The Experts below are selected from a list of 44313 Experts worldwide ranked by ideXlab platform
Haruaki Ninomiya - One of the best experts on this subject based on the ideXlab platform.
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Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4
Journal of Cell Science, 2006Co-Authors: Yuki Ohsaki, Joanna P Davies, Marie T. Vanier, Yuko Sugimoto, Michitaka Suzuki, Hiroshi Hosokawa, Tamotsu Yoshimori, Yiannis Ioannou, Kousaku Ohno, Haruaki NinomiyaAbstract:Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(δLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.
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Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4.
Journal of cell science, 2006Co-Authors: Yuki Ohsaki, Yiannis A. Ioannou, Joanna P Davies, Marie T. Vanier, Yuko Sugimoto, Michitaka Suzuki, Hiroshi Hosokawa, Tamotsu Yoshimori, Kousaku Ohno, Haruaki NinomiyaAbstract:Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(deltaLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.
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genotype phenotype relationship of niemann pick disease type c a possible correlation between clinical onset and levels of NPC1 protein in isolated skin fibroblasts
Journal of Medical Genetics, 2000Co-Authors: Toshiyuki Yamamoto, Marie T. Vanier, Haruaki Ninomiya, Gilles Millat, Michika Matsumoto, Yasutoshi Ohta, Eiji Nanba, Yukie Tsutsumi, Kazuhiro Yamakawa, Peter G PentchevAbstract:Editor—Niemann-Pick disease type C (NP-C, MIM 257220) is a fatal autosomal recessive disorder characterised by progressive neurological deterioration and hepatosplenomegaly. NP-C patients can be classified into four major groups according to the onset of neurological symptoms, that is, early infantile, late infantile, juvenile, and adult forms, and the earlier the clinical onset the more quickly progressive are the symptoms and the shorter is the life span.1-4Complementation analysis using cultured skin fibroblasts indicated the presence of at least two subgroups of NP-C, NPC1 (the major subgroup that comprises >90% of NP-C patients) and NPC2 (the minor subgroup).2-4 In 1997, the NPC1 gene ( NPC1 ) (accession No AF002020) that is responsible for the NPC1 subgroup was identified by positional cloning.5 6 The number of NPC1 mutations known to date is not far off 100,7-11 taking into account the accumulated data from seven groups presented in a recent international workshop (International Workshop, The Niemann-Pick C Lesion and the Role of Intracellular Lipid Sorting in Human Disease, Bethesda, USA, October 1999). Because the genomic structure of NPC1 was unknown, initial mutation screening was performed on RT-PCR products or partial genomic amplicons. In our previous study using RT-PCR products, we identified 14 different mutations in 19 alleles from 11 patients, and failed to detect mutations in the remaining three alleles.8 Mutation screening using RT-PCR products has several drawbacks compared with screening using genomic amplicons. For example, mutations that reduce the mRNA stability may escape the screening.12 13 To refine the screening method, we screened a CITB human BAC library (Research Genetics, Huntsville, AL) and isolated a clone 386K10 that contained all the 25 exons of NPC1 and a 2 kb fragment of 5′UTR. Our analysis using 386K10 confirmed the exon/intron boundary sequences reported by Morris et al …
Yiannis Ioannou - One of the best experts on this subject based on the ideXlab platform.
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Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4
Journal of Cell Science, 2006Co-Authors: Yuki Ohsaki, Joanna P Davies, Marie T. Vanier, Yuko Sugimoto, Michitaka Suzuki, Hiroshi Hosokawa, Tamotsu Yoshimori, Yiannis Ioannou, Kousaku Ohno, Haruaki NinomiyaAbstract:Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(δLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.
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NPC1 late endosomes contain elevated levels of non esterified free fatty acids and an abnormally glycosylated form of the npc2 protein
Biochemical Journal, 2005Co-Authors: Fannie W. Chen, Ronald E Gordon, Yiannis IoannouAbstract:NPC (Niemann–Pick type C) disease is a rare lipidosis characterized by the accumulation of LDL (low-density lipoprotein)-derived non-esterified cholesterol in the E/L (endosomal/lysosomal) system. The gene products that are responsible for the two NPC complementation groups are distinct and dissimilar, yet their cellular and disease phenotypes are virtually indistinguishable. To investigate the relationship between NPC1 and NPC2 and their potential role in NPC disease pathogenesis, we have developed a method for the rapid and efficient isolation of late endocytic vesicles from mouse liver by magnetic chromatography. Late endosomes from Wt (wild-type) and NPC1 mice were found to differ not only in their cholesterol and sphingomyelin content, as expected, but also in their non-esterified (‘free’) fatty acid content, with NPC1 vesicles showing an approx. 7-fold increase in non-esterified fatty acid levels compared with Wt vesicles. Furthermore, we show that the NPC2 protein is in an incompletely deglycosylated form in NPC1 late endosomes by a mechanism that is specific to the NPC2 protein and not a global aberration of protein glycosylation/deglycosylation or trafficking, since NPC2 secreted from NPC1 cells is indistinguishable from that secreted from Wt cells. Also, a greater proportion of the normally soluble cellular NPC2 protein partitions with detergent-insoluble late endosomal internal membrane domains in NPC1 vesicles. In addition, we show that, although a small amount of the NPC2 protein associates with these membranes in Wt vesicles, this localization becomes much more pronounced in NPC1 vesicles. These results suggest that the function of the NPC2 protein may be compromised as well in NPC1 endosomes, which might explain the paradoxical phenotypic similarities of the two NPC disease complementation groups.
