The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters
The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters
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DOI：
10.1074/jbc.m115.675579
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发表时间：
2015-10-02
影响因子：
4.8
通讯作者：
Gelissen, Ingrid C.
中科院分区：
文献类型：
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作者：
Aleidi, Shereen M.;Howe, Vicky;Gelissen, Ingrid C.
三磷酸腺苷结合盒转运体Abcg1在细胞内胆固醇稳态中起着重要的作用，调节失调与许多高负担疾病有关。以往的研究报道，Abcg1通过泛素蛋白酶体系统被泛素化和降解。然而，到目前为止，其分子机制，包括任何限速泛素化酶或E3连接酶的身份，都是未知的。我们用液相色谱质谱鉴定了与Abcg1相关的两个Hect结构域E3连接酶，命名为HUWE1(Hect、UBA和WWE结构域包含1，E3泛素蛋白连接酶)和NEDD4-1(神经前体细胞表达发育下调基因4)，后者是NEDD4泛素连接酶家族的创始成员。在过量表达人Abcg1的细胞中沉默HUWE1和NEDD4-1显著增加Abcg1单体和二聚体蛋白的水平，而ABCA1蛋白的表达不受影响。此外，连接酶沉默增加了Abcg1介导的胆固醇向高密度脂蛋白的输出，在过度表达转运蛋白的细胞中以及在THP-1巨噬细胞中。反过来，两种连接酶的过度表达导致Abcg1单体和二聚体的蛋白质水平显著降低。与Abcg1一样，在HUWE1和NEDD4-1过度表达的细胞中，ABCG4蛋白水平和胆固醇输出活性在沉默后显著增加。综上所述，我们首次确定了两个E3连接酶，它们是翻译后调节Abcg1和ABCG4蛋白水平和细胞胆固醇输出活性的基础酶。
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.