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  • Letter
  • Published:

Insights into ubiquitin chain architecture using Ub-clipping

Nature volume 572, pages 533–537 (2019)Cite this article

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This article has been updated

Abstract

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1,2,3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10–20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.

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Fig. 1: Lbpro cleaves ubiquitin and generates GlyGly-modified proteins.
Fig. 2: Dissecting branched ubiquitin chains with Ub-clipping.
Fig. 3: Ub-clipping in cells.
Fig. 4: Exploring mitophagy with Ub-clipping.

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Data availability

All data have been deposited to the Mass Spectrometry Interactive Virtual Environment (MassIVE) (ftp://massive.ucsd.edu/MSV000083662) or included as Supplementary Information. Source data for all gels have been included in Supplementary Fig. 1.

Change history

  • 27 August 2019

    Owing to a technical error, this Letter was not published online on 14 August 2019, as originally stated, and was instead first published online on 15 August 2019. The Letter has been corrected online.

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Acknowledgements

We thank M. Skehel, S. Maslen, A. Webb, W. Harper and A. Ordureau for reagents, help and discussion on mass spectrometry, and members of the D.K. laboratory for reagents and discussions. We are grateful for the support of B. Schulman, M. Mann and the Max Planck Institute in the final stages of manuscript preparation. This work was supported by the Medical Research Council (U105192732), the European Research Council (724804), the Lister Institute for Preventive Medicine (D.K.), and grants P 24038 and P 28183 from the Austrian Science Fund (T.S.). J.L.U. is funded by a Gates Cambridge Scholarship.

Author information

Author notes

  1. Kirby N. Swatek

    Present address: Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany

  2. Christina Gladkova

    Present address: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

  3. Tycho E. T. Mevissen

    Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

  4. Jonathan N. Pruneda

    Present address: Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Kirby N. Swatek, Joanne L. Usher, Anja F. Kueck, Christina Gladkova, Tycho E. T. Mevissen, Jonathan N. Pruneda & David Komander

  2. Department of Medical Biochemistry, Max Perutz Labs, Vienna Biocenter, Medical University of Vienna, Vienna, Austria

    Tim Skern

  3. Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    David Komander

  4. Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia

    David Komander

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Contributions

K.N.S. conceived and designed the study, performed most of the experiments, interpreted results and wrote the manuscript. J.L.U. performed mitochondrial experiments, helped to annotate fragmentation patterns of branched ubiquitin, and performed control experiments. A.F.K. helped to perform the quantification of ubiquitin species from whole-cell lysates. C.G. contributed to the kinetic characterization of Lbpro∗. T.E.T.M. provided critical reagents. J.N.P. contributed to the characterization of Lbpro and Lbpro∗, and provided structural and ubiquitin biology expertise. T.S. provided Lbpro, protocols and acquired funding. D.K. conceived and designed the study, interpreted results, wrote the manuscript and acquired funding.

Corresponding author

Correspondence to David Komander.

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Peer review information Nature thanks Don Kirkpatrick, Miratul Muqit and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Characterization of Lbpro ubiquitin cleavage.

a, Representative raw spectrum of Lbpro-treated Lys48-linked diubiquitin analysed by electrospray ionization MS. Two species arise owing to internal cleavage of ubiquitin after Arg74. One scan is shown from experiments performed in technical triplicate. b, After 24 h of Lbpro treatment, diubiquitin was further supplemented with fresh Lbpro and incubated for an additional 24 h. There are no changes in the intensities of ubiquitin bands, which suggests that Lbpro products are stable. Lys27 diubiquitin in this panel and in Fig. 1a was generated chemically from synthetically produced ubiquitin and was refolded; this generates a variable fraction of substrate that cannot be hydrolysed by deubiquitinases (DUBs), leading to apparent lower activity due to incomplete hydrolysis. Diubiquitin cleavage assays were performed independently in duplicate. c, Model of ubiquitin cleavage by Lbpro. Ubiquitin (green) was modelled on the basis of the crystal structure of Lbpro (blue) bound to the C-terminal domain of ISG15 (PDB: 6FFA19). A close-up view shows the C terminus of ubiquitin placed across the active site, enabling cleavage between Arg74 and Gly75.

