Abstract
Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.
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References
Ding, F., Xiao, H., Wang, M., Xie, X. & Hu, F. The role of the ubiquitin–proteasome pathway in cancer development and treatment. Front. Biosci. 19, 886–895 (2014).
Mani, A. & Gelmann, E. P. The ubiquitin–proteasome pathway and its role in cancer. J. Clin. Oncol. 23, 4776–4789 (2005).
Wang, J. & Maldonado, M. A. The ubiquitin–proteasome system and its role in inflammatory and autoimmune diseases. Cell. Mol. Immunol. 3, 255–261 (2006).
Atkin, G. & Paulson, H. Ubiquitin pathways in neurodegenerative disease. Front. Mol. Neurosci. 7, 63 (2014).
Ciechanover, A. & Brundin, P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 40, 427–446 (2003).
Olsen, J. V. & Mann, M. Status of large-scale analysis of post-translational modifications by mass spectrometry. Mol. Cell. Proteom. 12, 3444–3452 (2013).
Rigbolt, K. T. & Blagoev, B. Quantitative phosphoproteomics to characterize signaling networks. Semin. Cell Dev. Biol. 23, 863–871 (2012).
Dikic, I., Wakatsuki, S. & Walters, K. J. Ubiquitin-binding domains—from structures to functions. Nat. Rev. Mol. Cell Biol. 10, 659–671 (2009).
Akimov, V., Rigbolt, K. T., Nielsen, M. M. & Blagoev, B. Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics. Mol. Biosyst. 7, 3223–3233 (2011).
Hjerpe, R. et al. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep. 10, 1250–1258 (2009).
Danielsen, J. M. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol. Cell. Proteom. 10, M110.003590 (2011).
Kliza, K. et al. Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry. Nat. Methods 14, 504–512 (2017).
Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926 (2003).
Akimov, V. et al. StUbEx: stable tagged ubiquitin exchange system for the global investigation of cellular ubiquitination. J. Proteome Res. 13, 4192–4204 (2014).
Xu, G., Paige, J. S. & Jaffrey, S. R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat. Biotechnol. 28, 868–873 (2010).
Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).
Udeshi, N. D. et al. Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition. Mol. Cell. Proteom. 11, 148–159 (2012).
Udeshi, N. D. et al. Refined preparation and use of anti-diglycine remnant (K-ε-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments. Mol. Cell. Proteom. 12, 825–831 (2013).
Wagner, S. A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell. Proteom. 10, M111.013284 (2011).
Na, C. H. et al. Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. J. Proteome Res. 11, 4722–4732 (2012).
Wagner, S. A. et al. Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol. Cell. Proteom. 11, 1578–1585 (2012).
Sylvestersen, K. B., Young, C. & Nielsen, M. L. Advances in characterizing ubiquitylation sites by mass spectrometry. Curr. Opin. Chem. Biol. 17, 49–58 (2013).
Ciechanover, A. & Ben-Saadon, R. N-terminal ubiquitination: more protein substrates join. Trends Cell Biol. 14, 103–106 (2004).
Vittal, V. et al. Intrinsic disorder drives N-terminal ubiquitination by Ube2w. Nat. Chem. Biol. 11, 83–89 (2015).
Gilda, J. E. et al. Western blotting inaccuracies with unverified antibodies: need for a western blotting minimal reporting standard (WBMRS). PLoS One 10, e0135392 (2015).
Hornbeck, P. V. et al. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–D520 (2015).
Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).
Adams, J. et al. Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. Bioorg. Med. Chem. Lett. 8, 333–338 (1998).
Mazurkiewicz, M. et al. Acute lymphoblastic leukemia cells are sensitive to disturbances in protein homeostasis induced by proteasome deubiquitinase inhibition. Oncotarget 8, 21115–21127 (2017).
Dammer, E. B. et al. Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease. J. Biol. Chem. 286, 10457–10465 (2011).
Rose, C. M. et al. Highly multiplexed quantitative mass spectrometry analysis of ubiquitylomes. Cell Syst. 3, 395–403 (2016).
Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).
Kelstrup, C. D. et al. Performance evaluation of the Q Exactive HF-X for shotgun proteomics. J. Proteome Res. 17, 727–738 (2018).
Sap, K. A., Bezstarosti, K., Dekkers, D. H. W., Voets, O. & Demmers, J. A. A. Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA-mediated subunit knockdown in Drosophila. J. Proteome Res. 16, 2848–2862 (2017).
