Identification of Surface Residues on Niemann-Pick C2 Essential for Hydrophobic Handoff of Cholesterol to NPC1 in Lysosomes

Wang, Michael L.; Motamed, Massoud; Infante, Rodney E.; Abi-Mosleh, Lina; Kwon, Ho-Keun; Brown, Michael S.; Goldstein, Joseph L.

doi:10.1016/j.cmet.2010.05.016

Cited by 204 publications

(244 citation statements)

References 19 publications

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“…In summary, a model has been proposed for the transfer of cholesterol from NPC2 to NPC1 proteins (6,14,15). The biochemical findings reported here support the proposed directionality of this transfer process and provide human genetic evidence regarding the importance of the interaction between NPC1 and NPC2 proteins to accomplish cholesterol delivery from lysosomes into the cytoplasm.…”

Section: Discussionsupporting

confidence: 64%

“…Wang et al (15) also identified NPC1 residues L175/L176 and E191/Y192 as being critical for receipt of cholesterol from NPC2. Moreover, when introduced into cells, the L175Q/L176Q mutant NPC1 protein cannot restore egress of cholesterol from lysosomes (15). This observation is consistent with a requirement for interaction between NPC1 and NPC2 proteins as part of the normal cholesterol export process.…”

supporting

confidence: 71%

“…This observation is consistent with a requirement for interaction between NPC1 and NPC2 proteins as part of the normal cholesterol export process. Despite strong genetic and biochemical indications for interactions between NPC1 and NPC2 proteins, direct binding has not been demonstrated to date; moreover, gel filtration and surface plasmon resonance failed to detect interactions between NPC1 N-terminal domain and NPC2 protein (15). We show here that NPC1's second lumenal domain binds NPC2 in a cholesteroldependent manner, a process that may facilitate directional transfer of cholesterol from NPC2 onto NPC1's N-terminal domain.…”

mentioning

confidence: 85%

“…Specific features of the transfer reaction and the NPC1 N-terminal domain structure have led to the proposal that NPC1 exists in a closed conformation that can be opened by interaction with NPC2 to facilitate cholesterol receipt (6,14). Cholesterol transfer requires a cluster of NPC2 surface residues (79, 81, and 83) that are located immediately adjacent to the sterol-binding pocket (15). Wang et al (15) also identified NPC1 residues L175/L176 and E191/Y192 as being critical for receipt of cholesterol from NPC2.…”

mentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 64%

supporting

confidence: 71%

mentioning

confidence: 85%

mentioning

confidence: 99%

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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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“…no. 134113; Abcam) or 2 μg/mL mouse monoclonal IgG-13G4 against NPC2 (36). Bound antibodies were visualized by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Thermo Scientific) after a 1 h incubation with a 1:5,000 dilution of either donkey anti-mouse or goat anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch).…”

Section: Methodsmentioning

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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Sterol uptake by the NPC system in eukaryotes: a Saccharomyces cerevisiae perspective

et al. 2021

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Sterols are an essential component of membranes in all eukaryotic cells and the precursor of multiple indispensable cellular metabolites. After endocytotic uptake, sterols are integrated into the lysosomal membrane by the Niemann-Pick type C (NPC) system before redistribution to other membranes. The process is driven by two proteins that, together, compose the NPC system: the lysosomal sterol shuttle protein NPC2 and the membrane protein NPC1 (named NCR1 in fungi), which integrates sterols into the lysosomal membrane. The Saccharomyces cerevisiae NPC system provides a compelling model to study the molecular mechanism of sterol integration into membranes and sterol homeostasis. This review summarizes recent advances in the field, and by interpreting available structural data, we propose a unifying conceptual model for sterol loading, transfer and transport by NPC proteins.

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Identification of Surface Residues on Niemann-Pick C2 Essential for Hydrophobic Handoff of Cholesterol to NPC1 in Lysosomes

Cited by 204 publications

References 19 publications

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

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

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

Sterol uptake by the NPC system in eukaryotes: a Saccharomyces cerevisiae perspective

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