Abstract
Several ATP-Binding Cassette (ABC) transporters, including ABCG1 and the related ABCG4, are essential regulators of cellular lipid homeostasis. ABCG1 is expressed ubiquitously and is functional in the context of atherosclerosis. However, ABCG4 is expressed almost exclusively in brain and has been linked to Alzheimer’s disease (AD). These transporters are highly regulated post-translationally by E3 ubiquitin ligases, with the ligase NEDD4-1 (Neural precursor cell-expressed developmentally downregulated gene 4) implicated in their protein stability. In this study, we investigated interacting partners of ABCG1 using peptide-mass spectrometry and identified the potential adaptor protein, Alix (apoptosis-linked gene 2-interacting protein X). In this paper, we hypothesized and investigated whether Alix could facilitate the interaction between NEDD4-1 and the ABC transporters. We showed that Alix and NEDD4-1 proteins were co-expressed in several commonly used cell lines. Knockdown of Alix in cells overexpressing ABCG1 or ABCG4 increased transporter protein expression while co-immunoprecipitation experiments showed interaction between NEDD4-1, Alix, and ABC transporters. In summary, we provide evidence that Alix serves as a co-factor for the interaction between the E3-ubiquitin ligase NEDD4-1 and the ABC transporter targets, ABCG1 and ABCG4.
Highlights
Cholesterol is essential for many cellular processes, including the production of cell membranes and synthesis of hormones
We hypothesized that potential adaptor proteins containing proline-rich consensus sequences might mediate the interaction between the ligases that we identified and our ATP-Binding Cassette (ABC) lipid transporter targets. In this follow-up study, we identified one potential adaptor protein, Alix, from a mass-spectrometry screen of proteins interacting with ABCG1
Alix was previously shown to be capable of serving as an adaptor between NEDD4-1 and another target protein [20], so we subsequently investigated whether Alix could facilitate the interaction between ABCG1, ABCG4 and NEDD4-1
Summary
Cholesterol is essential for many cellular processes, including the production of cell membranes and synthesis of hormones. Previous work from our laboratory has identified and investigated the roles of three E3 ligases belonging to the Homologous to E6-AP Carboxyl Terminus (HECT)-domain subfamily (namely HECTD1, HUWE1, and Neural Precursor Cell-expressed Developmentally Downregulated Gene 4 or NEDD4-1) in the regulation of ABC lipid transporter activity and stability [14,15]. Considering that these E3 ligases already have important functions in the regulation of other proteins, any mechanistical understanding of how they interact with ABC transporters could provide a more specific approach to future targeting strategies. Alix was previously shown to be capable of serving as an adaptor between NEDD4-1 and another target protein [20], so we subsequently investigated whether Alix could facilitate the interaction between ABCG1, ABCG4 and NEDD4-1
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