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Guilty until proven innocent: the case of NPC1 and cholesterol.
Trends in biochemical sciences, 2005Co-Authors: Yiannis IoannouAbstract:Cholesterol accumulation in the endosomes and lysosomes of Niemann-Pick C (NPC) cells is considered to be the hallmark of this disorder, so the main focus of NPC research has revolved around cholesterol and its role in disease pathogenesis. However, recent data indicate that cholesterol is not the primary culprit in this human lipidosis. I propose a new hypothesis for the potential action or function of the NPC1 protein in the endosome. In this context, the relationship of NPC2 and NPC1 is also discussed.
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targeting of NPC1 to late endosomes involves multiple signals including one residing within the putative sterol sensing domain
Journal of Biological Chemistry, 2004Co-Authors: Catherine Scott, Joanna P Davies, Maureen E Higgins, Yiannis IoannouAbstract:The NPC1 protein is a multipass transmembrane protein whose deficiency causes the autosomal recessive lipid storage disorder Niemann-Pick type C1. NPC1 localizes predominantly to late endosomes and has a dileucine motif located within a small cytoplasmic tail thought to target the protein to this location. Our data have suggested previously that the protein can reach its correct location in the absence of its cytoplasmic tail, suggesting that other signals contribute to NPC1 targeting. By using various FLAG-tagged and CD32-NPC1 chimeric fusion constructs, we show that multiple signals are responsible for the trafficking of NPC1 to the endosomal compartment, including the dileucine motif and a previously unidentified signal residing within the putative sterol-sensing domain transmembrane domain 3. Neither region alone was capable of directing heterologous CD32 fusions to late endosomes exclusively via the trans-Golgi network to the late endosome route taken by wild-type NPC1; transmembrane domain 3 was unable to maintain CD32 in late endosomes, indicating that two or more signals work in concert to target and retain NPC1 in this compartment. In addition we confirm that the tail dileucine motif is not essential for NPC1 targeting to late endosomes, and we discuss the implications of this finding along with the previously unappreciated role for transmembrane domain 3 in NPC1 localization and function.
Marie T. Vanier - One of the best experts on this subject based on the ideXlab platform.
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Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4.
Journal of cell science, 2006Co-Authors: Yuki Ohsaki, Yiannis A. Ioannou, Joanna P Davies, Marie T. Vanier, Yuko Sugimoto, Michitaka Suzuki, Hiroshi Hosokawa, Tamotsu Yoshimori, Kousaku Ohno, Haruaki NinomiyaAbstract:Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(deltaLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.
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Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4
Journal of Cell Science, 2006Co-Authors: Yuki Ohsaki, Joanna P Davies, Marie T. Vanier, Yuko Sugimoto, Michitaka Suzuki, Hiroshi Hosokawa, Tamotsu Yoshimori, Yiannis Ioannou, Kousaku Ohno, Haruaki NinomiyaAbstract:Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(δLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.
