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.
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