title: The multidrug resistance pump ABCB1 is a substrate for the ubiquitin ligase NEDD4-1.
abstract: The ATP Binding Cassette transporter ABCB1 can export the neurotoxic peptide β-amyloid from endothelial cells that line the blood-brain barrier (BBB). This has the potential to lower cerebral levels of β-amyloid, but ABCB1 expression in the BBB appears to be progressively reduced in patients with Alzheimer's disease. The surface density of many membrane proteins is regulated by ubiquitination catalyzed by ubiquitin E3 ligases. In brain capillaries of mice challenged with β-amyloid ex vivo, we show that the level of the ubiquitin ligase Nedd4 increases concomitant with reduction in Abcb1. In vitro we show that human ABCB1 is a substrate for human NEDD4-1 ligase. Recombinant ABCB1 was purified from Sf21 insect cells and incubated with recombinant NEDD4-1 purified from Escherichia coli. The treated ABCB1 had reduced mobility on SDS-PAGE, and mass spectrometry identified eight lysine residues, K271, K272, K575, K685, K877, K885, K887 and K1062 that were ubiquitinated by NEDD4-1. Molecular modelling showed that all of the residues are exposed on the surface of the intracellular domains of ABCB1. K877, K885 and K887 in particular, are located in the intracellular loop of transmembrane helix 10 (TMH10) in close proximity, in the tertiary fold, to a putative NEDD4-1 binding site in the intracellular helix extending from TMH12 (PxY motif, residues 996-998). Transient expression of NEDD4-1 in HEK293 Flp-In cells stably expressing ABCB1 was shown to reduce the surface density of the transporter. Together, the data identify this ubiquitin ligase as a potential target for intervention in the pathophysiology of Alzheimer's disease.
authors.affiliation: Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London , London , UK .
authors.forename: Begum G
authors.initials: BG
authors.lastname: Akkaya
authors.forename: Joseph K
authors.initials: JK
authors.lastname: Zolnerciks
authors.forename: Tasha K
authors.initials: TK
authors.lastname: Ritchie
authors.forename: Bjoern
authors.initials: B
authors.lastname: Bauer
authors.forename: Anika M S
authors.initials: AM
authors.lastname: Hartz
authors.forename: James A
authors.initials: JA
authors.lastname: Sullivan
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Linton
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000544:Alzheimer Disease / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001812:Blood-Brain Barrier / Q000378:metabolism; D001921:Brain / Q000098:blood supply / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008822:Mice, Transgenic; D008958:Models, Molecular; D000075702:Nedd4 Ubiquitin Protein Ligases; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D061987:Sf9 Cells; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D016229:Amyloid beta-Peptides; D056827:Endosomal Sorting Complexes Required for Transport; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D008239:Lysine
keywords: ABCB1; Alzheimer’s disease; Nedd4; P-glycoprotein; amyloid protein; blood-brain barrier; ubiquitin ligase