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.

Keywords: ABCB1; Alzheimer’s disease; Nedd4; P-glycoprotein; amyloid protein; blood-brain barrier; ubiquitin ligase.

Figure 1

(A) Representative western analysis of protein lysates prepared from isolated brain capillaries from matched wild-type and hAPP mice at age 3 months, and (B) capillaries of wild-type mice before and after challenge with 100nM human Aβ40. (C) Purification profile by metal affinity chromatography of human ABCB1. Lanes 1, membrane fraction (100μg of protein or 0.25% of volume); 2, solubilised membrane fraction (0.25%); 3, column flow-through (0.25%); 4-13, washes from 20 mM to 200 mM imidazole in 20 mM increments (each 4% of volume); 14, EDTA 50 mM (4% of volume). (D) Western analysis of purified ABCB1 before and after ubiquitination. NEDD4-1 reaction mixture in the absence (lane 1) and presence (lane 2) of methylated ubiquitin.

Figure 2

Cartoon representation of two conformations of ABCB1. (A) Homology model based on Sav1866 (pdb 2HYD) from S. aureus. (B) Model of mouse Abcb1a (pbd 35GU). In each case the domains are coloured from the amino terminus as follows TMD1, red; NBD1, orange; TMD2, blue; NBD2, cyan. The side chains of the putative NEDD4-1 binding motif (PxY) are shown as magenta spheres (P996 and Y998). The ubiquitinated lysines are shown as green spheres. The linker region linking NBD1 to TMD2 could not be modelled in either conformation therefore the precise position of K685 is not known but must be close to the first resolved residue in the amino-terminal region of TMD2, which in the closed conformation is W698, and in the open conformation is equivalent to L688 of human ABCB1. The C-alphas of W698 and the equivalent of L688 are shown as yellow spheres in the blue domain.

Figure 3

Flow cytometric analysis of ABCB1 in the plasma membrane of Flp-In-ABCB1-12His cells. Left dotplot, cells were gated for normal size and granularity (the same gate was used for populations); Middle dotplot, two colour analysis of mock-transfected cells; Right dotplot, two colour analysis of cells transfected transiently with pcDNA3-NEDD4-1-mCherry. Each dot represents a single cell and the density of cells is colour coded from blue (few cells) to red (many cells). The numbers represent the percentage of cells in a given gate, and the coloured letters define the analytical gates used to plot the histogram. The histogram shows ABCB1 expression in NEDD4-1-expressing (orange and red) and non-expressing cells (blue and green). Histogram colour coding is consistent with the lettering in the dotplots.