Deubiquitinase USP19 extends the residual enzymatic activity of phenylalanine hydroxylase variants
Cell culture and treatments
Human embryonic kidney (HEK293) cells (Korean Cell Bank, Seoul, Korea) were cultured at 37 °C in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (GIBCO BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (GIBCO BRL) and 1% penicillin and streptomycin (GIBCO BRL). One day before transfection, the cells were seeded in a 6-well culture dish, and then about 1 μg of HA-PAHwt, HA-R241C, and HA-R243Q plasmids were added to each well for transfection. The concentration ranging from 100 to 500 ng of Flag-USP19 was transfected, depending on the experiment. Untransfected HEK293 cells were used as the control. Polyethyleneimine (Polysciences, Warrington, PA, USA) was used according to the manufacturer's protocol to transfect the HEK293 cells. After 48 hours, the cells were harvested. For the CHX assay, cells were transfected with the indicated constructs and incubated for two days. After 48 h of transfection, the cells were treated with CHX (150 μg/mL) and harvested at various time points. For the proteasomal or lysosomal inhibition, cells were transfected with indicated plasmid. After 48 h of transfection the cells were treated with MG132 for 12 h or ammonium chloride (NH4Cl) for 24 h respectively.
Plasmids, antibodies, and reagents
HA-tagged PAH and the pLVX-IRES-ZsGreen1-PAHwt construct were kindly provided by Prof. Shen Nan (University Children's Hospital, Heidelberg, Germany)33,52. PAH R241C and R243Q were generated in HA-tagged PAH and pLVX-IRES-ZsGreen1-PAHwt constructs by site-directed mutagenesis using the primers indicated in Supplementary Table S1. Flag-tagged USP19 and Flag-tagged ubiquitin were purchased from Addgene (Watertown, MA, USA). The following primary antibodies were used in our study: mouse monoclonal antibody against the HA tag (sc-7392, 1:1000), GAPDH (sc-32233, 1:1000), ubiquitin (SC-8017, 1:1000) and rabbit polyclonal antibody against ubiquitin (sc-9133, 1:500). Protein A/G Plus Agarose beads (sc-2003) were purchased from Santa Cruz Biotech (Dallas, TX, USA). Mouse anti-PH8 antibody (MAB5278, 1:1000, Merck Millipore, Kenilworth, NJ, USA), rabbit polyclonal antibodies against USP19 (Cat no. #25,768-1-AP, 1:1000, Proteintech, Rosemont, IL, USA) , rabbit polyclonal antibody against LC3B (Cat No. #2775S, Cell signaling technology, Denvers, MA, USA) and mouse monoclonal antibody against the Flag tag (Anti-DDDDK-tag, M185-3L, 1:1000, MBL Life Science, Woburn, MA, USA) were used. In addition, we used IP lysis buffer (Cat. no. #87787; Thermo Fisher), cell lysis buffer (Cat. no. #R2002, Biosesang), and a protease inhibitor cocktail (Cat. no. #11836153001, Roche, South San Francisco, CA, USA), the proteasomal inhibitor MG132 (Cat. no. #S2619, Selleckchem, Houston, TX, USA), the protein translation inhibitor CHX (Cat no. #239765, Merck, Kenilworth, NJ, USA), the DUB inhibitor PR-619 (Cat. no. #S7130, Selleckchem, Houston, TX, USA) and Lysosomal inhibitor NH4Cl (Sigma-Aldrich, St. Louis, MO, USA).
Stable expression of PAH and variants in HEK293 cells
To generate HEK293 cells that stably expressing PAHwt, R241C, and R243Q, we used the pLVX-IRES-ZsGreen1 plasmid encoding PAHwt, R241C and R243Q for lentiviral production. One day prior to transfection, 1 × 106 HEK293 cells were seeded and cultured in a 100 mm culture dish. The HEK293 cells were then co-transfected with pLVX-IRES-ZsGreen1 plasmids encoding PAHwt, R241C, or R243Q and packaging vectors (pLP1, pLP2 and pLP-VSVG) in a 4:1:1:1 ratio. Cell supernatants were harvested 48 h after transfection and either used to infected cells or stored at − 80 °C. Cells were infected for 6 h with the lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 10 μg/mL of polybrene (Sigma-Aldrich) to obtain stable HEK293 cell lines expressing PAHwt or a PAH variant. The transduction efficiency of pLVX-ZsGreen1 plasmid encoding PAHwt, R241C and R243Q was checked after 72 h (Supplementary Fig. S1a and S1b). The cells transduced with empty vector were used as control.
