Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination

Abstract

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Sensor design and characterization.
Fig. 2: Quantitative, real-time DUB activity assays with a Ub sensor.
Fig. 3: Effects of cellular stresses on Ub pools.
Fig. 4: Free Ub staining in fixed and permeabilized HeLa, U2OS, MEF and RPE1 cells.

Similar content being viewed by others

Data availability

The data that support the findings of this paper are available from the corresponding author on reasonable request.

References

  1. Komander, D. & Rape, M. The ubiquitin code. Annu Rev. Biochem. 81, 203–229 (2012).

    Article  CAS  Google Scholar 

  2. Park, C. W. & Ryu, K. Y. Cellular ubiquitin pool dynamics and homeostasis. BMB Rep. 47, 475–482 (2014).

    Article  Google Scholar 

  3. Ryu, K. Y., Garza, J. C., Lu, X. Y., Barsh, G. S. & Kopito, R. R. Hypothalamic neurodegeneration and adult-onset obesity in mice lacking the Ubb polyubiquitin gene. Proc. Natl Acad. Sci. USA 105, 4016–4021 (2008).

    Article  CAS  Google Scholar 

  4. Ryu, K. Y. et al. The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance. EMBO J. 26, 2693–2706 (2007).

    Article  CAS  Google Scholar 

  5. Ryu, K. Y. et al. The mouse polyubiquitin gene Ubb is essential for meiotic progression. Mol. Cell Biol. 28, 1136–1146 (2008).

    Article  CAS  Google Scholar 

  6. Kimura, Y. et al. An inhibitor of a deubiquitinating enzyme regulates ubiquitin homeostasis. Cell 137, 549–559 (2009).

    Article  CAS  Google Scholar 

  7. Wang, C. H. et al. USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin. eLife 6, e26886 (2017).

    Article  Google Scholar 

  8. Crimmins, S. et al. Transgenic rescue of ataxia mice with neuronal-specific expression of ubiquitin-specific protease 14. J. Neurosci. 26, 11423–11431 (2006).

    Article  CAS  Google Scholar 

  9. Chen, P. C. et al. The proteasome-associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions. J. Neurosci. 29, 10909–10919 (2009).

    Article  CAS  Google Scholar 

  10. Oh, C., Park, S., Lee, E. K. & Yoo, Y. J. Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention. Sci. Rep. 3, 2623 (2013).

    Article  Google Scholar 

  11. Kedves, A. T. et al. Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C. J. Clin. Invest 127, 4554–4568 (2017).

    Article  Google Scholar 

  12. Hallengren, J., Chen, P. C. & Wilson, S. M. Neuronal ubiquitin homeostasis. Cell Biochem. Biophys. 67, 67–73 (2013).

    Article  CAS  Google Scholar 

  13. Dantuma, N. P., Groothuis, T. A., Salomons, F. A. & Neefjes, J. A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling. J. Cell Biol. 173, 19–26 (2006).

    Article  CAS  Google Scholar 

  14. Yau, R. & Rape, M. The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586 (2016).

    Article  CAS  Google Scholar 

  15. Kaiser, S. E. et al. Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools. Nat. Methods 8, 691–696 (2011).

    Article  CAS  Google Scholar 

  16. Reyes-Turcu, F. E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006).

    Article  CAS  Google Scholar 

  17. Swanson, K. A., Kang, R. S., Stamenova, S. D., Hicke, L. & Radhakrishnan, I. Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation. EMBO J. 22, 4597–4606 (2003).

    Article  CAS  Google Scholar 

  18. Lee, S. et al. Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5. Nat. Struct. Mol. Biol. 13, 264–271 (2006).

    Article  CAS  Google Scholar 

  19. Penengo, L. et al. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin. Cell 124, 1183–1195 (2006).

    Article  CAS  Google Scholar 

  20. Maupin-Furlow, J. A. Ubiquitin-like proteins and their roles in archaea. Trends Microbiol. 21, 31–38 (2013).

    Article  CAS  Google Scholar 

  21. Choi, Y. S., Jeon, Y. H., Ryu, K. S. & Cheong, C. 60th residues of ubiquitin and Nedd8 are located out of E2-binding surfaces, but are important for K48 ubiquitin-linkage. FEBS Lett. 583, 3323–3328 (2009).

    Article  Google Scholar 

  22. Zhang, D., Raasi, S. & Fushman, D. Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains. J. Mol. Biol. 377, 162–180 (2008).

    Article  CAS  Google Scholar 

  23. Sokratous, K. et al. Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry. J. Am. Chem. Soc. 134, 6416–6424 (2012).

