Proteome-wide analysis of USP14 substrates revealed its role in hepatosteatosis via stabilization of

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n.s. 2.0 1.5 1.0 Plasma TG level (mM) 0.5 Liver TG level (mg/g) 10 20 30 40 0 0.0 2.0 1.5 1.0 Plasma TG level (mM) 0.5 0.0 * *** ** ** *** P = 0.062 shFASN Ad-GFP Ad-USP14 WB: USP14 WB: FASN WB: β -actin 250 42 Ad- USP14 Ad- GFP Ad- GFP Ad- USP14 shNC shFASN Fig. 6 Overexpression of USP14 promotes hepatosteatosis. a Overexpression of hepatic USP14 in C57BL/6 mice infected with adenoviral wild-type (WT) USP14, catalytic inactive (CI) USP14, or green uorescent protein (GFP) by western blot (up) and quantitative real-time PCR (bottom) ( n = 6 for each group). The two lanes are two representative biological replicates. Each lane represents the tissue protein sample from one mouse. A representative western blot was shown and with the quanti fi cation plot based on scanning densitometry analysis (right). *** p < 0.001 versus indicated groups (Student s t -test). b , c Liver weight b , hepatic TG contents c in three groups of mice. d Representative hepatic histology by Oil Red O staining from mice overexpressing GFP, CI-USP14, or WT-USP14, scale bar 100 μ m. e Plasma TG levels in three groups of mice. f Representative protein levels of USP14 in mice administrated with adenovirus as indicated. g , h Hepatic and plasma TG contents in four groups of mice. After a 16-h fast, mice were killed at day 10. Two-way ANOVA was used for statistical analysis. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07185-y ARTICLE NATURE COMMUNICATIONS | (2018)9:4770 | DOI: 10.1038/s41467-018-07185-y | 7
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(PPAR α ) play crucial roles in the regulation of genes responsible for hepatic lipids metabolism 36 38 . Therefore, MPH were treated with agonists of these nuclear receptors. As a result, we found that USP14 expression was speci fi cally induced by TO901317, a LXR agonist (Fig. 8 a), whereas agonists of other nuclear receptors, including GW4064, dexamethasone and WY14643, did not affect its expression. Therefore, we speculate that USP14 might be a molecular target of LXR. To test it, we examined the promoter region of USP14 and found that a potential LXR-binding motif exists in the proximal promoter region of USP14 gene (Fig. 8 b). Luciferase reporter assays further showed that LXR increased the transcriptional activity of this promoter, especially in the presence of TO901317. However, mutation of the motif largely attenuated the trans-activated roles of LXR (Fig. 8 c). Chromatin immuno- precipitation (ChIP) assays further showed that TO901317 could facilitate the recruitment of LXR and RNA Pol II to the LXRE region of the USP14 gene (Fig. 8 d). Therefore, we demonstrate that LXR signaling may be an important mechanism leading to upregulation of USP14 in obese livers. Discussion USP14 is a well-known negative regulator for proteasomal activity. Recent studies have shown that USP14 participates in cell signaling transduction via regulating the turnover of its speci fi c substrates, which in turn have important functional consequences in neuroglial diseases and cancers. Despite these effects, the direct substrates and additional roles of USP14 remain largely unknown. Theoretically, a true proteolysis-associated substrate of
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  • Winter '19
  • Robert S Kiss
  • Fatty acid metabolism, Fatty acid synthase, FASN

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