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niemann pick c disease use of denaturing high performance liquid chromatography for the detection of NPC1 and npc2 genetic variations and impact on management of patients and families
Molecular Genetics and Metabolism, 2005Co-Authors: Gilles Millat, Nathalie Bailo, Sabine Molinero, Celine Rodriguez, Karim Chikh, Marie T. VanierAbstract:Abstract Niemann–Pick disease type C (NPC), a neurovisceral disorder characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. While the corresponding proteins appear essential for proper cellular cholesterol trafficking, their precise function and relationship are still unclear. Mutational analysis of patients, useful for the study of structure/function relationships, is especially valuable for proper management of affected families. Correlations have been found between genotypes and the severity of the neurological outcome of the patients, and molecular genetics constitutes the optimal approach for prenatal diagnosis. However, mutation detection in NPC disease is a challenge. The NPC1 gene, affected in >95% of the families, is large in size (≈50 kb), and the already known disease-causing mutations and numerous polymorphisms are scattered over 25 exons. Furthermore, detection of NPC2 patients by complex genetic complementation tests is unpractical. In the present study, we describe a rapid and reliable strategy for detecting NPC genetic variations using DHPLC analysis. Conditions of analysis were optimized for all the NPC1 and NPC2 30 exons and validated using 38 previously genotyped patients. These conditions were then applied to screen a panel of 35 genetically uncharacterized, unrelated NPC patients. Pathogenic mutations were identified in 68/70 alleles. Among the mutations identified, 29 were novel, including two of the NPC2 gene. We conclude that DHPLC is a rapid, low-cost, highly accurate, and efficient technique for the detection of NPC genetic variants.
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Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: David E. Sleat, Jennifer A. Wiseman, Mukarram El-banna, Sandy Price, Lucie Verot, Michael M. Shen, G. Stephen Tint, Marie T. Vanier, Steven U. Walkley, Peter LobelAbstract:Niemann–Pick C (NPC) disease is a fatal neurodegenerative disorder characterized by a lysosomal accumulation of cholesterol and other lipids within the cells of patients. Clinically identical forms of NPC disease are caused by defects in either of two different proteins: NPC1, a lysosomal–endosomal transmembrane protein and NPC2, a soluble lysosomal protein with cholesterol binding properties. Although it is clear that NPC1 and NPC2 are required for the egress of lipids from the lysosome, the precise roles of these proteins in this process is unknown. To gain insight into the normal function of NPC2 and to investigate its interactions, if any, with NPC1, we have generated a murine NPC2 hypomorph that expresses 0–4% residual protein in different tissues and have examined its phenotype in the presence and absence of NPC1. The phenotypes of NPC1 and NPC2 single mutants and an NPC1;NPC2 double mutant are similar or identical in terms of disease onset and progression, pathology, neuronal storage, and biochemistry of lipid accumulation. These findings provide genetic evidence that the NPC1 and NPC2 proteins function in concert to facilitate the intracellular transport of lipids from the lysosome to other cellular sites.
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genotype phenotype relationship of niemann pick disease type c a possible correlation between clinical onset and levels of NPC1 protein in isolated skin fibroblasts
Journal of Medical Genetics, 2000Co-Authors: Toshiyuki Yamamoto, Marie T. Vanier, Haruaki Ninomiya, Gilles Millat, Michika Matsumoto, Yasutoshi Ohta, Eiji Nanba, Yukie Tsutsumi, Kazuhiro Yamakawa, Peter G PentchevAbstract:Editor—Niemann-Pick disease type C (NP-C, MIM 257220) is a fatal autosomal recessive disorder characterised by progressive neurological deterioration and hepatosplenomegaly. NP-C patients can be classified into four major groups according to the onset of neurological symptoms, that is, early infantile, late infantile, juvenile, and adult forms, and the earlier the clinical onset the more quickly progressive are the symptoms and the shorter is the life span.1-4Complementation analysis using cultured skin fibroblasts indicated the presence of at least two subgroups of NP-C, NPC1 (the major subgroup that comprises >90% of NP-C patients) and NPC2 (the minor subgroup).2-4 In 1997, the NPC1 gene ( NPC1 ) (accession No AF002020) that is responsible for the NPC1 subgroup was identified by positional cloning.5 6 The number of NPC1 mutations known to date is not far off 100,7-11 taking into account the accumulated data from seven groups presented in a recent international workshop (International Workshop, The Niemann-Pick C Lesion and the Role of Intracellular Lipid Sorting in Human Disease, Bethesda, USA, October 1999). Because the genomic structure of NPC1 was unknown, initial mutation screening was performed on RT-PCR products or partial genomic amplicons. In our previous study using RT-PCR products, we identified 14 different mutations in 19 alleles from 11 patients, and failed to detect mutations in the remaining three alleles.8 Mutation screening using RT-PCR products has several drawbacks compared with screening using genomic amplicons. For example, mutations that reduce the mRNA stability may escape the screening.12 13 To refine the screening method, we screened a CITB human BAC library (Research Genetics, Huntsville, AL) and isolated a clone 386K10 that contained all the 25 exons of NPC1 and a 2 kb fragment of 5′UTR. Our analysis using 386K10 confirmed the exon/intron boundary sequences reported by Morris et al …
Rodney E. Infante - One of the best experts on this subject based on the ideXlab platform.
