Functional Characterization of the NPC Homologue NPC1L1

描述（由申请人提供）：最近，我们鉴定了一种大的多位膜蛋白NPC 1 L1，其与尼曼-匹克C1（NPC 1）疾病蛋白具有显著的同源性（42%氨基酸同一性）。NPC 1疾病是一种严重的溶酶体变性，其中胆固醇和其他脂质从内体/溶酶体（E/L）系统的排出是有缺陷的，导致神经变性和过早死亡。负责NPC疾病的两种形式，NPC 1和NPC 2的蛋白质，最近被确定。这些蛋白质的初步表征表明，NPC 1可能作为一种脂质渗透酶的膜上的晚期内体，而NPC 2是一个小的，可溶性的，胆固醇结合的溶酶体蛋白。我们新鉴定的NPC 1同源物NPC 1 L1的作用目前尚不清楚，并且没有疾病归因于其功能丧失。基于NPC 1和NPC 1 L1之间的同源性和我们的初步数据，我们假设NPC 1 L1具有与NPC 1相似的脂质通透酶功能，但位于不同的亚细胞位置。因此，拟议研究的总体目标是表征NPC 1 L1的功能，并确定其在亚细胞脂质和/或胆固醇转运中的作用。努力将首先指向的拓扑结构和细胞内定位的NPC 1 L1蛋白的分析和表征，以确定其功能的细胞位置。其膜拓扑结构的解决方案将建立其潜在的泵活性的方向，以及其与新鉴定的真核渗透酶的抗性-增殖-分裂（RND）家族的关系。接下来，将进行NPC 1 L1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的分析和表征，以确定NPC 1 L1是否表现出脂肪酸或其他脂质、透化酶活性。这些研究将通过在原核生物中表达NPC 1 L1来完成，所述原核生物经工程改造以在其内源性RND通透酶基因中含有与我们的研究相关的突变。根据需要，还将利用在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，NPC 1 L1基因敲除小鼠模型的产生和表征应该为我们提供必要的数据，以完全表征NPC 1 L1的生理功能及其参与脂质/胆固醇转运或稳态。

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.