BIOLOGICAL FUNCTION OF THE NIEMANN PICK C PROTEIN

NIEMAN-PICK C型(NPC)是一种常染色体隐性遗传性溶酶体储存疾病，可导致幼儿进行性神经变性。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聚和细胞胆固醇稳态的异常调节。NPC1基因是最近克隆的。它编码一个1245aa的膜蛋白，与两个参与胆固醇稳态的蛋白序列同源。NPC1的生物学功能是什么？我们的假设是，NPC1控制着来自溶酶体的胆固醇携带囊泡的靶向。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IM1：研究NPC1在各种胆固醇转运途径中的作用。NPC1将在受调控的启动子的控制下表达；胆固醇运输的动力学将被测量。具体目标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。具体目的3：确定Ced-1基因是否为NPC1。具体目的4：分析NPC1的细胞内定位。NPC1的分布将通过密度梯度和免疫荧光显微镜进行分析。具体目的5：研究NPC1的膜取向。随着致病突变图谱的绘制，NPC1的结构域组织将成为结构/功能分析的重要内容。对NPC1生物学功能的了解对于指导对患病儿童的可能治疗方法的研究至关重要。它还与控制全身胆固醇水平有关，还将获得有关胆固醇控制、胆固醇反向转运和胆汁酸代谢的信息。

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.