Tuesday, September 26, 2023

USP14 governs CYP2E1 to promote nonalcoholic fatty liver disease through deubiquitination and stabilization of HSP90AA1 – Cell Death & Disease

Ethical compliance

All animal experiments were approved by the Northwest A&F University Ethics Committee and followed the principles of the NIH Guide for Care and Use of Laboratory Animals.

Animal models and treatment

Male C57BL/6 J wild-type mice aged 8 weeks were purchased from the Laboratory Animal Center of the Fourth Military Medical University (Xi’an, China). Mice were reared in a temperature-controlled environment (25 ± 1 °C) with a 12-hour light/dark cycle and free access to water and food throughout the experiment. To model steatosis, mice were fed an HFD (protein, 20%; fat, 60%; carbohydrate, 20%) for 22 weeks or an HFHC (protein, 14%; fat, 42%; carbohydrate, 44%; cholesterol, 0.2%) for 16 weeks. Mice fed a normal chow (NC) diet served as controls. The recombinant adenovirus carrying the mouse USP14 promoter was constructed by the Miaoling Plasmid Platform (Wuhan, China) and named pAd-USP14. The primers used in this study are shown in Table S1. To achieve hepatic USP14 overexpression, mice were injected with pAd-USP14 (1 × 109 PFU) via the tail vein after 16 weeks of HFD feeding, once a week for 6 weeks, and pAd-CMV was injected into control mice. USP14-specific shRNAs were also purchased from the Miaoling Plasmid Platform. The sense target sequence for shUSP14 is 5′-CCTGCTTACTTAACTATTCAA-3′. To silence USP14 expression in the liver, mice treated with HFD or HFHC for 16 weeks were injected with shUSP14 and shCtrl adenoviruses once a week for 6 weeks (1 × 108 PFU). All viruses were purified by the cesium chloride method and dialyzed in PBS containing 10% glycerol before injection.

Furthermore, to explore whether the function of USP14 is dependent on CYP2E1, we administered clomethiazole (CMZ, 50 mg/kg) intraperitoneally to HFD-induced USP14-overexpressing mice or pAd-CMV control mice every other day for four weeks [33]. CMZ was purchased from MedChemExpress (New Jersey, USA).

Cell culture and treatment

Primary mouse hepatocytes were isolated and cultured according to the method previously described [34] in our laboratory. Primary hepatocytes, AML12 cells, and HEK-293T cells were cultured in a 5% thermostatic CO2 incubator supplemented with 10% fetal bovine serum (FBS, 10100147, Gibco, CA, USA) and 1% penicillin‒streptomycin (15140122, Gibco). To induce NAFLD models in vitro, hepatocytes were treated with medium containing 0.25 mM palmitic acid (PA, P0500, Sigma‒Aldrich, St. Louis, MO, USA) and 0.5 mM oleic acid (OA, 112-80-1, MedChemExpress) for the indicated times, and bovine serum albumin without fatty acids was used as a control. To enhance or interfere with USP14 expression, AML12 cells were infected with adenovirus produced from the above constructs pAd-USP14, pAd-CMV, shUSP14, and shCtrl, and cells were collected to determine the appropriate metrics.

Mouse experiments

Food intake and body weight data were recorded every other day for all mice. The glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed one week before the end of the test. Briefly, mice were injected intraperitoneally with glucose (1.5 g/kg) or insulin (1 U/kg) after fasting, and then blood glucose was measured at the indicated times (0, 15, 30, 60, and 120 min) after the injection. Serum ALT and AST activities were measured using commercial kits according to the manufacturer’s instructions (BC1555 for ALT and BC1565 for AST, Solarbio, Beijing, China).

Histological analysis

Mouse livers or eWAT were fixed overnight in 4% paraformaldehyde solution and paraffin-embedded. The embedded samples were cut to 5 µm thickness and then stained with hematoxylin and eosin (H&E) to assess cellular morphological changes in the tissue. Next, paraffin-embedded sections of the liver were stained with picrosirius red (PSR) to assess collagen deposition. Alternatively, liver tissue was embedded at the optimal cutting temperature, cut to 8 µm thickness, and stained using Oil Red O to observe liver lipid accumulation. Finally, stained images were obtained using light microscopy.

TG and TC measurement

TG and TC were extracted from mouse liver or cells according to methods previously described in our laboratory [34]. In brief, appropriate amounts of samples were homogenized or lysed in a solution of chloroform and methanol (3:2), dried under a stream of nitrogen, and resuspended in isopropanol. The samples were then assayed for TG and TC levels using a commercial kit (MAK040 for TG and MAK043 for TC, Sigma‒Aldrich) according to the instructions. Mouse serum samples were assayed for TG and TC directly according to the kit instructions.

MDA and SOD analysis

Both MDA and SOD in the liver or cells were analyzed using commercial kits (S0131S for MDA and S0101S for SOD, Beyotime, Shanghai, China). Liver tissues or cells were homogenized or lysed in a solution containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 1% Triton X-100, followed by protein quantification. The homogenate or lysate was further centrifuged at 12,000 × g for 10 min to obtain the supernatant for subsequent assays. The analysis was carried out according to the kit instructions.

Intracellular ATP and NAD+/NADH assays

Intracellular ATP and NAD+/NADH levels were measured using the ATP Assay Kit (S0026, Beyotime) and the NAD+/NADH Assay Kit (S0175, Beyotime) according to the manufacturer’s instructions.

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential was measured using the jc-1 probe (ab113850, Abcam, Cambridge, UK). Cells were washed with PBS followed by the addition of JC1 working solution and incubated for 10 min at 37 °C. After three washes, cells were stained with DAPI (C1002, Beyotime) under light-protected conditions to visualize the nuclei. Finally, fluorescence microscopy was used to observe the intensity of the red and green fluorescence signals and to calculate the ratio of the two fluorescence intensities.

Cellular ROS and mitochondrial ROS assays

To assess total cellular ROS and mitochondrial ROS levels, AML12 cells were stained with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, YEASEN, Shanghai, China) and a mitochondrial superoxide indicator (MitoSOX Red, YEASEN), respectively, and incubated at 37 °C for 1 h in the dark. The nuclei were then infiltrated with DAPI, washed with PBS, and then observed by fluorescence microscopy.

BODIPY staining

After treatment of primary mouse hepatocytes or AML12 cells with PAOA (0.25/0.5 mM) for 12 h, the cells were incubated for 30 min with 10 μM BODIPY 493/503 (216434-81-0, MedChemExpress) working solution and washed three times with PBS and nuclei were stained with DAPI for 3 min. Cells were washed again 3 times with PBS and imaged by fluorescence microscopy.


HEK-293T cells were cotransfected with Flag-USP14 and Myc-HSP90AA1 plasmids and cells were collected and fixed using 4% paraformaldehyde for 5 min. Cells were permeabilized using 0.25% Triton X-100 and blocked with 1% BSA for 2 h. Cells were sequentially incubated with primary antibody (USP14, ab235960, 1:100 dilution, Abcam; MYC, 60003-2, 1:500 dilution, Proteintech) and fluorescently conjugated secondary antibody (goat anti-rabbit IgG, A78953, 1:2000 dilution, Invitrogen; goat anti-mouse IgG, A32727, 1:2000 dilution, Invitrogen). Nuclei were stained with DAPI. Fluorescence microscopy was used to obtain images.

Western blot analysis

After protein extraction from the liver and cells, protein samples were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight with the specific primary antibody and then incubated for 2 h with the appropriate horseradish peroxidase-labeled secondary antibody. Specific bands were detected by chemiluminescence assay. All antibodies used in this study are shown in Supplementary Table S2.

RNA extraction and quantitative RT‐PCR analysis

Total RNA was extracted from the liver and cells using TRIzol (TaKaRa, Japan) and then reverse transcribed using a PrimeScript RT kit (TaKaRa, Japan). The complementary DNA obtained was subjected to quantitative RT‒PCR using SYBR Premix Ex Taq (TaKaRa, Japan). The expression levels of genes relative to β-actin mRNA levels were calculated in each sample according to the 2−ΔΔCt method. All primers are shown in Supplementary Table S3.

Immunoprecipitation assays and ubiquitination analysis

Immunoprecipitation was performed according to the Pierce™ Immunoprecipitation Kit (26149, Thermo Fisher) instructions. After HEK-293T cells were cotransfected with the corresponding plasmids, cell lysates were prepared using lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 1% NP-40). The lysates were then incubated overnight at 4 °C with anti-USP14 rabbit antibody, anti-HSP90AA1 rabbit antibody, anti-CYP2E1 rabbit antibody, and protein G-conjugated agarose, or Flag and Myc affinity agarose. Beads containing bound protein were washed six times using IP Wash Buffer and eluted with glycine. The eluate was mixed with 5× loading buffer and boiled at 95 °C for 10 min for immunoblotting. For ubiquitination analysis, the indicated plasmids were cotransfected into 293 T cells, which were then lysed in precooled IP lysis buffer containing SDS. The subsequent steps were identical to those for co-IP.

Plasmid constructs

The cDNAs of mouse USP14, HSP90AA1, and CYP2E1 were amplified using PCR and cloned into the pEnCMV-MCS-3 × FLAG-SV40-Neo vector or pCMV-MCS-3 × Myc-Neo vector to obtain overexpression vectors with different tags. An active site mutant of USP14 (USP14 C114A) and a truncation mutant of USP14 lacking the UBL were constructed with reference to previously described methods [24]. Primer sequences for plasmid constructs are listed in Table S1. Ubiquitin (Ub) expression vectors with HA tags were obtained from the Miaoling Plasmid Platform (Wuhan, China).

RNA sequencing and data processing

Total RNA was extracted from each group of livers using TRIzol reagent (Ambion/Invitrogen, USA), followed by stringent quality control of the RNA samples and construction of cDNA libraries. After passing library inspection, the library was pooled and sequenced by illumina NovaSeq 6000 (illumina, USA) according to the effective concentration and the target downstream data volume required. Gene expression values (FPKM) of all samples were analyzed by PCA. Hierarchical clustering was displayed using the weighted two-group arithmetic method (UPGMA) and the HLLUST function of the R package. KEGG pathway enrichment analysis of all differential genes was performed by Fisher’s exact test. pathways with P < 0.05 were defined as significantly enriched pathways.

Gene set enrichment analysis used KEGG as a predefined gene set to compare HFD-pAd-CMV and HFD-pAd-USP14 based on their expression levels. Enrichment scores (ES) were then calculated to estimate the significance level (P value) of the ES. The ES of each gene set was standardized to obtain a normalized enrichment score (NES). The false positive rate was controlled by calculating the false discovery rate (FDR) value; gene sets with an FDR < 0.25 were considered statistically significant.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 or SPSS 21.0 (IBM Corp., Armonk). After the data passed the normal distribution test, Student’s t test was used to analyze significant differences between the two groups, and by one-way analysis of variance (ANOVA) followed by Tukey–Kramer test or Dunnett’s test for more than two groups. Data are shown as the mean ± standard deviation (SD). p values < 0.05 were considered statistically significant. No statistical methods were used to predetermine the sample size. No blinding was used during experiments and outcome analysis.

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