Extended Data Fig. 2 Engineered Lbpro has enhanced activity against ubiquitin.

a, Left, structural model of ubiquitin-bound Lbpro as in Extended Data Fig. 1c with ubiquitin under a green surface. Top right, close-up view of the ubiquitin Ile44 patch (Leu8, Ile44, His68, Val70) and its predicted interactions with Lbpro. Differences in the equivalent surface in ISG15 explain the higher affinity of ISG15 than ubiquitin for Lbpro19. Bottom right, modelling of an improved hydrophobic contact between Lbpro and ubiquitin. The corresponding Lbpro mutant (with a L102W mutation) is denoted as Lbpro∗. b, Diubiquitin cleavage assays, as in Fig. 1a. The cleavage of each diubiquitin (diUb) linkage type was compared for Lbpro and Lbpro∗. Assays were performed independently in duplicate. c, Example ubiquitin-KG-TAMRA cleavage assays comparing Lbpro (left) and Lbpro∗ (right), the difference in polarization relative to a substrate-only negative control is shown. The average trace of assays performed in technical triplicate is shown. TAMRA-KG represents a cleaved product positive control. d, Catalytic efficiencies derived from two independent sets of ubiquitin-KG-TAMRA cleavage measurements as in c. Slope and error values derived from linear regression are reported for each replicate individually. Fold improvement of Lbpro∗ over Lbpro in catalytic efficiency towards this substrate is indicated. e, Example diubiquitin K63-FlAsH cleavage assays as in c. f, Catalytic efficiencies derived from two independent sets of diubiquitin K63-FlAsH cleavage measurements as in e. Slope and error values derived from linear regression are reported for each replicate individually. Fold improvement of Lbpro∗ over Lbpro in catalytic efficiency towards this substrate is indicated. The Rep1 [Lbpro] = 7.81 μM data point has been excluded, as reliable exponential decay parameters could not be fitted for this point. g, Branched ubiquitin cleavage assays. Three different branched ubiquitin chains (Lys6/Lys48; Lys11/Lys48; Lys11/Lys63, described in ref. 49) were used in Lbpro (left) and Lbpro∗ (right) cleavage assays. Assays were performed independently in triplicate. h, Catalytic efficiencies derived from gel-based analysis of three independent Lys6/Lys48 branched triubiquitin cleavage assays performed at three different enzyme concentrations. The fold improvement of Lbpro∗ over Lbpro in catalytic efficiency towards this substrate is indicated. The centre values correspond to the mean of independent experiments performed in triplicate (n = 3); error bars represent s.d. from the mean. Slope and error values derived from linear regression based on the mean values are reported. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 3 Lbpro cleavage of ubiquitin chains from in vitro assembly reactions.

To test the activity of Lbpro on different chain topologies, ubiquitin chain assembly systems that produce various chain types in vitro were analysed using Ub-clipping. Cleavage of polyubiquitin by Lbpro was followed by SDS–PAGE with silver staining and anti-ubiquitin western blotting (top), and the produced monoubiquitin band was excised from a Coomassie-stained gel and analysed by trypsin digest and AQUA MS (bottom). Gel-based assays were performed independently in duplicate and AQUA MS was performed in technical duplicate. a, The bacterial HECT-like E3 ligase NleL was used with UBE2L3 and ubiquitin(K48R) to produce Lys6-linked chains39. b, The E2 enzyme UBE2S lacking the C-terminal Lys-rich sequence (UBE2S(ΔC)) assembles predominantly Lys11 linkages38. c, The HECT E3 ligase AREL1 with UBE2L3 assembles Lys11-, Lys33- and Lys48-linked polyubiquitin37. d, Reaction with NleL/UBE2L3 as in a but using the ubiquitin(K6R) mutant39. e, The HECT E3 ligase NEDD4L assembles Lys63-linked polyubiquitin50. Also see Extended Data Fig. 5a. f, The RBR E3 ligase HOIP assembles Met1-linked polyubiquitin51.

