Intracellular cholesterol transporter which acts in concert with NPC1 and plays an important role in the egress of cholesterol from the endosomal/lysosomal compartment. Both NPC1 and NPC2 function as the cellular 'tag team duo' (TTD) to catalyze the mobilization of cholesterol within the multivesicular environment of the late endosome (LE) to effect egress through the limiting bilayer of the LE. NPC2 binds unesterified cholesterol that has been released from LDLs in the lumen of the late endosomes/lysosomes and transfers it to the cholesterol-binding pocket of the N-terminal domain of NPC1. Cholesterol binds to NPC1 with the hydroxyl group buried in the binding pocket and is exported from the limiting membrane of late endosomes/ lysosomes to the ER and plasma membrane by an unknown mechanism. The secreted form of NCP2 regulates biliary cholesterol secretion via stimulation of ABCG5/ABCG8-mediated cholesterol transport.
Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.
Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann-Pick C1 (NPC1) and soluble Niemann-Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25-264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [(3)H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.
Interacting selectively and non-covalently with cholesterol (cholest-5-en-3-beta-ol); the principal sterol of vertebrates and the precursor of many steroids, including bile acids and steroid hormones.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.
Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann-Pick C1 (NPC1) and soluble Niemann-Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25-264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [(3)H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.
Dehydrodolichyl diphosphate (DedolPP) synthase catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize DedolPP, a biosynthetic precursor for dolichol which plays an important role as a sugar-carrier lipid in the biosynthesis of glycoprotein in eukaryotic cells. During certain pathological processes like Alzheimer's disease or some neurological disorders, dolichol has been shown to accumulate in human brain. In order to understand the regulatory mechanism of dolichol in eukaryotes, we performed a yeast two-hybrid screen using full length human DedolPP synthase gene [Endo et al. BBA 1625 (2003) 291] as a bait to find some proteins specifically interacting with the enzyme. We identified Niemann-Pick Type C2 protein (NPC2) to show a specific interaction with human DedolPP synthase. This interaction was further confirmed by in vitro co-immunoprecipitation experiment, indicating the possible physiological interaction between NPC2 and DedolPP synthase proteins in human.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
The hepatic expression of Niemann-Pick C1-like 1 (NPC1L1), which is a key molecule in intestinal cholesterol absorption, is high in humans. In addition to NPC1L1, Niemann-Pick C2 (NPC2), a secretory cholesterol-binding protein involved in intracellular cholesterol trafficking and the stimulation of biliary cholesterol secretion, is also expressed in the liver. In this study, we examined the molecular interaction and functional association between NPC1L1 and NPC2. In vitro studies with adenovirus-based or plasmid-mediated gene transfer systems revealed that NPC1L1 negatively regulated the protein expression and secretion of NPC2 without affecting the level of NPC2 messenger RNA. Experiments with small interfering RNA against NPC1L1 confirmed the endogenous association of these proteins. In addition, endocytosed NPC2 could compensate for the reduction of NPC2 in NPC1L1-overexpressing cells, and this demonstrated that the posttranscriptional regulation of NPC2 was dependent on a novel ability of NPC1L1 to inhibit the maturation of NPC2 and accelerate the degradation of NPC2 during its maturation. Furthermore, to confirm the physiological relevance of NPC1L1-mediated regulation, we analyzed human liver specimens and found a negative correlation between the protein levels of hepatic NPC1L1 and hepatic NPC2. CONCLUSION: NPC1L1 down-regulates the expression and secretion of NPC2 by inhibiting its maturation and accelerating its degradation. NPC2 functions as a regulator of intracellular cholesterol trafficking and biliary cholesterol secretion; therefore, in addition to its role in cholesterol re-uptake from the bile by hepatocytes, hepatic NPC1L1 may control cholesterol homeostasis via the down-regulation of NPC2.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Epithelial sodium channels (ENaC) located along the aldosterone-sensitive distal nephron (ASDN) play a pivotal role in the homeostasis of sodium balance. Among various modulating proteins, E3 ubiquitin ligase, Nedd4L, binds the PY motif of ENaC COOH terminals and catalyzes ubiquitination of the NH(2) terminal of the protein for subsequent degradation. One of the isoforms of human Nedd4L with evolutionarily new C2 domain was reported to play a significant role for degradating cell surface ENaC with interfering with wild isoforms by us previously. We focused the current analyses on isolating the binding molecules with C2 domain using yeast two-hybrid screening to elucidate further molecular interactions between ENaC and Nedd4L. We found NPC2, also known as HE-1, bind C2 domain of human Nedd4L and express along ASDN colocalized with Nedd4L. Additionally, in experiments using a model of salt-sensitive hypertension in Dahl rats, we provided evidence suggesting that transcriptional regulation and activation of NPC2 protein depends on sodium intake. NPC2 might regulate sodium reabsorption in the terminal nephron by interacting with ENaC-Nedd4L system.
