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

In this study by carrying out mass spectrometry based

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is enhanced in obesity remain poorly understood. In this study, by carrying out mass spectrometry-based deep proteome, ubiquitinome, and interactome analysis, we system- atically screened the USP14 substrates. This study not only identi fi ed new possible substrates of USP14, but also revealed the broad new cellular pathways that USP14 were highly associated with, especially in lipid and carbohydrate metabolism. Con- sistently, we found that USP14 is increased in obese livers. We further demonstrated that fatty acid synthase (FASN), the term- inal enzyme in DNL, is a speci fi c substrate of USP14. USP14- mediated deubiquitination and stabilization of FASN promotes TG accumulation, revealing a novel mechanism of hepatosteatosis pathogenesis. Results Upregulation of USP14 in livers of obese mice . We previously carried out Affymetrix arrays using livers of mice fed a high-fat diet (HFD) or a normal chow diet (ND) 25 , 26 , and by reanalysis of these data we found that the mRNA levels of several USP family members were signi fi cantly upregulated ( p < 0.05 by Student's t - test) in HFD mouse livers (Supplementary Fig. 1). Among these USP members, we found that the expression of USP14 was increased in HFD-fed mice (Supplementary Fig. 1). The upre- gulation of USP14 mRNA level was further con fi rmed by quan- titative real-time PCR (Fig. 1 a). In agreement, USP14 protein expression was increased in the livers of mice subjected to HFD (Fig. 1 b). Next, livers from leptin receptor-de fi cient mice ( db/db ) and NAFLD patients were examined. As a result, USP14 expression was higher in the livers of db/db mice and NAFLD patients, compared with the corresponding non-steatotic controls (Fig. 1 c f). Importantly, mRNA levels of USP14 correlated well with hepatic TG content (Fig. 1 g). Together, our results demonstrate that upregulation of USP14 is a conserved feature of hepatosteatosis, suggesting that it may have an important role in the progression of NAFLD. Quantitative proteomic pro fi ling of USP14 regulated proteins . To system-wide identify the degradation substrates of USP14, we carried out mass spectrometry-based stable isotope labeling with amino acids in cell culture (SILAC) quantitative proteomics analysis (Fig. 2 a). First, we chose HeLa cells as a cell line model, which contains relatively high level of endogenous USP14 and successfully constructed USP14 knockdown (KD) cells by speci fi c shRNA transfection. Then, the proteome of USP14 KD cells was labeled with heavy ( 13 C 6 -Lys and 13 C 6 15 N 4 -Arg) amino acids, whereas proteome of control cells was labeled with light ( 12 C 6 - Lys and 12 C 6 13 N 4 -Arg) amino acids in cell culture. An equal amount of cell lysates extracted from the light and heavy cells was mixed, digested, fractionated, and analyzed by MS for pro- teome quanti fi cation. We identi fi ed 7647 protein groups in four biological replicates including a pair of reverse labeled cells (Fig. 2 b). We de fi ned signi fi cantly different ( p < 0.05 by Student s t -test) proteins and used a criterion of 1.2-fold change or greater between these two groups as differential protein candidates.
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  • Winter '19
  • Robert S Kiss
  • Fatty acid metabolism, Fatty acid synthase, FASN

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