Functional Characterization of the NPC Homologue NPC1L1

描述（由申请人提供）：最近，我们发现了一个大的多聚膜蛋白NPC1L1，它与Niemann-Pick C1 （NPC1）疾病蛋白具有显著的同源性（42%的氨基酸同源性）。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）系统输出有缺陷，导致神经变性和过早死亡。最近发现了导致两种鼻咽癌疾病（NPC1和NPC2）的蛋白质。这些蛋白的初步表征表明，NPC1可能在晚期核内体的膜上作为脂质渗透酶，而NPC2是一种小的、可溶的、胆固醇结合的溶酶体蛋白。我们新发现的NPC1同源物NPC1L1的作用目前尚不清楚，也没有疾病归因于其功能丧失。基于NPC1和NPC1L1的同源性以及我们的初步数据，我们假设NPC1L1具有与NPC1相似的脂质渗透酶功能，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/或胆固醇运输中的作用。努力将首先指向NPC1L1蛋白的拓扑结构和细胞内位置的分析和表征，以确定其功能的细胞位置。其膜拓扑结构的解决将确定其潜在泵活性的方向，以及它与新发现的真核渗透膜抗性-结节-分裂（RND）家族的关系。接下来，将分析和表征NPC1L1蛋白的功能及其受亚细胞胆固醇和/或脂质水平的潜在调节，以确定NPC1L1是否表现出脂肪酸或其他脂质渗透酶活性。这些研究将通过在原核生物中表达NPC1L1来完成，这些原核生物被改造成包含与我们研究相关的内源性RND渗透酶基因突变。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的功能。最后，NPC1L1敲除小鼠模型的建立和表征将为我们提供必要的数据，以完全表征NPC1L1的生理功能及其在脂质/胆固醇转运或体内平衡中的作用。

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.