title: The E3 ubiquitin ligases, HUWE1 and NEDD4-1, are involved in the post-translational regulation of the ABCG1 and ABCG4 lipid transporters.
abstract: 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.
authors: From the Faculty of Pharmacy, The University of Sydney, Sydney NSW 2006 and.
authors: Shereen M
authors: SM
authors: Aleidi
authors: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney NSW 2052 Australia.
authors: Vicky
authors: V
authors: Howe
authors: From the Faculty of Pharmacy, The University of Sydney, Sydney NSW 2006 and School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney NSW 2052 Australia.
authors: Laura J
authors: LJ
authors: Sharpe
authors: From the Faculty of Pharmacy, The University of Sydney, Sydney NSW 2006 and.
authors: Alryel
authors: A
authors: Yang
authors: From the Faculty of Pharmacy, The University of Sydney, Sydney NSW 2006 and.
authors: Geetha
authors: G
authors: Rao
authors: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney NSW 2052 Australia aj.brown@unsw.edu.au.
authors: Andrew J
authors: AJ
authors: Brown
authors: From the Faculty of Pharmacy, The University of Sydney, Sydney NSW 2006 and ingrid.gelissen@sydney.edu.au.
authors: Ingrid C
authors: IC
authors: Gelissen
mesh_terms: D000070996:ATP Binding Cassette Transporter, Subfamily G; D000070998:ATP Binding Cassette Transporter, Subfamily G, Member 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000737:chemistry; D002853:Chromatography, Liquid; D003412:Cricetulus; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008264:Macrophages / Q000378:metabolism; D013058:Mass Spectrometry; D000075702:Nedd4 Ubiquitin Protein Ligases; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: C419353:ABCG1 protein, human; C439766:ABCG4 protein, human; D000070996:ATP Binding Cassette Transporter, Subfamily G; D000070998:ATP Binding Cassette Transporter, Subfamily G, Member 1; D018528:ATP-Binding Cassette Transporters; D056827:Endosomal Sorting Complexes Required for Transport; D008055:Lipids; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D002784:Cholesterol; C502830:HUWE1 protein, human; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: ABC transporter; E3 ubiquitin ligase; cholesterol regulation; membrane lipid; post-transcriptional regulation