These authors contributed equally to this work.
Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease
Article first published online: 18 DEC 2013
© 2013 by the American Association for the Study of Liver Diseases
Volume 59, Issue 2, pages 423–433, February 2014
How to Cite
Morikawa, K., Gouttenoire, J., Hernandez, C., Dao Thi, V. L., Tran, H. T.L., Lange, C. M., Dill, M. T., Heim, M. H., Donzé, O., Penin, F., Quadroni, M. and Moradpour, D. (2014), Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease. Hepatology, 59: 423–433. doi: 10.1002/hep.26671
Potential conflict of interest: Nothing to report.
Supported by grants 3100A0-122447 and 31003A-138484 from the Swiss National Science Foundation as well as by grant 09C53 from the Novartis Foundation.
- Issue published online: 29 JAN 2014
- Article first published online: 18 DEC 2013
- Accepted manuscript online: 8 AUG 2013 08:16AM EST
- Manuscript Accepted: 30 JUL 2013
- Manuscript Received: 23 JAN 2013
Additional Supporting Information may be found in the online version of this article.
|hep26671-sup-0002-suppfig1.tif||9944K||Supporting Figure 1. Virtual electropherogram with quantitative values produced by “slice-SILAC”. A representative subset of the data from the screen #1 (without IFN-α stimulation) is shown, including GPx8 (Suppl. Table 2). Only proteins found in at least two gel slices with diverging heavy-over-light (H/L) ratios were prefiltered and are represented here. Only the region below a molecular weight of 50 kDa is shown for ease of representation. Bubble diameters are proportional to the number of quantified peptide matches, while the gradient color represents the H/L ratio, as indicated in the legend. GPx8 is highlighted in yellow. A representative gene name for every protein group is given at the bottom (standard abbreviations as used, e.g., in the Swiss-Prot database). Of note, histone proteins H3F3A and HIST2H2AA3 showed shifts to lower molecular weight upon NS3-4A expression. However, given their broad distribution and known high degree of modification these were not further investigated. Rab13 appeared as a potential candidate (although at less significant H/L ratios) but was not found to be a NS3-4A protease substrate in the subsequent experimental validation process.|
|hep26671-sup-0003-suppfig2.tif||2463K||Supporting Figure 2. Cell lysates subjected to protein separation and mass spectrometry. A portion of the lysates from the heavy (H) or light (L) isotope-labeled UNS3-4A-24 cells subjected to protein separation and mass spectrometry in the present study was analyzed by immunoblot using monoclonal antibody (mAb) 1B6 against NS3 (Wölk B et al. J Virol 2000;74:2293-2304), mAb Adri-1 against MAVS (Adipogen, Epalinges, Switzerland), polyclonal antibody E23 against Stat1 p84/p91 (Santa Cruz, Santa Cruz, CA) or mAb AC-15 against β-actin (Sigma-Aldrich, St. Louis, MO) (lanes 3-6). Lysates from naïve Huh-7.5 cells and Huh-7.5 cells harboring a subgenomic HCV Con1 (sg1b, genotype 1b) replicon were included as reference (lanes 1 and 2). A total of 10 μg protein was loaded in each lane. aa, amino acid; IFN-α, interferon-α; tet, tetracycline.|
|hep26671-sup-0004-suppfig3.tif||1183K||Supporting Figure 3. Basis for the three-dimensional model of the N-terminal cytosolic tip and the transmembrane segment of GPx8 illustrated in Figure 2C. Similarity of the GPx8 N-terminal sequence to the sequence of amino acids 155-171 of filamin A immunoglobulin-like repeat 10 (PDB entry 3RGH; Page RC et al. Acta Crystallogr Sect F Struct Biol Cryst Commun 2011;67:871-876) (top row) and to the transmembrane α-helix 20-41 of FXYD protein of the sodium-potassium ATPase (PDB entry 2ZXE, chain G; Shinoda T et al. Nature 2009;459:446-450) (middle row). The degree of amino acid physicochemical conservation at each position relatively to GPx8 sequence is indicated with the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar; Thompson JD et al. Nucleic Acids Res 1994;22:4673-4680). The GPx8 sequence displays 71% similarity (47% identity) within the 17 amino acid overlap for the 3RGH sequence, and 89% similarity (26% identity) with the 19 amino acid overlap for the 2ZXE sequence. “Struct.” represents the secondary structure of the different protein segments deduced from the corresponding three-dimensional structures: h, helix; s, bend; t, hydrogen-bonded turn. The beginning of the X-ray structure of GPx8 luminal domain is also indicated (PDB entry 3KIJ; Nguyen VD et al. J Mol Biol 2011;406:503-515; bottom row). #, GPx8 transmembrane segment predicted by algorithms PHDhtm (http://www.predictprotein.org/), TMHMM (http://www.cbs.dtu.dk/services/TMHMM), DAS (http://www.sbc.su.se/miklos/DAS/), TopPred2 (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form_toppred), Tmpred (http://www.ch.embnet.org/software/TMPRED_form.html), HMMTOP (http://www.enzim.hu/hmmtop/), and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/).|
|hep26671-sup-0005-suppfig4.tif||1875K||Supporting Figure 4. siRNA-mediated silencing of GPx8. Huh-7.5 cells overexpressing GPx8 (Huh-7.5-GPx8) were transfected with GPx8 siRNAs #1 or #2 or a non-targeting control, as described in the Materials and Methods section. Cell lysates obtained 96 h later were analyzed by 15% SDS-PAGE and immunoblot using polyclonal antibody IN103 against GPx8. A total of 10 μg protein was loaded in each lane.|
|hep26671-sup-0006-suppfig5.tif||16052K||Supporting Figure 5. Subcellular localization of GPx8 and mutants. Huh-7.5 cells transduced with recombinant viruses expressing GPx8, GPx8C11A, GPx8S12 and GPx8C79T were infected with Jc1 HCVcc and stained 48 h later with polyclonal antibody IN103 against GPx8 and monoclonal antibody #337 against HCV NS3 (kindly provided by Thomas Pietschmann, Twincore, Hannover, Germany) as well as Alexa Fluor® 488- and 594-conjugated secondary antibodies (Life Technologies), respectively. Nuclei were stained with DAPI (AppliChem). Representative confocal laser scanning microscopy images are shown for each construct. GPx8 (green) and NS3 (red) are shown separately and as merged images including the DAPI staining (blue). An inset is shown at higher magnification for GPx8-transduced cells. GPx8 is mostly expressed at the ER membrane including the perinuclear membrane and partially colocalizes with HCV NS3. No appreciable difference is observed in the subcellular localization of GPx8 and the different GPx8 mutants.|
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