Insights into the Molecular Composition of Endogenous Unanchored

Article pubs.acs.org/jpr

Insights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains Joanna Strachan,†,∥ Lucy Roach,‡,∥ Kleitos Sokratous,‡ David Tooth,† Jed Long,‡,§ Thomas P. Garner,‡,§ Mark S. Searle,‡,§ Neil J. Oldham,‡ and Robert Layfield*,† †

School of Biomedical Sciences, ‡School of Chemistry, and §Centre for Biomolecular Sciences, University of Nottingham, United Kingdom S Supporting Information *

ABSTRACT: The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC−MS and LC−MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC−MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo. KEYWORDS: ubiquitin, unanchored polyubiquitin, top-down mass spectrometry, ZNF216

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been identified within these ubiquitin receptors,4 which are capable of recognizing different interaction faces or sites on ubiquitin as well as distinct polyubiquitin topologies.5 Intrinsic binding specificities of ubiquitin receptors and their UBDs can be exploited in the purification of ubiquitin-modified proteins,6,7 in some cases affording proteomic analyses of targets of ubiquitination.8 Existing protein MS approaches, based on bottom-up MS/MS analyses of tryptic peptides, have been employed to identify the presence of different isopeptide linkages in polyubiquitin modifications.9,10 Those lysine residues in ubiquitin involved in isopeptide bonds are not cleaved by trypsin and remain attached to the diglycine fragment derived from the linked C-terminus of the neighboring ubiquitin, producing a characteristic signature peptide for each linkage. Although this approach has demonstrated the presence of all seven isopeptide linkages of polyubiquitin in yeast11 and mammalian cells,12 it does not permit mapping of the absolute topology of polyubiquitin chains in terms of the isopeptide linkages present and the order in which they assembled, since there is a loss of connectivity

INTRODUCTION Typically the small protein ubiquitin acts as a covalent and selective post-translational modifier of other proteins. The precise nature of the ubiquitin modification determines the fate of the target protein and the biological process regulated.1 The archetypal ubiquitin-mediated process involves modification with polyubiquitin chains linked via isopeptide bonds through ubiquitin’s lysine residue 48 (K48-linked polyubiquitination), which can signal the target for degradation by the proteasome.2 Notably, however, all seven of ubiquitin’s lysine residues can participate in polyubiquitin chain formation, involving homotypic (all identical) isopeptide linkages, as well as mixed or forked linkages where a single ubiquitin links to multiple other ubiquitins. In addition ubiquitin’s N-terminal Met can form peptide linkages with other ubiquitins to generate linear polyubiquitin chains. This complex polyubiquitin code also regulates diverse processes such as NF-κB signaling (often involving K63 or linear polyubiquitination), DNA repair,1 and protein degradation by autophagy.3 Ubiquitin modifications are transduced by ubiquitin receptors, intracellular proteins that interact noncovalently with ubiquitin and ubiquitin-modified proteins. More than 20 different families of ubiquitin-binding domains (UBDs) have © 2012 American Chemical Society

Received: November 25, 2011 Published: January 23, 2012 1969

dx.doi.org/10.1021/pr201167n | J. Proteome Res. 2012, 11, 1969−1980

Journal of Proteome Research

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the ability of unanchored K48-linked polyubiquitin chains to competitively inhibit the 26S proteasome was indicated from earlier in vitro studies.24 The notion of the existence of biologically relevant unanchored polyubiquitin chains was also previously suggested from the characterization of enzymes (in addition to isoT) that specifically regulate the assembly/ disassembly of free polyubiquitin; in yeast a protein Rfu1 (regulator of free ubiquitin chains 1) acts as an inhibitor of the Doa4 DUB enzyme.25 Overexpression of Rfu1 causes accumulation of “short” unanchored polyubiquitin chains, whereas deletion of Rfu1 results in depletion of these chains and accumulation of free monoubiquitin, indicating that some forms of unanchored polyubiquitin could act as a regulated ubiquitin reservoir.25 In summary, the existence of physiologically relevant pools of unanchored polyubiquitin is supported by all of the studies described above. However, to date no direct molecular analyses of endogenous unanchored polyubiquitin chains to identify isopeptide linkage types and chain lengths have been performed. Here we describe the affinity purification of endogenous unanchored polyubiquitin chains of different lengths from tissue extracts using affinity chromatography strategies based on the use of different UBDs. Further, MS analysis is shown to be a powerful technique in the determination of their composition.

