BIOLOGICAL FUNCTION OF THE NIEMANN PICK C PROTEIN

C型尼曼匹克（NPC）是一种常染色体隐性遗传性溶酶体贮积病，可导致幼儿进行性神经退行性变。培养的NPC细胞表达脂蛋白衍生的胆固醇的缺陷转运，导致胆固醇的溶酶体积累和细胞胆固醇稳态的异常调节。NPC 1基因最近被克隆。它编码一个1245个氨基酸的膜蛋白，与参与胆固醇稳态的两个蛋白质具有序列同源性。NPC 1的生物学功能是什么？我们的假设是NPC 1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检验这一假设。特异性im 1：研究NPC 1在胆固醇转运途径中的作用。NPC 1将在受调节的启动子的控制下表达;将测量胆固醇转运的动力学。具体目标2：确定细胞胆固醇水平是否调节NPC 1。将研究通过细胞胆固醇水平对NPC 1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NPC 1。具体目标4：分析NPC 1的细胞内定位。将通过密度梯度和免疫荧光显微镜分析NPC 1分布。具体目标5：研究NPC 1的膜取向。随着致病突变的定位，NPC 1的结构域组织对于结构/功能分析将变得重要。NPC 1的生物学功能的知识对于指导研究受折磨儿童的可能疗法至关重要。它还与控制全身胆固醇水平有关，并将获得关于控制胆固醇逆向转运和胆汁酸代谢的胆固醇可用性的信息。

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.