acriflavine and Niemann-Pick-Diseases

Niemann-Pick type C (NPC) is an autosomal recessive lipid storage disorder characterized by lysosomal accumulation of cholesterol and gangliosides resulting from a defect in intracellular lipid trafficking. The NPC1 gene encodes a 1278-amino acid integral membrane protein involved in the sub-cellular trafficking of lipids. The exact biological function of NPC1 remains unclear. Recent evidence suggests that NPC1 is a eukaryotic member of the RND permease family of transport proteins, which when expressed in bacteria is capable of transporting fatty acids. The goal of this project was to assess the role of NPC1 in the transport of fatty acids in primary human fibroblasts using normal fibroblasts and fibroblasts from patients with three lysosomal storage diseases: NPC, mucolipidosis IV, and Sandhoff disease. If NPC1 is a fatty acid transporter, we expect to find fatty acid accumulation only in NPC fibroblasts. We used three experimental approaches to assess the role of NPC1 as a fatty acid transporter. First, we evaluated the accumulation versus metabolism of low density lipoprotein-derived oleic acid. Second, we assessed the amount of free fatty acid present after growth in lipoprotein-containing media. Third, we assessed the cellular accumulation of acriflavine, a fluorescent substrate for a number of resistance-nodulation-cell division permease transporters. Our results indicate that fatty acid flux through NPC1-deficient lysosomes is normal.

Topics: Acriflavine; Carrier Proteins; Cells, Cultured; Cholesterol; Chromatography, Gas; Dextrans; Fatty Acids; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Lipid Metabolism; Lipoproteins, LDL; Lysosomes; Membrane Glycoproteins; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Oleic Acid; Protein Transport

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.

Topics: Acriflavine; Amino Acid Motifs; Amino Acid Sequence; Biological Transport; Carrier Proteins; Cell Membrane; Cells, Cultured; Cholesterol; Cholesterol Esters; Endosomes; Escherichia coli; Fibroblasts; Fluorescence; Fluorescent Dyes; Humans; Intracellular Signaling Peptides and Proteins; Lysosomes; Membrane Glycoproteins; Membrane Proteins; Membrane Transport Proteins; Molecular Sequence Data; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Oleic Acid; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Proton-Motive Force; Recombinant Proteins; Sequence Alignment

Topics: Acriflavine; Biological Transport; Carrier Proteins; Cholesterol; Endoplasmic Reticulum; Glycoproteins; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Lysosomes; Membrane Glycoproteins; Membrane Transport Proteins; Mutation; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Proteins; Skin; Vesicular Transport Proteins