    Article  CAS  Google Scholar 

  24. Swaney, D. L., Rodriguez-Mias, R. A. & Villen, J. Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover. EMBO Rep. 16, 1131–1144 (2015).

    Article  CAS  Google Scholar 

  25. Kane, L. A. et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J. Cell Biol. 205, 143–153 (2014).

    Article  CAS  Google Scholar 

  26. Ordureau, A. et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc. Natl Acad. Sci. USA 112, 6637–6642 (2015).

    Article  CAS  Google Scholar 

  27. Koyano, F. et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510, 162–166 (2014).

    Article  CAS  Google Scholar 

  28. Harper, J. W., Ordureau, A. & Heo, J. M. Building and decoding ubiquitin chains for mitophagy. Nat. Rev. Mol. Cell Biol. 19, 93–108 (2018).

    Article  CAS  Google Scholar 

  29. Wauer, T. et al. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. EMBO J. 34, 307–325 (2015).

    Article  CAS  Google Scholar 

  30. Dang, L. C., Melandri, F. D. & Stein, R. L. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry 37, 1868–1879 (1998).

    Article  CAS  Google Scholar 

  31. Geurink, P. P. et al. Development of diubiquitin-based FRET probes to quantify ubiquitin linkage specificity of deubiquitinating enzymes. Chem. Bio. Chem. 17, 816–820 (2016).

    Article  CAS  Google Scholar 

  32. Yao, T. & Cohen, R. E. Ubiquitin-ovomucoid fusion proteins as model substrates for monitoring degradation and deubiquitination by proteasomes. Methods Enzym. 398, 522–540 (2005).

    Article  CAS  Google Scholar 

  33. Wang, T. et al. Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1. J. Mol. Biol. 386, 1011–1023 (2009).

    Article  CAS  Google Scholar 

  34. Wiener, R. et al. E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1. Nat. Struct. Mol. Biol. 20, 1033–1039 (2013).

    Article  CAS  Google Scholar 

  35. Gates, Z. P., Stephan, J. R., Lee, D. J. & Kent, S. B. Rapid formal hydrolysis of peptide-alphathioesters. Chem. Commun. 49, 786–788 (2013).

    Article  CAS  Google Scholar 

  36. Shahnawaz, M., Thapa, A. & Park, I. S. Stable activity of a deubiquitylating enzyme (Usp2-cc) in the presence of high concentrations of urea and its application to purify aggregation-prone peptides. Biochem. Biophys. Res. Commun. 359, 801–805 (2007).

    Article  CAS  Google Scholar 

  37. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

    Article  CAS  Google Scholar 

  38. Yang, X. et al. Absolute quantification of E1, ubiquitin-like proteins and Nedd8-MLN4924 adduct by mass spectrometry. Cell Biochem. Biophys. 67, 139–147 (2013).

    Article  CAS  Google Scholar 

  39. Chen, J. J. et al. Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues. J. Biol. Chem. 286, 40867–40877 (2011).

    Article  CAS  Google Scholar 

  40. Bianchi, M. et al. Dynamic transcription of ubiquitin genes under basal and stressful conditions and new insights into the multiple UBC transcript variants. Gene 573, 100–109 (2015).

    Article  CAS  Google Scholar 

  41. Liu, Z. et al. Noncovalent dimerization of ubiquitin. Angew. Chem. Int. Ed. Engl. 51, 469–472 (2012).

    Article  CAS  Google Scholar 

  42. Joo, H. Y. et al. Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature 449, 1068–1072 (2007).

    Article  CAS  Google Scholar 

  43. Jencks, W. P. On the attribution and additivity of binding energies. Proc. Natl Acad. Sci. USA 78, 4046–4050 (1981).

    Article  CAS  Google Scholar 

  44. Scott, D., Oldham, N. J., Strachan, J., Searle, M. S. & Layfield, R. Ubiquitin-binding domains: mechanisms of ubiquitin recognition and use as tools to investigate ubiquitin-modified proteomes. Proteomics 15, 844–861 (2015).

    Article  CAS  Google Scholar 

  45. Hicke, L., Schubert, H. L. & Hill, C. P. Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 6, 610–621 (2005).

    Article  CAS  Google Scholar 

  46. Morrow, M. E. et al. Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins. EMBO Rep. 19, e45680 (2018).

    Article  Google Scholar 

  47. Harrigan, J. A., Jacq, X., Martin, N. M. & Jackson, S. P. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat. Rev. Drug Disco. 17, 57–78 (2018).

    Article  CAS  Google Scholar 

  48. Majetschak, M. Extracellular ubiquitin: immune modulator and endogenous opponent of damage-associated molecular pattern molecules. J. Leukoc. Biol. 89, 205–219 (2011).

