPC1L1 来完成，这些原核生物被设计为包含与我们的研究相关的内源 RND 通透酶基因的突变。根据需要，还将利用酵母和哺乳动物细胞中的表达来进一步表征该蛋白质的功能。最后，NPC1L1 敲除小鼠模型的生成和表征应该为我们提供必要的数据，以完整表征 NPC1L1 的生理功能及其参与脂质/胆固醇转运或稳态的作用。