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Identification of Surface Residues on Niemann-Pick C2 Essential for Hydrophobic Handoff of Cholesterol to NPC1 in Lysosomes
Cell metabolism, 2010Co-Authors: Michael L. Wang, Hyock Joo Kwon, Lina Abi-mosleh, Michael S. Brown, Rodney E. Infante, Massoud Motamed, Joseph L. GoldsteinAbstract:Water-soluble Niemann-Pick C2 (NPC2) and membrane-bound NPC1 are cholesterol-binding lysosomal proteins required for export of lipoprotein-derived cholesterol from lysosomes. The binding site in NPC1 is located in its N-terminal domain (NTD), which projects into the lysosomal lumen. Here we perform alanine-scanning mutagenesis to identify residues in NPC2 that are essential for transfer of cholesterol to NPC1(NTD). Transfer requires three residues that form a patch on the surface of NPC2. We previously identified a patch of residues on the surface of NPC1(NTD) that are required for transfer. We present a model in which these two surface patches on NPC2 and NPC1(NTD) interact, thereby opening an entry pore on NPC1(NTD) and allowing cholesterol to transfer without passing through the water phase. We refer to this transfer as a hydrophobic handoff and hypothesize that this handoff is essential for cholesterol export from lysosomes.
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Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.
Cell, 2009Co-Authors: Hyock Joo Kwon, Lina Abi-mosleh, Michael L. Wang, Johann Deisenhofer, Joseph L. Goldstein, Michael S. Brown, Rodney E. InfanteAbstract:LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3beta-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3beta-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.
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69 npc2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers a potential step in cholesterol egress from lysosomes
Molecular Genetics and Metabolism, 2009Co-Authors: Rodney E. Infante, Hyock Joo Kwon, Michael L. Wang, Michael S. Brown, Arun Radhakrishnan, Joseph L. GoldsteinAbstract:Egress of lipoprotein-derived cholesterol from lysosomes requirestwo lysosomal proteins, polytopic membrane-bound Niemann–PickC1 (NPC1) and soluble Niemann–Pick C2 (NPC2). The reason for thisdual requirement is unknown. Previously, we showed that the solu-ble luminal N-terminal domain (NTD) of NPC1 (amino acids 25–264)binds cholesterol. This NTD is designated NPC1(NTD). We and othersshowed that soluble NPC2 also binds cholesterol. Here, we establishan
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npc2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers a step in cholesterol egress from lysosomes
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Rodney E. Infante, Hyock Joo Kwon, Michael L. Wang, Michael S. Brown, Arun Radhakrishnan, Joseph L. GoldsteinAbstract: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 [3H]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.
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Purified NPC1 Protein II. LOCALIZATION OF STEROL BINDING TO A 240-AMINO ACID SOLUBLE LUMINAL LOOP
The Journal of biological chemistry, 2007Co-Authors: Rodney E. Infante, Lina Abi-mosleh, Michael L. Wang, Joseph L. Goldstein, Arun Radhakrishnan, Lisa N. Kinch, Nick V. Grishin, Michael S. BrownAbstract:Abstract Defects in Niemann-Pick, Type C-1 protein (NPC1) cause cholesterol, sphingolipids, phospholipids, and glycolipids to accumulate in lysosomes of liver, spleen, and brain. In cultured fibroblasts, NPC1 deficiency causes lysosomal retention of lipoprotein-derived cholesterol after uptake by receptor-mediated endocytosis. NPC1 contains 1278 amino acids that form 13 membrane-spanning helices and three large loops that project into the lumen of lysosomes. We showed earlier that NPC1 binds cholesterol and oxysterols. Here we localize the binding site to luminal loop-1, a 240-amino acid domain with 18 cysteines. When produced in cultured cells, luminal loop-1 was secreted as a soluble dimer. This loop bound [3H]cholesterol (Kd, 130 nm) and [3H]25-hydroxycholesterol (25-HC, Kd, 10 nm) with one sterol binding site per dimer. Binding of both sterols was competed by oxysterols (24-, 25-, and 27-HC). Unlabeled cholesterol competed strongly for binding of [3H]cholesterol, but weakly for [3H]25-HC binding. Binding of [3H]cholesterol but not [3H]25-HC was inhibited by detergents. We also studied NPC2, a soluble protein whose deficiency causes a similar disease phenotype. NPC2 bound cholesterol, but not oxysterols. Epicholesterol and cholesteryl sulfate competed for [3H]cholesterol binding to NPC2, but not NPC1. Glutamine 79 in luminal loop-1 of NPC-1 is important for sterol binding; a Q79A mutation abolished binding of [3H]cholesterol and [3H]25-HC to full-length NPC1. Nevertheless, the Q79A mutant restored cholesterol transport to NPC1-deficient Chinese hamster ovary cells. Thus, the sterol binding site on luminal loop-1 is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities.
