title: A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.
abstract: Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.
authors.affiliation: Laboratory for Membrane Trafficking, VIB Center for Brain &Disease Research, KU Leuven, Department of Neurosciences, Leuven, Belgium.|Department of Life Science Technology, imec, Leuven, Belgium.|VIB Medical Biotechnology Center &Department of Biochemistry, UGent, Ghent, Belgium.
authors.forename: Arun Kumar
authors.initials: AK
authors.lastname: Tharkeshwar
authors.affiliation: Department of Life Science Technology, imec, Leuven, Belgium.|Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Jesse
authors.initials: J
authors.lastname: Trekker
authors.affiliation: Laboratory for Membrane Trafficking, VIB Center for Brain &Disease Research, KU Leuven, Department of Neurosciences, Leuven, Belgium.
authors.forename: Wendy
authors.initials: W
authors.lastname: Vermeire
authors.affiliation: VIB Medical Biotechnology Center &Department of Biochemistry, UGent, Ghent, Belgium.
authors.forename: Jarne
authors.initials: J
authors.lastname: Pauwels
authors.affiliation: Laboratory for Membrane Trafficking, VIB Center for Brain &Disease Research, KU Leuven, Department of Neurosciences, Leuven, Belgium.
authors.forename: Ragna
authors.initials: R
authors.lastname: Sannerud
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Priestman
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, United Kingdom.
authors.forename: Danielle
authors.initials: D
authors.lastname: Te Vruchte
authors.affiliation: VIB Bio-imaging core VIB Center for Brain &Disease Research, Leuven, Belgium.
authors.forename: Katlijn
authors.initials: K
authors.lastname: Vints
authors.affiliation: VIB Bio-imaging core VIB Center for Brain &Disease Research, Leuven, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Baatsen
authors.affiliation: Laboratory for Membrane Trafficking, VIB Center for Brain &Disease Research, KU Leuven, Department of Neurosciences, Leuven, Belgium.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Decuypere
authors.affiliation: Laboratory for Membrane Trafficking, VIB Center for Brain &Disease Research, KU Leuven, Department of Neurosciences, Leuven, Belgium.
authors.forename: Huiqi
authors.initials: H
authors.lastname: Lu
authors.affiliation: Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: Shaun
authors.initials: S
authors.lastname: Martin
authors.affiliation: Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Vangheluwe
authors.affiliation: Laboratory of Lipid Metabolism and Cancer, Department of Oncology, KU Leuven, Leuven, Belgium.
authors.forename: Johannes V
authors.initials: JV
authors.lastname: Swinnen
authors.affiliation: Department of Life Science Technology, imec, Leuven, Belgium.|Department of Physics, Solid State Physics and Magnetism, KU Leuven, Leuven, Belgium.
authors.forename: Liesbet
authors.initials: L
authors.lastname: Lagae
authors.affiliation: VIB Medical Biotechnology Center &Department of Biochemistry, UGent, Ghent, Belgium.|Proteomics Expertise Center, VIB Medical Biotechnology Center, Ghent, Belgium.
authors.forename: Francis
authors.initials: F
authors.lastname: Impens
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, United Kingdom.
authors.forename: Frances M
authors.initials: FM
authors.lastname: Platt
authors.affiliation: VIB Medical Biotechnology Center &Department of Biochemistry, UGent, Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.affiliation: Laboratory for Membrane Trafficking, VIB Center for Brain &Disease Research, KU Leuven, Department of Neurosciences, Leuven, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Annaert
mesh_terms: D000071182:Autophagosomes / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry; D011992:Endosomes / Q000378:metabolism; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D050356:Lipid Metabolism; D008247:Lysosomes / Q000378:metabolism / Q000473:pathology*; D058185:Magnetite Nanoparticles / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000172:deficiency* / Q000378:metabolism; D053758:Nanoparticles / Q000648:ultrastructure; D000077296:Niemann-Pick C1 Protein; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D013261:Sterols / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002352:Carrier Proteins; D003911:Dextrans; D047908:Intracellular Signaling Peptides and Proteins; D058185:Magnetite Nanoparticles; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D020543:Proteome; D013261:Sterols; C065507:ferumoxides