The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters

• Published in: The Journal of biological chemistry Vol. 290; no. 40; pp. 24604 - 24613
• Main Authors: Aleidi, Shereen M., Howe, Vicky, Sharpe, Laura J., Yang, Alryel, Rao, Geetha, Brown, Andrew J., Gelissen, Ingrid C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.10.2015; American Society for Biochemistry and Molecular Biology
• Subjects: ABC transporter; Animals; ATP Binding Cassette Transporter, Subfamily G; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters - metabolism; Biological Transport; Cell Line; CHO Cells; Cholesterol - chemistry; cholesterol regulation; Chromatography, Liquid; Cricetulus; E3 ubiquitin ligase; Endosomal Sorting Complexes Required for Transport - metabolism; Gene Expression Regulation; Gene Silencing; Humans; Lipids; Lipids - chemistry; Macrophages - metabolism; Mass Spectrometry; membrane lipid; Nedd4 Ubiquitin Protein Ligases; post-transcriptional regulation; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Protein Transport; RNA, Small Interfering - metabolism; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases - metabolism; E3 ubiquitin ligase; cholesterol regulation; ABC transporter; membrane lipid; post-transcriptional regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.675579

The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity. Background: The ABCG1 lipid transporter is regulated via protein ubiquitination. Results: We identify two E3 ligases that regulate the protein stability and activity of ABCG1 and ABCG4. Conclusion: The ligases, HUWE1 and NEDD4-1, are involved in the regulation of cholesterol export from cells. Significance: Understanding the fine tuning of cholesterol homeostasis will help to understand how dysregulation can cause disease.