ARTICLE DOI: 10.1038/s41467-018-07185-y OPEN Proteome-wide analysis of USP14 substrates revealed its role in hepatosteatosis via stabilization of FASN Bin Liu1,2,3, Shangwen Jiang3,4, Min Li1, Xuelian Xiong1, Mingrui Zhu3,4, Duanzhuo Li2, Lei Zhao3, Lili Qian3,4, Linhui Zhai3, Jing Li 5, Han Lu6, Shengnan Sun3, Jiandie Lin7, Yan Lu 1, Xiaoying Li1 & Minjia Tan 3,4 fi 1234567890():,; Ubiquitin-speci c protease 14 (USP14) is one of the major proteasome-associated deubi- quitinating enzymes critical for proteome homeostasis. However, substrates of USP14 remain largely unknown, hindering the understanding of its functional roles. Here we conduct a comprehensive proteome, ubiquitinome and interactome analysis for USP14 substrate screening. Bioinformatics analysis reveals broad new potential roles of USP14, especially in lipid and carbohydrate metabolism. Among the potential substrates identified, we show that fatty acid synthase (FASN), a key enzyme involved in hepatic lipogenesis, is a bona fide substrate of USP14. USP14 directly interacts with and increases FASN stability. As a result, overexpression of USP14 promotes liver triglyceride accumulation in C57BL/6 mice, whereas genetic ablation or pharmacological inhibition of USP14 ameliorates hepatosteatosis, hyperglycemia and insulin resistance in obese mice. In conclusion, our findings reveal for the first time an indispensable role of USP14 in hepatosteatosis through FASN stabilization. 1 Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan Institute of Metabolic Diseases, Key Laboratory of Metabolism and Molecular Medicine, the Ministry of Education, Fudan University, Shanghai 200032, PR China. 2 Hubei Key Laboratory for Kidney Disease Pathogenesis and Intervention, Hubei Polytechnic University School of Medicine, Huangshi, Hubei 435003, PR China. 3 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China. 4 University of Chinese Academy of Sciences, Beijing, PR China. 5 Department of Bioinformatics and Biostatistics, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, PR China. 6 Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025, PR China. 7 Life Sciences Institute and Department of Cell and Developmental Biology, University of Michigan Medical Center, Ann Arbor, MI, USA. These authors contributed equally: Bin Liu, Shangwen Jiang, Min Li. Correspondence and requests for materials should be addressed to Y.L. (email: [email protected]) or to X.L. (email: [email protected]) or to M.T. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4770 | DOI: 10.1038/s41467-018-07185-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07185-y he ubiquitin-proteasome system (UPS) controls intracel- mediated deubiquitination and stabilization of FASN promotes Tlular protein degradation through a series of steps, TG accumulation, revealing a novel mechanism of hepatosteatosis including substrate recognition, ubiquitin conjugation, and pathogenesis. ubiquitinated substrates degradation by proteasomes1. In mam- mals, E3 ubiquitin ligases and deubiquitylating enzymes (DUBs) Results play central roles in protein degradation and turnover through 2 Upregulation of USP14 in livers of obese mice. We previously protein ubiquitination and deubiquitination . DUBs catalyze the carried out Affymetrix arrays using livers of mice fed a high-fat removal of ubiquitins from their target proteins and render diet (HFD) or a normal chow diet (ND)25,26, and by reanalysis of ubiquitin homeostasis a highly dynamic process. There are 79 these data we found that the mRNA levels of several USP family DUBs encoded by the mammalian genome3. Among them, the fi fi members were signi cantly upregulated (p < 0.05 by Student's t- ubiquitin-speci c proteases (USPs) are the largest family of test) in HFD mouse livers (Supplementary Fig. 1). Among these DUBs, which participates in diverse cellular processes, including USP members, we found that the expression of USP14 was cell cycle progression, cell proliferation and differentiation, increased in HFD-fed mice (Supplementary Fig. 1). The upre- transcriptional regulation, and modulation of plasma membrane fi 4 gulation of USP14 mRNA level was further con rmed by quan- receptors . titative real-time PCR (Fig. 1a). In agreement, USP14 protein USP14 is the only USP family of DUBs that reversibly associate 5 expression was increased in the livers of mice subjected to HFD with the proteasomal 19S regulatory particle . USP14 serves as a (Fig. 1b). quality control component to rescue proteins from degradation fi 6 Next, livers from leptin receptor-de cient mice (db/db) and by dissembling the ubiquitin chain from its substrate distal tip . NAFLD patients were examined. As a result, USP14 expression Many studies have shown that USP14 plays critical roles in cel- was higher in the livers of db/db mice and NAFLD patients, lular signaling, neurological functions, and tumorigenesis. USP14 compared with the corresponding non-steatotic controls is a mediator of Dishevelled (Dvl) deubiquitination for Wnt (Fig. 1c–f). Importantly, mRNA levels of USP14 correlated well signaling pathway regulation7, and inhibit nuclear factor (NF)-κB 8 with hepatic TG content (Fig. 1g). Together, our results signaling through NLRC5 deubiquitination . In neurological demonstrate that upregulation of USP14 is a conserved feature systems, USP14 was reported to regulate long-term memory of hepatosteatosis, suggesting that it may have an important role formation, while alteration of USP14 contributes to loss-of- in the progression of NAFLD. mobility and early postnatal lethality in ataxia (axJ) mice9,10. USP14 was overexpressed in many cancers and promoted tumor fi cell proliferation through enhancing β-catenin accumulation and Quantitative proteomic pro ling of USP14 regulated proteins. inhibiting Bcl-xl-mediated cell apoptosis11. Although these stu- To system-wide identify the degradation substrates of USP14, we dies have demonstrated the importance of USP14 in cell phy- carried out mass spectrometry-based stable isotope labeling with siology and diseases, the question of whether USP14 participates amino acids in cell culture (SILAC) quantitative proteomics in other biological events, especially in energy metabolism, analysis (Fig. 2a). First, we chose HeLa cells as a cell line model, which contains relatively high level of endogenous USP14 and remains largely unexplored. Moreover, the global substrates of fi USP14 are still to be elucidated, representing a major bottleneck successfully constructed USP14 knockdown (KD) cells by speci c toward the functional characterization of USP14 and under- shRNA transfection. Then, the proteome of USP14 KD cells was “ ” 13 13 15 standing of the complexity of proteasome-associated deubiquiti- labeled with heavy ( C6-Lys and C6 N4-Arg) amino acids, “ ” 12 12,13 whereas proteome of control cells was labeled with light ( C6- nation events . 12 13 Lys and C6 N4-Arg) amino acids in cell culture. An equal Non-alcoholic fatty liver disease (NAFLD) is a major type of “ ” “ ” metabolic disorders, which has become a severe public health amount of cell lysates extracted from the light and heavy cells was mixed, digested, fractionated, and analyzed by MS for pro- problem due to its high association with metabolic syndrome and fi fi progression of liver diseases, including non-alcoholic steatohe- teome quanti cation. We identi ed 7647 protein groups in four fi biological replicates including a pair of reverse labeled cells patitis, liver brosis, cirrhosis, and eventually hepatocellular fi fi ’ carcinoma14–16. Aberrant triglycerides (TGs) accumulation in the (Fig. 2b). We de ned signi cantly different (p < 0.05 by Student s liver promoted by obesity, is a hallmark feature of NAFLD14,15. t-test) proteins and used a criterion of 1.2-fold change or greater Hepatic TG content is tightly regulated by de novo lipogenesis between these two groups as differential protein candidates. Subsequently, 108 downregulated proteins and 50 upregulated (DNL), fatty acid uptake, fatty acid oxidation and very low fi density lipoprotein (VLDL) export17. Steatosis develops when the proteins in the USP14 KD group were identi ed (Supplementary amount of TG input is greater than the amount of TG output17. Data 1). For example, the contribution of DNL to total hepatic TG con- tents in normal subjects is small and is much higher in obese Characterization of ubiquitinome in response to USP14 KD. patients18,19. Besides, hepatic overexpression of SREBP-1c or To further characterize the deubiquitination substrates of USP14, ChREBP, two master regulators of DNL, can result in we employed an affinity-based ubiquitinated peptide enrichment hepatosteatosis20,21. Consistently, increased hepatic expression of approach to systematically quantify the change of ubiquitinome several genes involved in DNL were observed in obese human and in USP14 KD cells. As lysine residues modified with di-glycine rodents22–24. However, the molecular mechanisms by which DNL remnant (K-ε-GG) will be derived from ubiquitinated proteins is enhanced in obesity remain poorly understood. after tryptic digestion, we carried out affinity capture of ubiquitin- In this study, by carrying out mass spectrometry-based deep modified peptides using anti-di-glycine remnant pan antibody in proteome, ubiquitinome,