Source data

Extended Data Fig. 4 Characterization of branched ubiquitin chains with Ub-clipping.

a, Quantitative intact MS analysis for a branched triubiquitin, in which one ubiquitin moiety is modified on both Lys11 and Lys48 (ref. 49). Lbpro cleavage generates the expected ratio of 1:2 for double-GlyGly and unmodified ubiquitin species. Intact MS analysis was performed independently in triplicate. b, In vitro assembly reactions from Fig. 2b, c. Experiments were performed independently in triplicate. c, Isotopic distribution of unmodified and different GlyGly-modified ubiquitin species from a Lbpro-treated TRAF6 assembly reaction with UBE2D3 (see b and Fig. 2b). Select spectra are shown from independent experiments performed in triplicate. d, Intact MS analysis of in vitro TRAF6 assembly reactions from b. Samples were separated by liquid chromatography (LC) before MS analysis and spectra deconvolution. LC–MS allowed for the detection of a ubiquitin species with four GlyGly modifications. These experiments were performed three times independently with similar results. e, LC–MS parallel reaction monitoring analysis of double-GlyGly-modified ubiquitin as produced by TRAF6 in b, to confirm complete clipping of the C-terminal GlyGly by Lbpro. Green, y ions; red, b ions; purple, internal ions; yellow, neutral loss ions. Experiments were performed independently in triplicate.

Extended Data Fig. 5 TUBE-purified ubiquitin from in vitro assembly reactions.

a, NEDD4L-assembled polyubiquitin chains (see Extended Data Fig. 3e) were purified by glutathione S-transferase (GST)-TUBE pull-downs (left) and treated with Lbpro∗ (right). TUBE pull-down enriches chains and removes mono- and short polyubiquitin from the reaction (left, bound), and Lbpro∗ cleaves TUBE-bound ubiquitin as monitored by anti-ubiquitin western blots. Experiments were performed independently in triplicate. b, As in a, using NleL for an E3 ligase known to make branched polyubiquitin39. Coomassie-stained assays were performed independently in triplicate and western blots independently in duplicate. c, Intact MS analysis of samples from a shows the relative amounts of each identified ubiquitin species. A ratio of 3:1 for GlyGly-modified:unmodified ubiquitin from the NEDD4L-assembled polyubiquitin smear suggests that the average chain length in the reaction is four ubiquitin molecules. d, Samples from b were analysed as in c. A substantial fraction of branched ubiquitin suggests that a large percentage of all chains in the reaction are branched. Relative quantities of roughly 2:5:1 for unmodified:GlyGly-modified:double-GlyGly modified ubiquitin lead to potential ubiquitinated species including the one depicted schematically. The caveat with this model is that chains in the reaction account for a range of lengths. Assays from c and d were performed independently in duplicate.

Source data

Extended Data Fig. 6 Characterization of Ub-clipping in cells.

a, b Whole-cell lysates of HeLa (a) or HEK293 (b) cells were treated with Lbpro and analysed by anti-ubiquitin western blots (compare with Fig. 3a). Experiments were performed independently in triplicate. c, Assessment of endogenous ubiquitin ligase or deubiquitinase activity during Lbpro treatment. HEK293 and HeLa lysates were incubated with fluorescently labelled monoubiquitin, Lys11-linked diubiquitin, and Lys48-linked diubiquitin for the indicated time points. Despite being competent for ligation and susceptible to cleavage by DUB (left), after 24 h incubation there was no visible DUB or ligase activity, even at high exposures (right). This experiment was performed independently in triplicate. d, Workflow of Lbpro ubiquitin purifications from whole-cell lysates. e, Recovery of monoubiquitin after precipitation (dialysis in water) as shown in the workflow from d. Western blots were performed on one of three independent experiments. f, Representative purification of ubiquitin from HCT116 cells. Samples were analysed by intact MS enabled by the lack of background protein. These experiments were performed three times independently with similar results. g, Ubiquitin purified from HeLa and HEK293 cells in this manner (see d–f) was analysed by AQUA MS. A representative example of independent experiments performed in triplicate is shown. Relative amounts of ubiquitin chain linkages are very similar to whole-cell lysate tryptic digests23. h, Lbpro-generated monoubiquitin bands purified as in f were excised and analysed using a shotgun proteomics approach to identify contaminant proteins. Experiments were performed independently in triplicate.