Joseph L. Goldstein - One of the best experts on this subject based on the ideXlab platform.
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Amino acid substitution in NPC1 that abolishes cholesterol binding reproduces phenotype of complete NPC1 deficiency in mice
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Xuefen Xie, Joseph L. Goldstein, Michael S. Brown, John M. Shelton, James A. Richardson, Guosheng LiangAbstract:Substitution mutations in adjacent amino acids of the N-terminal domain of NPC1, a lysosomal membrane protein, abolish its cholesterol binding activity and impair its ability to export cholesterol from lysosomes of cultured cells lacking NPC1 [Kwon HJ, et al. (2009) Cell 137:1213–1224]. Here, we show that the same two mutations (proline-202 and phenylalanine-203, both changed to alanine) reproduce the phenotype of complete NPC1 deficiency when knocked into the mouse NPC1 gene by homologous recombination. Homozygous NPC1pf/pf mice exhibited neurodegeneration beginning at day 49 and died at a median age of 84 d, as previously reported for mice that lack NPC1. Liver and other organs of the NPC1pf/pf mice accumulated excess cholesterol in lysosomes. In liver, mRNAs encoding several lysosomal proteins were elevated, including NPC1 and NPC2 and several digestive enzymes (acid lipase, β-glucuronidase, and cathepsins B and D). Weekly treatment with hydroxypropyl-β-cyclodextrin (HPCD) beginning at 7 wk reduced hepatic cholesterol accumulation and diminished the lysosomal mRNAs. We conclude that the cholesterol binding site in the N-terminal domain of NPC1 is essential for cholesterol export from lysosomes in living animals as it is in cultured cells. The HPCD-mediated reduction of excess lysosomal enzymes may contribute to the ability of this drug to delay the progression of NPC disease in mice.
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Identification of Surface Residues on Niemann-Pick C2 Essential for Hydrophobic Handoff of Cholesterol to NPC1 in Lysosomes
Cell metabolism, 2010Co-Authors: Michael L. Wang, Hyock Joo Kwon, Lina Abi-mosleh, Michael S. Brown, Rodney E. Infante, Massoud Motamed, Joseph L. GoldsteinAbstract:Water-soluble Niemann-Pick C2 (NPC2) and membrane-bound NPC1 are cholesterol-binding lysosomal proteins required for export of lipoprotein-derived cholesterol from lysosomes. The binding site in NPC1 is located in its N-terminal domain (NTD), which projects into the lysosomal lumen. Here we perform alanine-scanning mutagenesis to identify residues in NPC2 that are essential for transfer of cholesterol to NPC1(NTD). Transfer requires three residues that form a patch on the surface of NPC2. We previously identified a patch of residues on the surface of NPC1(NTD) that are required for transfer. We present a model in which these two surface patches on NPC2 and NPC1(NTD) interact, thereby opening an entry pore on NPC1(NTD) and allowing cholesterol to transfer without passing through the water phase. We refer to this transfer as a hydrophobic handoff and hypothesize that this handoff is essential for cholesterol export from lysosomes.
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Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.
Cell, 2009Co-Authors: Hyock Joo Kwon, Lina Abi-mosleh, Michael L. Wang, Johann Deisenhofer, Joseph L. Goldstein, Michael S. Brown, Rodney E. InfanteAbstract:LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3beta-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3beta-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.
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69 npc2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers a potential step in cholesterol egress from lysosomes
Molecular Genetics and Metabolism, 2009Co-Authors: Rodney E. Infante, Hyock Joo Kwon, Michael L. Wang, Michael S. Brown, Arun Radhakrishnan, Joseph L. GoldsteinAbstract:Egress of lipoprotein-derived cholesterol from lysosomes requirestwo lysosomal proteins, polytopic membrane-bound Niemann–PickC1 (NPC1) and soluble Niemann–Pick C2 (NPC2). The reason for thisdual requirement is unknown. Previously, we showed that the solu-ble luminal N-terminal domain (NTD) of NPC1 (amino acids 25–264)binds cholesterol. This NTD is designated NPC1(NTD). We and othersshowed that soluble NPC2 also binds cholesterol. Here, we establishan
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npc2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers a step in cholesterol egress from lysosomes
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Rodney E. Infante, Hyock Joo Kwon, Michael L. Wang, Michael S. Brown, Arun Radhakrishnan, Joseph L. GoldsteinAbstract: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 [3H]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.