Lu, Y., Lee, B. H., King, R. W., Finley, D. & Kirschner, M. W. Substrate degradation by the proteasome: a single-molecule kinetic analysis. Science 348, 1250834 (2015).
Haglund, K. et al. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat. Cell Biol. 5, 461–466 (2003).
Huang, F., Kirkpatrick, D., Jiang, X., Gygi, S. & Sorkin, A. Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol. Cell 21, 737–748 (2006).
Antonioli, M. et al. AMBRA1 interplay with cullin E3 ubiquitin ligases regulates autophagy dynamics. Dev. Cell 31, 734–746 (2014).
Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011).
Schwertman, P., Bekker-Jensen, S. & Mailand, N. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers. Nat. Rev. Mol. Cell Biol. 17, 379–394 (2016).
Ditzel, M. et al. Inactivation of effector caspases through nondegradative polyubiquitylation. Mol. Cell 32, 540–553 (2008).
Breitschopf, K., Bengal, E., Ziv, T., Admon, A. & Ciechanover, A. A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. EMBO J. 17, 5964–5973 (1998).
Taylor, A. Aminopeptidases: structure and function. FASEB J. 7, 290–298 (1993).
Akimov, V. et al. StUbEx PLUS—a modified stable tagged ubiquitin exchange system for peptide level purification and in-depth mapping of ubiquitination sites. J. Proteome Res. 17, 296–304 (2018).
Drazic, A., Myklebust, L. M., Ree, R. & Arnesen, T. The world of protein acetylation. Biochim. Biophys. Acta 1864, 1372–1401 (2016).
Bloom, J., Amador, V., Bartolini, F., DeMartino, G. & Pagano, M. Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation. Cell 115, 71–82 (2003).
Coulombe, P., Rodier, G., Bonneil, E., Thibault, P. & Meloche, S. N-terminal ubiquitination of extracellular signal–regulated kinase 3 and p21 directs their degradation by the proteasome. Mol. Cell. Biol. 24, 6140–6150 (2004).
Francavilla, C. et al. Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking. Nat. Struct. Mol. Biol. 23, 608–618 (2016).
Hoeller, D. et al. Regulation of ubiquitin-binding proteins by monoubiquitination. Nat. Cell Biol. 8, 163–169 (2006).
Hicke, L., Schubert, H. L. & Hill, C. P. Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 6, 610–621 (2005).
Psakhye, I. & Jentsch, S. Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair. Cell 151, 807–820 (2012).
Shen, T. H., Lin, H. K., Scaglioni, P. P., Yung, T. M. & Pandolfi, P. P. The mechanisms of PML–nuclear body formation. Mol. Cell 24, 331–339 (2006).
Chi, K. R. Drug developers delve into the cell’s trash-disposal machinery. Nat. Rev. Drug Discov. 15, 295–297 (2016).
Sheridan, C. Drug makers target ubiquitin proteasome pathway anew. Nat. Biotechnol. 33, 1115–1117 (2015).
D’Arcy, P. et al. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat. Med. 17, 1636–1640 (2011).
Brnjic, S. et al. Induction of tumor cell apoptosis by a proteasome deubiquitinase inhibitor is associated with oxidative stress. Antioxid. Redox Signal. 21, 2271–2285 (2014).
Nawrocki, S. T. et al. Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells. Cancer Res. 65, 11510–11519 (2005).
Poulsen, J. W., Madsen, C. T., Young, C., Poulsen, F. M. & Nielsen, M. L. Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry. J. Proteome Res. 12, 1020–1030 (2013).
Chylek, L. A. et al. Phosphorylation site dynamics of early T-cell receptor signaling. PLoS One 9, e104240 (2014).
Batth, T. S., Francavilla, C. & Olsen, J. V. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J. Proteome Res. 13, 6176–6186 (2014).
Bekker-Jensen, D. B. et al. An optimized shotgun strategy for the rapid generation of comprehensive human proteomes. Cell Syst. 4, 587–599 (2017).
Kelstrup, C. D. et al. Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer. J. Proteome Res. 13, 6187–6195 (2014).
Huang, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Colaert, N., Helsens, K., Martens, L., Vandekerckhove, J. & Gevaert, K. Improved visualization of protein consensus sequences by iceLogo. Nat. Methods 6, 786–787 (2009).
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).
Licata, L. et al. MINT, the molecular interaction database: 2012 update. Nucleic Acids Res 40, D857–D861 (2012).
Rigbolt, K. T., Vanselow, J. T. & Blagoev, B. GProX, a user-friendly platform for bioinformatics analysis and visualization of quantitative proteomics data. Mol. Cell. Proteom. 10, O110.007450 (2011).
Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
Ventura, A. et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl Acad. Sci. USA 101, 10380–10385 (2004).
Sanchez-Quiles, V. et al. Cylindromatosis tumor suppressor protein (CYLD) deubiquitinase is necessary for proper ubiquitination and degradation of the epidermal growth factor receptor. Mol. Cell. Proteom. 16, 1433–1446 (2017).
Acknowledgements
We would like to thank the PRO-MS Danish National Mass Spectrometry Platform for Functional Proteomics and the CPR Mass Spectrometry Platform for instrument support and assistance. This work was in part funded by the Lundbeck Foundation, the Danish National Research Foundation, the Danish Medical Research Council and the Danish Natural Science Research Council (DFF–1323-00191). The work was also supported in part by the Villum Foundation through the Villum Center for Bioanalytical Sciences (I.K. and B.B.) and by a research grant from the Danish Diabetes Academy funded by the Novo Nordisk Foundation (S.V.F.H.). Work at the Novo Nordisk Foundation Center for Protein Research (CPR) is funded in part by a generous donation from the Novo Nordisk Foundation (NNF14CC0001). The proteomics technology development applied was part of a project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 686547 (J.V.O.).
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V.A. and B.B. designed UbiSite and UbiSite experiments. J.V.O. and B.B. designed total-proteome experiments. V.A., I.B.-H., S.V.F.H., P.H., A.-K.P., C.D.K., M.P., S.D.K.C., J.T.V., M.M.N. and D.B.B.-J. performed the experiments. V.A., I.B.-H., S.V.F.H., P.H., M.P., A.-K.P., C.D.K., I.K., J.V.O. and B.B. analyzed data. V.A., I.B.-H., S.V.F.H., P.H., I.K., C.D.K., J.V.O. and B.B. prepared the manuscript.
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UbiSite is patented by the University of Southern Denmark (patent number US9476888B2; B.B., J.T.V., V.A., M.M.N.).
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Supplementary Figure 1 Enrichment of ubiquitinated peptides by UbiSite.
a, Sequence alignment of remnants after LysC digestion of NEDD8, ubiquitin and ISG-15. b, Comparison of UbiSite and VU1 antibodies in recognizing ubiquitinated species in Hep2 cells treated with vehicle (DMSO) or proteasomal inhibitor (5 μM, 8 h) by western blotting. Shown is a representative of three independent experiments. c, Overlap between unique ubiquitination sites in samples digested with LysC only or trypsin plus LysC. d, Proportions of identified ubiquitinated and non-ubiquitinated peptides in samples treated with LysC only or LysC followed by trypsin. e, Proportions of the summed peptide intensities in samples treated with LysC only or LysC followed by trypsin. f, Elution profiles of ESTLHLVLR and K48 ubiquitin chain peptides over all offline high-pH (HpH) fractions summarized for the three replicates of Hep2 cells treated with bortezomib. The bottom panel shows the number of identified ubiquitinated peptides within each fraction and replicate for the same cells and treatment. g, Drop in the number of peptide identifications during elution of the highly abundant ESTLHLVLR and K48 ubiquitin chain peptides. Top panels show the total ion chromatograms for HpH fractions 5 and 12, as indicated. Red dotted lines provide the number of peptide-to-spectrum matches (PSMs) throughout the LC gradient, binned in 5-min windows. The bottom panel shows the total MS1 spectrum for fraction 12 during the 20-min elution of the two abundant peptides.
Supplementary Figure 2 Ubiquitination sequence motifs and coverage of polyubiquitin chains.
a, Sequence motif analysis of ubiquitination sites from two different clones of the diGly-specific antibody (Mol. Cell. Proteomics 11, 1578–1585, 2012) and UbiSite (lower panel). b, Overlap of diGly-lysine-modified sites and proteins from Jurkat cells identified in Udeshi et al. (Mol. Cell. Proteomics 12, 825–831, 2013) with the ubiquitination sites and proteins identified in this study from the Jurkat cell line alone by the UbiSite approach. c, Ubiquitination sites found in ubiquitin and the number of times each peptide was identified with an Andromeda score higher than 25.
Supplementary Figure 3 Ubiquitination is distributed over the entire proteome.
a, Hierarchical clustering of pairwise Spearman’s correlation coefficients between each replica and experiment. The results are plotted as heat maps for the total proteomes (top) and for all ubiquitination sites from the LysC followed by trypsin dataset (bottom). b, GO term biological processes (GOBP), molecular functions (GOMF) and cellular component (GOCC) enrichment of the total proteome and the UbiSite-enriched ubiquitinome. c, SILAC-based quantification of ubiquitin chain peptides in response to bortezomib (BOR) and b-AP15 (AP15) relative to control (CTR) for Jurkat and Hep2 cells based on three and four biologically independent experiments, respectively. Bars represent the corresponding SILAC ratios (mean + s.d.)
Supplementary Figure 4 Correlation analyses between protein abundance changes and the deviation in their ubiquitination sites in response to proteasomal inhibition.
a, DIA analyses of the total proteomes of bortezomib- (Bort), b-AP15- and mock-treated (Ctrl) Jurkat and Hep2 cells. Scatterplots showing correlations among the five independent replicates for each condition and cell line. Minimum and maximum sample sizes for Jurkat cells were 6,021 and 6,433 proteins, respectively. Minimum and maximum sample sizes for Hep2 cells were 6,120 and 6,527 proteins, respectively. Numbers in each box show the corresponding Pearson correlation coefficients. b, Scatterplots displaying the change in protein abundance versus protein’s ubiquitination site with the biggest change in response to bortezomib (BOR) and b-AP15 (AP15) as compared to mock-treated cells (CTR). R refers to Pearson correlation. c, Scatterplot showing the change in protein abundance versus protein’s mean ubiquitination site in response to bortezomib (BOR) (data extracted from Mol. Cell 44, 325–340, 2011). R refers to Pearson correlation coefficient. d, Scatterplot displaying the change in protein abundance versus protein’s mean ubiquitination site in response to MG132 (data extracted from J. Proteome Res. 16, 2848–2862, 2017). R refers to Pearson correlation coefficient. e, Accumulation of high-molecular-weight protein–ubiquitin conjugates upon treatment of cells with proteasomal inhibitors detected by immunoblotting on the same cellular lysates that were used for the DIA measurements. Shown is a representative of three independent experiments.
Supplementary Figure 5 Extensive ubiquitination of proteins from the non-membrane-bound organelle proteasome and centrosome.
a, Correlation of ubiquitination site changes on proteasomal and centrosomal proteins between bortezomib (BOR) and b-AP15 (AP15) treatment within the same cell line. R refers to Spearman correlation. b, Correlation of bortezomib- or b-AP15-induced changes on the proteasomal and centrosomal ubiquitination sites between the two cell lines. R refers to Spearman correlation. c, An average number of ubiquitination sites per protein on the EUPs from the proteasome and centrosome in the current UbiSite dataset and in the study by Udeshi et al. (Mol. Cell. Proteomics 12, 825–831, 2013). The average numbers of significantly regulated sites per protein in response to proteasomal inhibition are indicated as well. d,e, Comparison of the significantly regulated ubiquitination sites on proteasomal (d) and centrosomal (e) EUPs from the two studies.
Supplementary Figure 6 N-terminal protein ubiquitination is not affected by proteasomal inhibition.
a, Prevalence of amino acids at the second position of N-terminally ubiquitinated proteins in Hep2 (top) and Jurkat (bottom) cells. b, Comparison of the N-terminally ubiquitinated proteins identified by the StUbEx PLUS approach in U2OS cells (blue) (data extracted from J. Proteome Res. 17, 296, 2018) with those identified in the current UbiSite study (red). c, log10 intensities of N-terminally ubiquitinated peptides in untreated cells or upon proteasomal inhibition with bortezomib (BOR) or b-AP15 (AP15). Peptides quantified in all three replicates of at least one treatment are shown.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 and Supplementary Notes 1,2
Supplementary Table 1
Lysine ubiquitination sites of the UbiSite-enriched ubiquitinomes of Hep2 and Jurkat cells
Supplementary Table 2
Doubly ubiquitinated peptides
Supplementary Table 3
Total proteomes of Hep2 and Jurkat cells
Supplementary Table 4
Quantification and annotation enrichments of ubiquitination sites
Supplementary Table 5
Extensively ubiquitinated proteins (EUPs)
Supplementary Table 6
Peptides from N-terminally ubiquitinated proteins
Supplementary Dataset 1
Uncropped western blot images
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Akimov, V., Barrio-Hernandez, I., Hansen, S.V.F. et al. UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites. Nat Struct Mol Biol 25, 631–640 (2018). https://doi.org/10.1038/s41594-018-0084-y
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DOI: https://doi.org/10.1038/s41594-018-0084-y
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