Source data

Extended Data Fig. 7 Intact mass spectrometry and parallel reaction monitoring analysis of Lbpro-treated whole-cell lysates.

a, Deconvolution of intact MS spectra for ubiquitin from HEK293 (left) and HeLa (right) cells, as in Fig. 3c. b–d, Parallel reaction monitoring assay of branched ubiquitin isolated from HCT116 (b), HEK293 (c) and HeLa (d) cells. The mass corresponding to a +12 charge state of branched species (2 × GG) was isolated and fragmented. Product ions are assigned to the amino acid sequence of ubiquitin, and coloured as in Extended Data Fig. 4e. This control shows that the double-GlyGly-modified ubiquitin originates from a branched species and not from single-GlyGly-modified ubiquitin with an intact C terminus. All experiments were performed three times independently with similar results.

Extended Data Fig. 8 Characterization of TUBE pull-downs from cells.

a, b Efficiency of polyubiquitin depletion by TUBE pull-down from HeLa (a) and HEK293 (b) cells. Before the addition of the glutathione affinity resin, an input sample was taken (lysate). Samples of the supernatant after incubation with the affinity resin and of the beads after washing with PBS were analysed by ubiquitin western blotting. Experiments in a and b were performed independently in triplicate. c, Intact MS analysis of TUBE-purified ubiquitin species. The isotopic distribution of the charge state z = +12 is shown. Distinct GlyGly-modified ubiquitin species are easily distinguishable by mass. d, Peak integration of the nominal mass of each ubiquitin species (unmodified, 1 × GG, 2 × GG) from LC–MS analysis. Distinct GlyGly-modified ubiquitin species are also distinguishable by their chromatographic behaviour. Experiments in c and d were performed independently in triplicate. These results are consistent with previous data using limited trypsinolysis18; we consistently identified around 2–3% more branched ubiquitin, possibly owing to differences in ubiquitin enrichment strategies or to partial digestion of ubiquitin by trypsin. e, Data from HeLa cells (Fig. 3f), when applied to a finite pool of 10 ubiquitin moieties, correspond roughly to a collection of 4 × unmodified ubiquitin (white), 5 × GlyGly modified ubiquitin (green) and 1 × double-GlyGly modified, branch-point ubiquitin. With these ratios, several distinct species can be assembled, some of which are depicted. It is clear that a single branch-point ubiquitin in this case requires 3 of the 10 ubiquitin molecules (that is, 30%) to be in a branched chain. This number can be higher if the branched chain also incorporates mono-GlyGly-modified species. For expanded systems (with, for example, double the number of ubiquitins, and 2 × double-GlyGly-modified ubiquitin) this number can also be lower when, for example, multiple branched species exist in the same polymer (in which case a chain can be built from 5 out of 20 ubiquitin molecules—that is, 25% of ubiquitin is in branched architectures). More definitive assessments of individual architectures would be possible when individual lengths of chains—for example, tetra-ubiquitin—could be analysed with this method. Centred values correspond to the mean of independent experiments performed in triplicate (n = 3). Error values represent s.d. from the mean.

Extended Data Fig. 9 Cleavage efficiency of phospho-polyubiquitin chains and MS analysis of parkin assembly reactions.

a, In vitro assemblies of polyubiquitin from ubiquitin and Ser65 phosphoubiquitin using cIAP1 and UBE2D3 as previously described11, and their cleavage by Lbpro. Cleavage efficiency was visualized by Coomassie staining. Experiments were independently performed in duplicate with similar results. b, Samples from a visualized by anti-ubiquitin western blotting. Experiments were independently performed in duplicate with similar results. c, Polyubiquitin assembled by Ser65-phosphorylated human parkin (pParkin) with UBE2L3 was purified by GST–TUBE pull-downs. Assembly reactions were performed in the absence (−) and presence (+) of 10% total phosphoubiquitin. Experiments were performed independently in triplicate. d, TUBE-purified parkin reactions from c were analysed by AQUA MS to determine chain-linkage composition. A representative example of independent experiments performed in triplicate is shown. e, Lbpro-treated parkin assembly reactions were analysed by intact MS and spectra deconvolution. A small phosphoubiquitin contamination is present owing to residual PINK1 kinase in the assembly reactions. In these assays, parkin predominantly assembles monoubiquitin and short chains. Intact MS analysis was performed independently in duplicate. f, To detect a non-ubiquitin substrate with multiple GlyGly modifications by intact MS, in vitro assemblies were performed as per e in the absence of phosphoubiquitin, and with a higher UBE2L3 concentration to facilitate the analysis of UBE2L3. Lbpro-treated assemblies were analysed by LC–MS, revealing up to four GlyGly modifications on a single UBE2L3 molecule. Inset, the distribution of GlyGly-modified UBE2L3 in the +21 charge state. The experiment was performed independently in triplicate.