between linkage sites in the chain. An AQUA (absolute quantification) method for the analysis of linkage types has been used to determine the percentage composition of polyubiquitin linkages attached to individual proteins or within mixtures;13,14 however, once again this relies on tryptic digestion. Although middle-down MS strategies using limited protease digestion have been developed for identifying the absolute topologies of polyubiquitin chains,15 the potential of top-down proteomics (MS analyses of intact proteins without the use of proteases) to study connectivity within ubiquitin modifications has not yet been explored. The common assumption that the influences of ubiquitin are mediated exclusively via the modification of other proteins has recently been challenged with the demonstration of a role for unanchored polyubiquitin chains in the direct activation of certain protein kinases. TRAF6 is an E3 ubiquitin-ligase essential for the activation of NF-κB in response to various cytokines, and the ubiquitin species responsible for activating TAK1 kinase in a TRAF6-dependent in vitro reconstituted system were identified as unanchored K63-linked polyubiquitin chains.16 In addition, evidence was presented that unanchored chains with isopeptide linkages other than K63 and K48 are also able to activate the I-κB kinase (IKK) complex. However the existence of unanchored polyubiquitin in a cellular context was only indirectly inferred from the use of the deubiquitinating (DUB) enzyme USP5/isopeptidase-T (isoT),16 a chain disassembling enzyme exhibiting high-affinity binding to the free C-terminal tail of ubiquitin.17 Subsequent work also established a role for unanchored polyubiquitin in innate immunity.18 In this case, endogenous unanchored polyubiquitin chains (shown to be K63-linked based on the expression of a K63R ubiquitin mutant) were found to bind directly to RIG-I and activate the IRF3 signaling cascade. These polyubiquitin chains were determined to be unanchored based on their ability to form thiolester intermediates with the E1 ubiquitin-activating enzyme. The presence of K63-linkages was further supported from their observed disassembly by CYLD, a DUB selective for K63-linked polyubiquitin, and the use of antibodies specific for the K63 linkage.18 More recently, an involvement for free polyubiquitin chains in TRIM5-mediated innate immune signaling has been demonstrated, which is again associated with activation of TAK1 by free chains.19 TRIM5 is an E3 ubiquitin-ligase, and in this case a TRIM5-CypA fusion protein was shown, using in vitro conjugation reactions, to generate polyubiquitin chains reactive with K63-ubiquitin-specific antibodies. This study also presented MS evidence for the K63linkage within the in vitro generated chains (MS/MS sequencing of tryptic peptides) and failed to detect signature peptides associated with other polyubiquitin linkages. Finally, a very recent study proposed a role for unanchored polyubiquitin in the recruitment of protein aggregates (via HDAC6) to intracellular aggresomes.20 In yeast, deletion of the UBP14 gene (a functional homologue of isoT) was previously shown to result in a striking accumulation of unanchored polyubiquitin with defects in ubiquitin-dependent proteolysis,21 and unanchored polyubiquitin chains have recently been noted to be abundant in yeast during vigorous growth, again evidenced through the use of isoT-mediated deubiquitination.22 Other work also confirmed that suppression of isoT results in the accumulation of unanchored polyubiquitin chains in mammalian cells, accompanied by an increase in the level and transcriptional activity of p53 due to impaired ubiquitin-dependent proteolysis.23 Indeed

