Is Alix a cofactor/adaptor for ABCG1/ABCG4 and NEDD4-1?

Alrosan A1, Sharpe L2, Yang A1, Brown AJ2 and Gelissen IC1

  1. School of Pharmacy, Faculty of Medicine and Health, University of Sydney.
  2. School of Biotechnology and Biomolecular Sciences, University of NSW, Sydney.

Several ABC transporters, including ABCA1, ABCG1 and its close relative ABCG4, are essential regulators of cellular lipid homeostasis. ABCA1 and ABCG1 are expressed ubiquitously and are known in the context of macrophage lipid homeostasis and atherosclerosis, with substrates including cholesterol and phospholipids. ABCG4 is expressed almost exclusively in the brain, with potential substrates including cholesterol, oxysterols and cholesterol synthesis intermediates. ABCG4 has been linked to Alzheimer’s disease due to its additional role in amyloid-beta peptide export from brain cells. The protein half-lives of these transporters are relatively short (in the vicinity of 1-5 hours, dependent upon isoform variations), and it has been suggested that they are highly regulated at the post-translational level. We have previously shown that the HECT-domain E3-ubiquitin ligase, NEDD4-1, regulates the protein stability and activity of ABCG1 and ABCG4. E3-ligases are becoming increasingly of interest as therapeutic targets, hence elucidating these pathways is of interest. NEDD4-1 has a number of other identified targets, and can make use of a cofactor. Using peptide-mass spectrometry, we identified a potential cofactor/adaptor protein that might facilitate the interaction between ABCG1 and NEDD4-1, named Alix (also known as Programmed cell dead 6-interacting protein, AIP1 or ALG-2-interacting protein). Intriguingly, Alix has independently been identified as a possible plasma marker for Alzheimer’s disease. We hypothesised that Alix may facilitate the interaction between NEDD4-1 and the ABC transporters. Silencing of Alix in CHOK1 cells overexpressing ABCG1 increased transporter protein levels. Co-immunoprecipitation experiments were able to pull down all three proteins, i.e ABCG1, Alix and NEDD4-1. Further experiments are currently performed with ABCG4 as well as effects of Alix silencing on transporter activity. These studies will elucidate whether Alix plays a role in regulating the activity of ABCG1 as well as ABCG4.