Source data

Extended Data Fig. 10 Characterization of parkin-overexpressing cells.

a, Experiments were performed with doxycycline-inducible HeLa Flp-In T-REx cells expressing parkin proteins (a gift from A. Ordureau and W. Harper, Harvard). Cells were treated with 0.1 μg ml−1 doxycycline for 16 h and parkin expression was monitored by anti-parkin western blots. Western blots were performed on one of three independent experiments. b, TUBE enrichment of polyubiquitin from a after CCCP treatment. Also see Fig. 4a. Western blots were performed on one of three independent experiments. c, Top, AQUA analysis for total ubiquitin, ubiquitin chains, and phosphoubiquitin using TUBE pull-downs from CCCP-treated parkin-overexpressing cell lines (see a, b, Fig. 4a). TUBE-purified ubiquitin chains were treated with Lbpro∗, separated by SDS–PAGE, and the monoubiquitin band excised and subjected to AQUA MS analysis. Bottom, the percentages calculated from the top panel. Values correspond to the mean of independent experiments performed in triplicate (n = 3). Error values represent s.d. from the mean. d, Quantification of ubiquitin species from the parkin(S65A) cell line, as in Fig. 4d. Centre values correspond to the mean of independent experiments performed in triplicate (n = 3). Error values represent s.d. from the mean. e, Sodium carbonate (Na2CO3) treatment of mitochondria and Lbpro cleavage assays. Addition of Na2CO3 releases peripheral mitochondria membrane proteins and unconjugated free monoubiquitin. Untreated and Na2CO3-treated mitochondria were digested with Lbpro for 2.5 h at 37 °C. After incubation for 2.5 h, the supernatant (S) and pellet (P) were analysed by anti-ubiquitin western blots. Without Na2CO3, incubation at 37 °C releases ubiquitin from mitochondria into the supernatant, presumably owing to residual DUB activity. Western blots were performed on one of three independent experiments. f, Left and middle, identification of non-ubiquitinated phosphoubiquitin by intact MS analysis from Lbpro-treated samples in e. The isotopic distribution of the charge state z = +12 is shown for parkin wild-type and parkin(C431S) cell lines. Right, the fold-increase in phosphoubiquitin comparing parkin(C431S) and wild-type cell lines, as measured by spectra deconvolution. A considerable amount of phosphoubiquitin (10% of the total ubiquitin) can be detected with this method in cells expressing inactive parkin. Centre values correspond to the mean of independent experiments performed in triplicate (n = 3). g, To exclude the possibility of contaminating phosphatase activity during incubation with Lbpro∗, sodium carbonate-extracted mitochondrial samples were treated as in Fig. 4h and spiked with 15N-labelled phosphoubiquitin, and formation of 15N-labelled unphosphorylated ubiquitin was monitored. As indicated, no detectable formation of a 15N-labelled unphosphorylated ubiquitin species was observed after overnight incubation with Lbpro∗. Experiments were performed in biological triplicate. h, As in Extended Data Fig. 8e, the data from Fig. 4h is applied to a pool of 20 ubiquitin molecules, in which eight are unmodified, five are GlyGly-modified, one is double-GlyGly modified, and six are phosphorylated. This simplified system would allow for a distribution of chains as depicted schematically, and would indicate that chains on mitochondria are short and phosphorylated on the tips of the chain. Recent site occupancy mapping—performed in the same cell system32—revealed abundant ubiquitination sites in particular on VDAC proteins, enabling us to model the ubiquitin coat during mitophagy, as depicted. Centre values correspond to the mean of independent experiments performed in triplicate. Error values represent s.d. from the mean.

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Swatek, K.N., Usher, J.L., Kueck, A.F. et al. Insights into ubiquitin chain architecture using Ub-clipping. Nature 572, 533–537 (2019). https://doi.org/10.1038/s41586-019-1482-y

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