immunoprecipitation
HEK293 cells were lysed with IP lysis buffer containing 150 mM sodium chloride, 1 mM EDTA 25 mM, Tris-HCl (pH 7.4), 1% NP-40, 5% glycerol, 1 mM PMSF, and protease inhibitor cocktail for 20 min. The cell lysates were incubated with the indicated antibodies at 4 °C overnight and immunoprecipitated for 2–4 h with 20 μL of Protein A/G Plus Agarose beads (Santa Cruz Biotechnology) with constant agitation at 4 °C. The beads were washed thrice with lysis buffer and resuspended in 2X denaturing SDS sample buffer (Cat. no. S3401, Sigma-Aldrich)): 5X SDS sample loading buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol , 0.004% bromophenol blue, and 0.125 M Tris–HCl (pH 6.8). The samples were boiled at 95–100 °C for 5 min, followed by immunoblotting. For the binding assay, mouse IgG (Cat. No. #18-8817-33, Rockland, Philadelphia, Pennsylvania, USA), a light chain-specific secondary antibody, was used to prevent interference from heavy immunoglobulin chains.
Duolink proximity ligation assay
A Duolink in situ proximity ligation assay (PLA) kit (DUO92101, Sigma-Aldrich) was used to detect the interaction between PAH and USP19 and to evaluate the ubiquitination status of PAH. HEK293 cells were fixed at room temperature in 4% paraformaldehyde for 10 min and blocked using 1X blocking solution. The cells were then incubated at 4 °C overnight with primary antibodies targeting PAH, USP19, or ubiquitin, followed by incubation with PLA probes at 37 °C for 1 h. After washing them three times, we added ligation-ligase solution and incubated them at 37 °C for 30 min. Next, the slides were incubated with amplification-polymerase solution at 37 °C in the dark for 100 min. Finally, the cells were stained with mounting medium containing DAPI. A Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Wetzlar, Germany) was used to capture the fluorescence images.
PAH activity assay
To measure PAH enzyme activity, transfected cells were harvested and lysed by three freeze-thaw cycles in lysis buffer (0.25 mol/L sucrose, 1X PBS, pH 7.2) containing a protease inhibitor. The samples were centrifuged at 15,800 g for 20 min at 4 °C to obtain clear lysates and determine PAH activity, as previously described53. Briefly, 120 μg of total protein was pre-incubated with 1 mmol/L of l-Phe (P17008, Sigma, St. Louis, MO, USA) and 2 µg of catalase (C1345, Sigma) in 0.1 mol/L Na-HEPES buffer (pH 7.0, T&I, BHE-9000, Gangwon, Korea) for 5 min followed by a 1 min incubation with 10 mM ferrous ammonium sulfate. The reaction was initiated by adding 200 µmol/L of BH4 (Cat no. #T4425, Sigma) in 5 mM DTT (Cat no. #10197777001, Sigma) and allowed to proceed for 30 min at 25 °C. The reaction was stopped by adding 50 µL of 2% (w/v) acetic acid in ethanol. All concentrations mentioned refer to the final concentration in a 100 µL reaction mixture. The amounts of Tyr were determined using a tyrosine assay kit (Cat no. #ab185435) according to the manufacturer's protocol. A standard curve for Tyr was used to determine the Tyr concentration in the samples (Fig. 6e).
For the HPLC–UV analysis, a Phenomenex EZ:Faast™ kit was used to prepare the samples. The amount of Phe was determined using an HPLC system with a UV detector at a wavelength of 210 nm. Acetonitrile (94:6 v/v), 20 mmol/L sodium acetate buffer (pH 6.5), and a Shiseido Capcell Pak MF C8 analytical column (4.6 mm × 150 mm) were used for the chromatography. The flow rate was 1 mL/min, and the sample injection volume was 20 µL. A standard curve for Phe was used to determine the Phe concentration in the samples (Fig. 6b). The chromatograms represent the peak height (in arbitrary units, AUV) against the retention time (Fig. 6d).
Statistics
The statistical analysis was conducted using GraphPad Prism 9 (GraphPad Software, Inc. San Diego, CA, USA), and the results are presented as the mean ± standard deviation of three independent experiments. One-way ANOVA and paired t testing were used to analyze the data, and multiple comparisons among groups were performed with Tukey's post hoc test or Šídák's multiple comparisons test. For comparisons between two groups, two-way ANOVA was used to analyze the data (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005, and ns denotes non-significant) .
Consent to participate
All individual authors included in the study provide consent for publication. The authors are responsible for the correctness of the statements provided in the manuscript.
continue reading
Comments
Post a Comment