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.

ATP结合盒转运蛋白ABCB1可以从衬有血脑屏障（BBB）的内皮细胞输出神经毒性肽β-淀粉样蛋白。这可能会降低脑内β-淀粉样蛋白的水平，但阿尔茨海默氏病患者的BBB中ABCB1表达似乎逐渐降低。许多膜蛋白的表面密度受泛素E3连接酶催化的泛素化作用调节。在体外受β淀粉样蛋白挑战的小鼠的脑毛细血管中，我们显示泛素连接酶Nedd4的水平随着Abcb1的减少而增加。在体外，我们显示人ABCB1是人NEDD4-1连接酶的底物。从Sf21昆虫细胞中纯化重组ABCB1，并与从大肠杆菌。经处理的ABCB1在SDS-PAGE上的迁移率降低，质谱分析鉴定了8个赖氨酸残基K271，K272，K575，K685，K877，K885，K887和K1062，它们被NEDD4-1泛素化。分子建模表明，所有残基都暴露在ABCB1胞内结构域的表面上。尤其是K877，K885和K887，位于跨膜螺旋10（TMH10）的细胞内环中，在三级折叠中与从TMH12（PxY基序，残基996–998）。NEDD4-1在稳定表达ABCB1的HEK293 Flp-In细胞中的瞬时表达显示降低转运蛋白的表面密度。总之，数据将这种泛素连接酶鉴定为干预阿尔茨海默氏病病理生理的潜在靶标。