BIOLOGICAL FUNCTION OF THE NIEMANN PICK C PROTEIN

尼曼-皮克C型（NPC）是一种常染色体隐性溶酶体贮积病，在幼儿中引起进行性神经变性。培养的鼻咽癌细胞表达脂蛋白来源的胆固醇运输缺陷，导致胆固醇的溶酶体积累和细胞胆固醇稳态的异常调节。NPC1基因最近被克隆出来。它编码一个1245aa膜蛋白，其序列与两个参与胆固醇稳态的蛋白同源。NPC1的生物学功能是什么？我们的假设是NPC1控制来自溶酶体的携带胆固醇的囊泡的靶向。我们将使用野生型和胆固醇转运缺陷的中国仓鼠卵巢细胞来验证这一假设。特异性目的：探讨NPC1在各胆固醇转运途径中的作用。NPC1将在调控启动子的控制下表达；将测量胆固醇运输的动力学。特异性目的2：确定细胞胆固醇水平是否调节NPC1。将研究细胞胆固醇水平对NPC1表达的转录、翻译和翻译后控制。特异性目的3：确定ced-1基因是否为NPC1。特异性目的4：分析NPC1在细胞内的定位。通过密度梯度和免疫荧光显微镜分析NPC1的分布。特异性目的5：研究NPC1的膜取向。随着致病突变的绘制，NPC1的结构域组织将对结构/功能分析变得重要。了解NPC1的生物学功能对于指导研究可能的治疗方法至关重要。它也与控制全身胆固醇水平有关，也将获得控制胆固醇可用性的信息，用于逆向胆固醇运输和胆汁酸代谢。

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disease that causes progressive neurological degeneration in young children. Cultured NPC cells express defective transport of lipoprotein-derived cholesterol, resulting in lysosomal accumulation of cholesterol and aberrant regulation of cellular cholesterol homeostasis. The NPC1 gene was recently cloned. It encodes a 1245 aa membrane protein, with sequence homology to two proteins involved in cholesterol homeostasis. What is the biological function of NPC1? Our hypothesis is that NPC1 governs the targeting of cholesterol- carrying vesicles derived from lysosomes. We will test this hypothesis using wild-type and cholesterol transport defective Chinese hamster ovary cells. Specific im 1: To investigate the role of NPC1 in each cholesterol transport pathway. NPC1 will be expressed under the control of a regulated promoter; kinetics of cholesterol transport will be measured. Specific Aim 2: To determine if cellular cholesterol levels regulate NPC1. Transcriptional, translational and post- translational control of NPC1 expression by cellular cholesterol levels will be investigated. Specific Aim 3: To determine if the ced-1 gene is NPC1. Specific Aim 4: To analyze the intracellular location of NPC1. NPC1 distribution will be analyzed by density gradients and immunofluorescence microscopy. Specific Aim 5: To investigate the membrane orientation of NPC1. The domain organization of NPC1 will become important for structure/function analysis as disease-causing mutations are mapped. Knowledge of the biological function of NPC1 is critical for guiding the investigation into possible therapies for afflicted children. It also has relevance to the control of whole body cholesterol levels as well will gain information on the control of cholesterol availability for reverse cholesterol transport and bile acid metabolism.