    Article  CAS  Google Scholar 

  49. Raasi, S. & Pickart, C. M. Ubiquitin chain synthesis. Methods Mol. Biol. 301, 47–55 (2005).

    CAS  PubMed  Google Scholar 

  50. Whitby, F. G., Xia, G., Pickart, C. M. & Hill, C. P. Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes. J. Biol. Chem. 273, 34983–34991 (1998).

    Article  CAS  Google Scholar 

  51. Wilkinson, K. D. et al. Metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase T. Biochemistry 34, 14535–14546 (1995).

    Article  CAS  Google Scholar 

  52. Wilkinson, K. D. Quantitative analysis of protein–protein interactions. Methods Mol. Biol. 261, 15–32 (2004).

    CAS  PubMed  Google Scholar 

  53. Pasupala, N. et al. OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination. J. Biol. Chem. 293, 18285–18295 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Ma (University of California, San Francisco) for MEF cells, C. Wolberger (Johns Hopkins University) for OTUB1 protein and B. Brasher (Boston Biochem) for phosphoubiquitin. We also thank O. Peersen for assistance with the rapid-kinetics experiments and use of the Bio-Logic stopped-flow spectrofluorometer, and R. Handa for use of the Imaris image analysis software. This research was supported by NIH-NIGMS grant no. R01 GM115997 (to R.E.C.) and NIH-NIEHS grant no. R21 ES029150 (to R.E.C. and T.Y.).

Author information

Authors and Affiliations

Authors

Contributions

Y.S.C. and R.E.C. conceived and designed the ubiquitin sensor reagents. Y.S.C. produced the sensors and characterized them in vitro. S.A.B., T.Y. and R.E.C. conceived the cell-based studies, which were done by S.A.B., L.F.P. and F.S. All authors contributed to writing or commenting on the manuscript.

Corresponding author

Correspondence to Robert E. Cohen.

Ethics declarations

Competing interests

U.S. patent no. 10,018,634 has been awarded to Colorado State University Research Foundation (R.E.C. and Y.C., inventors) for ubiquitin sensors and assays described in this paper.

Additional information

Peer review information: Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Primary sequences of the free Ub sensors.

The bold, underlined residues indicate mutated residues, and the cysteines in red indicate fluorophore conjugation sites. The residues highlighted in cyan are linkers introduced to connect the domains, and the yellow and green highlighted sequences show peptides added to provide 6-His and HA-epitope tags, respectively.

Supplementary Figure 2 Sensor binding assays.

a, Atto532-Ub(S20C) titrated with tIVR. b, Atto532-tIVR titrated with Ub. c, Alexa488-Ub(S20C) titrated to tISR. d, tISR competition binding assay performed by titration of Ub in presence of Alexa488-Ub(S20C). In each titration, the fluorescence intensity change from ligand binding was measured. Error bars in a, which are mostly hidden by the point symbols, show standard deviations of the assay repeated two times independently; titrations in b - d were performed once. Kd values ± s.d. shown in each panel were determined from non-linear fits to the data.

Supplementary Figure 3 Screening for an optimal fluorescent dye and conjugation site on the tIVR sensor.

a, Cys residue in the linker connecting IsoTBuz and Vps27UIM; b, N46C; and c, M105C in IsoTBuz were selected to conjugate various fluorescent dyes. In each panel, the arrow shows the conjugation site on the tIVR model and the histograms show performance with different fluorophores installed at that site. To determine which site and fluorescent dye gave the largest fluorescence change upon Ub binding, fluorescence intensities of the labeled sensors (2 nM) were measured with and without saturating Ub. F0 is fluorescence intensity of the labeled sensors without Ub, and ∆F is the intensity change upon addition of 200 µM Ub.

Supplementary Figure 4 Kinetics of Atto532-tUI binding to Ub.

a, To determine the dissociation rate of the Atto532-tUI•Ub complex, Atto532-tUI (1.0 nM) was pre-incubated at 25 °C with 1.0 nM Ub, and then excess unlabeled tUI (1 µM) was rapidly added and the fluorescence was measured every 0.25 s using a stopped-flow fluorimeter. Three independent reactions were performed, and all the data were fit with a single-exponential decay model to determine koff. The experimental data (blue) are shown superimposed with the fitted curve (red). b, The change in fluorescence intensity of 50 pM Atto532-tUI immediately after addition of 0.5, 1.0, or 2.0 nM Ub was monitored at 25 °C. The fluorescence was measured every 0.25 s. From each curve, an observed rate (kobs) was determined from the best-fitting exponential curve, and from these kon was determined from the equation kobs = kon * [Ub] + koff. Each reaction to generate the association data was performed once. c, The experimentally determined association and dissociation rates ± s.d. for the Atto532-tUI•Ub complex are shown together with the Kd ± s.d. calculated from their ratio ± s.d.

