Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding

Gelsthorpe, Mark E.; Baumann, N; Millard, Elizabeth E.; Gale, Sarah E.; Langmade, S. Joshua; Schaffer, Jean E.; Ory, Daniel S.

doi:10.1074/jbc.m708735200

Cited by 152 publications

(257 citation statements)

References 41 publications

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“…Our data suggest that these mutations may render the NPC1 protein unstable and rapidly degraded but they would not completely abolish the NPC1 functionality. These finding are in line with those described by Gelsthorpe et al (Gelsthorpe et al 2008) showing that a twofold overexpression of GFP-tagged NPC1 I1061T in transient transfected npc1-deficient CHO cells was enough to partially restore the cholesterol trafficking. These authors postulated that a percentage of 2-5% of the nascent NPC1 I1061T mutant protein would be able to fold properly, escape the ER quality control and reach the lysosome/LE compartment.…”

Section: Discussionsupporting

confidence: 92%

“…Transient transfection of npc1-deficient CHO cells with GFP-tagged NPC1-I1061T led to lysosomal localization of the mutant protein and functional complementation of the NPC mutant phenotype. Therefore, it has been suggested that therapeutic strategies directed to enhance the amount of mutated protein, such as the use of chemical chaperones, may rescue the pathologic phenotype in patients carrying the p.I1061T mutation (Gelsthorpe et al 2008).…”

Section: Introductionmentioning

confidence: 99%

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Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

et al. 2011

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Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by the lysosomal/ late endosomal (LE) accumulation of unesterified cholesterol and other lipids due to a defect in the intracellular lipid trafficking. About 95% of patients present mutations in the NPC1 gene. Among the 290 mutations reported in the NPC1 gene, about 70% are missense. However, little information is available regarding the impact of missense mutations on NPC1 protein stability and function. In this study, we in vitro characterized the pathogenic effect of 7 NPC1 missense mutations. In all cases, the basal levels of mutant NPC1 expression were reduced with respect to wild type. Treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity, with the proteasome inhibitor MG132 or glycerol 10%, a chemical chaperone known to stabilize misfolded proteins, resulted in a significant increase of NPC1 protein levels in all cell lines, indicating that these mutants are subjected to proteasomal degradation due to protein misfolding The increment of NPC1 mutant protein induced by the proteasome inhibitor was associated with a localization of NPC1 protein within lysosomal/LE compartment. In cell lines carrying mutations p.N1156S, p.L1191F, p.V1165M, and p.I1061T, the increment of NPC1 mutant protein resulted in an improvement of the intracellular trafficking of cholesterol and GM1. These findings showed that it is possible to correct the NPC cellular phenotype by increasing the amount of endogenous NPC1 mutated protein, suggesting that at least some NPC1 mutations might be potentially rescued by small molecules-based chaperone therapy.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

et al. 2011

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“…3A). It is essential to establish the localization of the mutants, as many NPC1 mutations lead to misfolding and ER-associated degradation (25,26). Although correctly localized, NPC1ΔΩ-A blocks cholesterol transport out of late endosomes, as does the NPC1-P691S mutant; NPC1ΔΨ loses 50% activity compared with NPC1-WT (Fig.…”

Section: Resultsmentioning

confidence: 99%

3.3 Å structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

Trinh

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

103

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Niemann-Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann-Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314-1,278), which-in contrast to previous lower resolution structures-features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909-C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD-NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.cholesterol transport | Niemann-Pick type C disease | cysteine-rich domain | sterol-sensing domain | crystal structure

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“…ref. 21). Thus, before concluding that the R518Q mutation was a function-blocking mutation, it was important to verify that the NPC1 R518Q protein is capable of transport to lysosomes.…”

Section: Figurementioning

confidence: 99%

Niemann–Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding

Deffieu

Pfeffer²

2011

Proc. Natl. Acad. Sci. U.S.A.

149

173

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Niemann-Pick type C1 (NPC1) protein is needed for cellular utilization of low-density lipoprotein-derived cholesterol that has been delivered to lysosomes. The protein has 13 transmembrane domains, three large lumenal domains, and a cytoplasmic tail. NPC1's lumenally oriented, N-terminal domain binds cholesterol and has been proposed to receive cholesterol from NPC2 protein as part of the process by which cholesterol is exported from lysosomes into the cytosol. Using surface plasmon resonance and affinity chromatography, we show here that the second lumenal domain of NPC1 binds directly to NPC2 protein. For these experiments, a soluble NPC1 lumenal domain 2 was engineered by replacing adjacent transmembrane domains with antiparallel coiled-coil sequences. Interaction of NPC2 with NPC1 lumenal domain 2 is only detected at acidic pH, conditions that are optimal for cholesterol binding to NPC2 and transfer to NPC1; the pH is also appropriate for the acidic environment where binding would take place. Binding to NPC1 domain 2 requires the presence of cholesterol on NPC2 protein, a finding that supports directional transfer of cholesterol from NPC2 onto NPC1's N-terminal domain. Finally, human disease-causing mutations in NPC1 domain 2 decrease NPC2 binding, suggesting that NPC2 binding is necessary for NPC1 function in humans. These data support a model in which NPC1 domain 2 holds NPC2 in position to facilitate directional cholesterol transfer from NPC2 onto NPC1 protein for export from lysosomes.cholesterol trafficking | Niemann-Pick type C disease A major source of cellular cholesterol is endocytosed as lowdensity lipoprotein, which is delivered to late endosomes and lysosomes where cholesterol is released (1). Within late endosomes and lysosomes, Niemann-Pick type C1 (NPC1) and NPC2 proteins are required for the subsequent delivery of cholesterol to other intracellular compartments (2). NPC1 is a large, 1,254 residue, integral membrane protein that is predicted to span the bilayer 13 times (3, 4); it contains a lumenally oriented, N-terminal cholesterol binding site that interacts with the 3β-hydroxyl end of the cholesterol molecule (5-7). NPC2 is a much smaller, soluble lysosomal protein of 132 amino acid residues (8) that binds cholesterol in an opposite orientation via cholesterol's isooctyl side chain (9-11). The importance of NPC1 and NPC2 for cholesterol and glycosphingolipid homeostasis is demonstrated in Niemann-Pick type C disease: patients carrying homozygous mutations in either of these proteins suffer neurodegeneration and die in childhood due to cholesterol and glycosphingolipid accumulation in the brain, liver, and lungs (12, 13).NPC2 is thought to play an important role in extracting cholesterol from intralysosomal membranes that are rich in cholesterol and bis(monoacylglycerol) phosphate. Transfer of that cholesterol to NPC1 is proposed to facilitate passage of this hydrophobic sterol across the glycocalyx that lines the inner lysosomal membrane.Recent studies using purified NPC2 and a soluble con...

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Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding

Cited by 152 publications

References 41 publications

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

3.3 Å structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

Niemann–Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding

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