Liver X receptors (LXRs) are nuclear receptors that regulate macrophage cholesterol efflux by inducing ATP-binding cassette transporter A1 (ABCA1) and ABCG1/ABCG4 gene expression. The Niemann-Pick C (NPC) proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant governing its availability for efflux to extracellular acceptors. Here we investigated the influence of LXR activation on intracellular cholesterol trafficking in primary human macrophages. Synthetic LXR activators increase the amount of free cholesterol in the plasma membrane by inducing NPC1 and NPC2 gene expression. Moreover, ABCA1-dependent cholesterol efflux induced by LXR activators was drastically decreased in the presence of progesterone, which blocks postlysosomal cholesterol trafficking, and reduced when NPC1 and NPC2 mRNA expression was depleted using small interfering RNA. The stimulation of cholesterol mobilization to the plasma membrane by LXRs led to a decrease in cholesteryl ester formation and Acyl-coenzyme A cholesterol acyltransferase-1 activity. These data indicate that LXR activation enhances cholesterol trafficking to the plasma membrane, where it becomes available for efflux, at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.
Mutations in the Niemann-Pick disease genes cause lysosomal cholesterol accumulation and impaired low density lipoprotein (LDL) cholesterol esterification. These findings have been attributed to a block in cholesterol movement from lysosomes to the site of the sterol regulatory machinery. In this study we show that Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) mutants have increased cellular cholesterol, yet they are unable to suppress LDL receptor activity and cholesterol biosynthesis. Cholesterol overload in both NPC1 and NPC2 mutants results from the failure of LDL cholesterol tobothsuppresssterolregulatoryelement-bindingprotein-dependent gene expression and promote liver X receptor-mediated responses. However, the severity of the defect in regulation of sterol homeostasis does not correlate with endoplasmic reticulum cholesterol levels, but rather with the degree to which NPC mutant fibroblasts fail to appropriately generate 25-hydroxycholesterol and 27-hydroxycholesterol in response to LDL cholesterol. Moreover, we demonstrate that treatment with oxysterols reduces cholesterol in NPC mutants and is able to correct the NPC1I1061T phenotype, the most prevalent NPC1 disease genotype. Our findings support a role for NPC1 and NPC2 in the regulation of sterol homeostasis through generation of LDL cholesterol-derived oxysterols and have important implications for the treatment of NPC disease.
The chemical reactions and pathways involving cholesterol, cholest-5-en-3 beta-ol, the principal sterol of vertebrates and the precursor of many steroids, including bile acids and steroid hormones. It is a component of the plasma membrane lipid bilayer and of plasma lipoproteins and can be found in all animal tissues.
The directed movement of cholesterol, cholest-5-en-3-beta-ol, into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.
Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann-Pick C1 (NPC1) and soluble Niemann-Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25-264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [(3)H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.
The directed movement of glycolipids, compounds containing (usually) 1-4 linked monosaccharide residues joined by a glycosyl linkage to a lipid, into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.
Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.
The Nogo-B receptor (NgBR) is a recently identified receptor for the N terminus of reticulon 4B/Nogo-B. Other than its role in binding Nogo-B, little is known about the biology of NgBR. To elucidate a basic cellular role for NgBR, we performed a yeast two-hybrid screen for interacting proteins, using the C-terminal domain as bait, and identified Niemann-Pick type C2 protein (NPC2) as an NgBR-interacting protein. NPC2 protein levels are increased in the presence of NgBR, and NgBR enhances NPC2 protein stability. NgBR localizes primarily to the endoplasmic reticulum (ER) and regulates the stability of nascent NPC2. RNAi-mediated disruption of NgBR or genetic deficiency in NgBR lead to a decrease in NPC2 levels, increased intracellular cholesterol accumulation, and a loss of sterol sensing, all hallmarks of an NPC2 mutation. These data identify NgBR as an NPC2-interacting protein and provide evidence of a role for NgBR in intracellular cholesterol trafficking.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.
The directed movement of phospholipids into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore. Phospholipids are any lipids containing phosphoric acid as a mono- or diester.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.
Dehydrodolichyl diphosphate (DedolPP) synthase catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize DedolPP, a biosynthetic precursor for dolichol which plays an important role as a sugar-carrier lipid in the biosynthesis of glycoprotein in eukaryotic cells. During certain pathological processes like Alzheimer's disease or some neurological disorders, dolichol has been shown to accumulate in human brain. In order to understand the regulatory mechanism of dolichol in eukaryotes, we performed a yeast two-hybrid screen using full length human DedolPP synthase gene [Endo et al. BBA 1625 (2003) 291] as a bait to find some proteins specifically interacting with the enzyme. We identified Niemann-Pick Type C2 protein (NPC2) to show a specific interaction with human DedolPP synthase. This interaction was further confirmed by in vitro co-immunoprecipitation experiment, indicating the possible physiological interaction between NPC2 and DedolPP synthase proteins in human.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus from a virus.
Evidence
1:
Inferred from Expression PatternUniProtKB
Insights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host- and virus-mediated transcriptional responses. An oligo-based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real-time reverse transcription polymerase chain reaction (RT-PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two-dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis-associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase-like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real-time RT-PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.
Protein which participates in the biochemical reactions where cholesterol is involved, including transport. Cholesterol is the major sterol of higher animals and an important component of cell membranes, especially of the plasma membrane.
Protein involved in the biochemical reactions of lipids. Lipids are a diverse class of compounds which are insoluble in water but soluble in organic solvents. They include fats, oils, triacylglycerols, fatty acids, glycolipids, phospholipids and steroids.
Protein involved in the biochemical reactions of steroids. Steroids are a large group of complex tetracyclic lipids that consist of a 17- carbon-ring system. Examples are bile acids, sterols, various hormones and saponins.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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