Supplementary Figure 5 Analysis of the DUB reaction products by SDS-PAGE.

Samples from the end-points of the real-time DUB reactions in Fig. 2 were separated by SDS-PAGE together with the following control samples: Atto532-tIVR in lane 1, Ub5-OM(LY) in lane 2, Ub1-OM(LY) in lane 3, and the Usp2cc-digested product in lane 7. Fluorescent gel bands were detected with a Typhoon laser scanner using excitation at either (a) 488 nm, (b) 532 nm, or (c) both to visualize Lucifer Yellow-labeled ovomucoid protein (OM(LY)) or Atto532-labeled tIVR. d, The bar graph compares the Ub released in the DUB reactions in Fig. 2 quantified by either the protein band fluorescence (panel a) or the sensor fluorescence (Fig. 2). e, Atto532-tIVR (2 nM) was titrated with Ub (2–32 nM) and the data were fit with a 1:1 binding model as described in Methods. The fitted equation was used to convert fluorescence intensities from the real-time DUB reaction curves (Fig. 2, left panel) to free Ub concentrations (Fig. 2, right panel).

Supplementary Figure 6 Generation of Ub-hydrazide from Ub thioesters.

a, SDS-PAGE was used to monitor E2-Ub thioester formation and subsequent hydrazinolysis. Ub was incubated with E1 and E2 (UbcH5c) enzymes without (lane 1) or with (lane 2) ATP to form Ub~UbcH5c thioester. After treatment with NEM (lane 3) to inactivate enzymatic activities, hydrazine was added at the concentrations indicated; lanes 4–6 show that hydrazinolysis appeared to be complete (i.e., E2-Ub thioester was gone) under all three conditions. b, Upper scheme shows the reaction to generate Ub-hydrazide (see Methods) used in Fig. 1b-d titrations. Lower panel shows overlayed MALDI-TOF mass spectra of Ub and products (i.e., Ub–MESNA and Ub–hydrazide) from each step in the synthesis. The results in a and b are representative of 3 independent experiments.

Supplementary Figure 7 Processing cell lysates with Usp2cc releases most conjugated Ub.

SDS-PAGE and immunoblotting with anti-Ub antibody (P4G7) shows the levels of conjugated Ub in HeLa cell lysates prepared as described in the Methods for in-solution measurements of Ub pools. The lysate was generated and incubated without or with Usp2cc for 1 h at 37 °C as described in Methods. This analysis was performed once.

Supplementary Figure 8 Ub pools in proteasome-inhibited HeLa cells do not change significantly after 1 h.

In-solution quantification of Ub pools in lysates after treatment with vehicle (DMSO) or the proteasome inhibitor, BTZ (1 µM). Statistical analysis of the sample mean was by one-way ANOVA with Bonferroni’s adjustment; error bars represent ± s.d. (n = 3).

Supplementary Figure 9 Competition by free Ub for cell staining by HA-tUI.

Quantitation of mean fluorescence in HeLa cells (maximum projection images) after incubation with HA-tUI with or without excess free Ub. For 100 nM HA-tUI, n = 131; for 100 nM HA-tUI + 100 μM Ub, n = 28. AU, arbitrary units; error bars indicate mean ± s.d. Statistical analysis used two-tailed unpaired Student's t-test with Welch's correction.

Supplementary Figure 10 Free Ub staining by HA-tUI shows a different subcellular distribution than K48-linked polyUb or total conjugated Ub.

HeLa cells were fixed with 4% PFA and stained with DAPI (blue), anti-K48 polyUb antibody (green), and HA-tUI (magenta) in panel a, or DAPI (blue), anti-Ub FK2 antibody (green), and HA-tUI (magenta) in panel b. For each cell, the images are from a single z-stack plane that shows both nucleus and cytoplasm. The cells shown are representative of more than 10 cells analyzed on each of two coverslips.

Supplementary Figure 11 Competition binding assays to measure tUI affinity for Ub.

Competition binding assays were performed with a, 70 pM Atto532-tUI or b, 50 pM Atto532-tUI in the presence of 80 pM Ub. tUI was titrated from 0.063 nM to 32 nM, from which a mean Ki of 194 pM was determined from fittings to the two independent data sets.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Choi, YS., Bollinger, S.A., Prada, L.F. et al. High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination. Nat Methods 16, 771–777 (2019). https://doi.org/10.1038/s41592-019-0469-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-019-0469-9

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing