Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein

Petersen, Daniel C.; Reinholdt, Peter; Szomek, Maria; Hansen, Selina; Poongavanam, Vasanthanathan; Dupont, Alice; Heegaard, Christian W.; Krishnan, Kathiresan; Fujiwara, Hideji; Covey, Douglas F.; Ory, Daniel S.; Kongsted, Jacob; Wüstner, Daniel

doi:10.1016/j.bbamem.2019.183063

Cited by 11 publications

(14 citation statements)

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“…Mammalian NPC2 has been shown to have a binding pocket that is smaller than that of yeast NPC2 and is known to bind multiple sterols. ,− Using a nitrocellulose strip binding assay, interaction of mammalian NPC2 with LBPA, PS, PC, PG, and PA has been recently demonstrated . Similarly, sterol transfer between membranes by mammalian NPC2 is enhanced in the presence of charged lipids, such as PS, PA, and LBPA .…”

Section: Discussionmentioning

confidence: 99%

“…Bovine NPC2 has been shown to bind a variety of sterols besides cholesterol, including cholesterol sulfate and, though with an affinity lower than that for cholesterol, certain oxysterols and, even more weakly, the hydrophobic amine U18666A. ,− Yeast NPC2 has also been shown to bind cholesterol, ergosterol, DHE, and U18666A, but also edelfosine, a phosphatidylcholine-like lysophospholipid, suggesting that its binding spectrum is rather broad . While the overall structures of mammalian and yeast NPC2 are similar, the binding site for yeast NPC2 is significantly larger and more open .…”

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confidence: 99%

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Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein

et al. 2020

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Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of binding of a lipid to yeast NPC2. We find that NPC2 binds fluorescenceand spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole. 56 oxysterols and, even more weakly, the hydrophobic amine 57 U18666A. 3,9−11 Yeast NPC2 has also been shown to bind 58 cholesterol, ergosterol, DHE, and U18666A, but also 59 edelfosine, a phosphatidylcholine-like lysophospholipid, sug-60 gesting that its binding spectrum is rather broad. 8 While the 61 overall structures of mammalian and yeast NPC2 are similar, 62 the binding site for yeast NPC2 is significantly larger and 63 more open. 8 This difference suggests that yeast NPC2 could 64 eventually bind ligands other than mammalian NPC2.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein

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“…Sidechain oxidized cholesterol derivatives, such as 25-or 27-hydroxycholesterol, can form from LDL-derived cholesterol upon export from endo-lysosomes and transport to the ER or mitochondria, respectively ( Axelson and Larsson, 1995 ; Frolov et al, 2003 ). Export of these oxysterols from endo-lysosomes depends on NPC1, and to a lower extent on NPC2 ( Frolov et al, 2003 ; Abi-Mosleh et al, 2009 ; Petersen et al, 2020 ). 25- hydroxycholesterol can activate LXRs to stimulate ABCA1 mediated cholesterol efflux, and this oxysterol is itself efficiently removed from cells by ABCA1 ( Venkateswaran et al, 2000 ; Tam et al, 2006 ).…”

Section: Formation and Efflux Of Oxysterolsmentioning

confidence: 99%

“…The same is probably the case for Wolman disease and cholesteryl ester hydrolase deficiency, which both lead to accumulation of CEs in endo-lysosomes due to defective or absent acid lipase and thereby impaired ABCA1 mediated cholesterol efflux ( Bowden et al, 2011 ). NPC2 binds 25-hydroxycholesterol with a much lower affinity than cholesterol and is probably less important for the export of oxysterols from endo-lysosomes because the water solubility of 25- and 27-hydroxycholesterol is sufficient for spontaneous transfer ( Frolov et al, 2003 ; Petersen et al, 2020 ). Sidechain oxidized cholesterol derivatives move rapidly across membranes and can increase the permeability of lipid membranes to small molecules and ions, which could contribute to lysosome destabilization by oxysterols ( Yuan et al, 2000 ; Kulig et al, 2018 ).…”

Section: Formation and Efflux Of Oxysterolsmentioning

confidence: 99%

“…This analogue named 25-hydroxycholestatrienol can suppress cholesterol synthesis, activate ACAT for sterol esterification, be esterified in cells and stimulate expression of ABCA1 via activation of LXRs ( Iaea et al, 2015 ). Its export from lysosomes depends on NPC1 and to a lower extent on NPC2 ( Iaea et al, 2015 ; Petersen et al, 2020 ). Based on the same design strategy, we have recently developed a novel derivative of 27-hydroxycholesterol, 27-hydroxycholestatrienol (27-OH-CTL), which differs from 27-hydroxycholesterol only by two additional double bonds in the steroid ring system ( Figure 5 ), allowing for spectroscopic analysis and live-cell imaging by ultraviolet-sensitive microscopy, similar to 25-hydroxycholestatrienol ( Szomek et al, 2020 ).…”

Section: Formation and Efflux Of Oxysterolsmentioning

confidence: 99%

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Pathways and Mechanisms of Cellular Cholesterol Efflux—Insight From Imaging

Juhl

Wüstner

2022

Front. Cell Dev. Biol.

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Cholesterol is an essential molecule in cellular membranes, but too much cholesterol can be toxic. Therefore, mammalian cells have developed complex mechanisms to remove excess cholesterol. In this review article, we discuss what is known about such efflux pathways including a discussion of reverse cholesterol transport and formation of high-density lipoprotein, the function of ABC transporters and other sterol efflux proteins, and we highlight their role in human diseases. Attention is paid to the biophysical principles governing efflux of sterols from cells. We also discuss recent evidence for cholesterol efflux by the release of exosomes, microvesicles, and migrasomes. The role of the endo-lysosomal network, lipophagy, and selected lysosomal transporters, such as Niemann Pick type C proteins in cholesterol export from cells is elucidated. Since oxysterols are important regulators of cellular cholesterol efflux, their formation, trafficking, and secretion are described briefly. In addition to discussing results obtained with traditional biochemical methods, focus is on studies that use established and novel bioimaging approaches to obtain insight into cholesterol efflux pathways, including fluorescence and electron microscopy, atomic force microscopy, X-ray tomography as well as mass spectrometry imaging.

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Computational analysis of altered one- and two-photon CD of sterols inside a protein binding pocket

2022

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Niemann Pick C2 (NPC2) is a small glycoprotein involved in cellular trafficking of cholesterol. Its dysfunction causes accumulation of cholesterol in the lysosomal organelles, a hallmark of Niemann Pick type C disease. Therefore, understanding cholesterol transport and the accumulation mechanism that may result is essential to understand the occurrence of this neurodegenerative disease. Cholestatrienol (CTL) is a fluorescent sterol widely used as an alternative to cholesterol because of its improved optical properties in spectroscopy and live-cell imaging. In this paper, the electronic circular dichroism (ECD) and the two-photon circular dichroism (TPCD) of CTL within the NPC2 binding pockets are simulated using response theory employing timedependent density functional theory (TD-DFT). Moreover, an explicit embedding method has been used to include the effect of the protein residues and the water solvent in calculating the spectroscopic properties. In both cases, an alteration of the CD signal is observed.

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Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein

Cited by 11 publications

References 69 publications

Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein

Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein

Pathways and Mechanisms of Cellular Cholesterol Efflux—Insight From Imaging

Computational analysis of altered one- and two-photon CD of sterols inside a protein binding pocket

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