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EXPERIMENTAL SECTION

Plasmids and Recombinant Protein Expression

The protein coding region of full length rat ZNF216 (residues 1−213), a ubiquitin receptor, was PCR amplified from rat skeletal muscle cDNA. The PCR product was ligated in to the BamHI/XhoI sites of pGEX-4T-1 (GE Healthcare) to allow expression of N-terminal glutathione S-transferase (GST)tagged full length ZNF216 protein. The A20 Znf construct was generated by introducing a premature stop codon after residue 60 using QuikChange site-directed mutagenesis (Stratagene).26 Primers were also designed to clone the Znf UBP (BUZ) domain (residues 163−291) of full-length human isoT. The Znf UBP domain was PCR amplified from human U20S (osteosarcoma cells) cDNA and was also ligated into the BamHI/XhoI sites of pGEX-4T-1. Constructs were verified using DNA sequencing. GST-tagged proteins were expressed in E. coli strain XL10-Gold cells (Stratagene) in Luria broth (Sigma). Cells were pelleted and lysed by sonication in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100 (TBST). After subsequent centrifugation (35,000g for 30 min at 4 °C), the pelleted insoluble material was discarded, and soluble GST-tagged proteins were purified using glutathione Sepharose 4B (GE Healthcare). The A20 Znf or Znf UBP peptides were eluted from glutathione Sepharose 4B via cleavage with thrombin, and protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific), according to the manufacturer’s protocol. A20 Znf Domain Affinity Chromatography

Thrombin-cleaved A20 Znf peptide was immobilized using Cyanogen bromide-activated Sepharose 4B (CNBr Seph 4B) (GE Healthcare; protein on beads at 5 mg/mL) and used to capture proteins from rat skeletal muscle. Polyubiquitinated proteins were removed by treatment with the USP2 DUB catalytic core (ENZO Life Sciences). Mixed hindlimb skeletal muscle, including soleus, extensor digitorum longus, gastrocnemius, and plantaris, was collected from normal male Lister1970



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hooded rats (125−150 g; Charles River, U.K.) immediately post mortem27 and stored at −80 °C; all procedures were approved by the University of Nottingham Ethical Review Committee and were performed under Home Office Project and Personal license authority. Typically ∼15 g of frozen tissue was homogenized in 100 mL of homogenizing buffer comprising 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% (v/v) NP-40, 5 mM N-ethylmaleimide (NEM), 20 μM MG-132, 0.1% (v/v) mammalian protease inhibitor cocktail (Sigma). Homogenates were centrifuged at 35,000g for 30 min at 4 °C, typically yielding a final protein concentration of ∼3 mg/mL in the clarified supernatant. DTT was added to the supernatant to a final concentration of 10 mM (to react with excess NEM) followed by mixing at 4 °C for 15 min. Supernatant (∼80 mL/ 240 mg) was passed twice through 800 μL of A20 ZnfSepharose at 4 °C. Beads were thoroughly washed in wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% (v/v) NP-40, 1 mM DTT) and incubated in DUB buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT; equal volume solution to beads) with or without 10 μg of USP2 catalytic core for 1 h at 37 °C. Eluant was collected from beads, two further equal volume DUB buffer washes were performed, and the washes were pooled. Proteins retained on beads were eluted with gel loading buffer and separated by SDS-PAGE using 5−20% gradient gels and then visualized by either Coomassie staining (protein from 100 μL of beads) or immunoblotted with anti-ubiquitin (in house antibody;6 protein from 10 μL of beads). USP2 eluates were also immunoblotted with anti-ubiquitin (30 μL of pooled USP2 eluant). Commercial K48- and K63-linked polyubiquitin standards were from ENZO Life Sciences. The muscle-derived Coomassie-stained diubiquitin band visualized by SDS-PAGE was tryptically digested (see Trypsin digestion, below) and directed for MS/MS analysis. Diubiquitin eluted from 50 μL of USP2-treated A20 Znf beads (not tryptically digested) was also submitted for intact mass/top-down MS analyses (see NanoLC−MS and MS/MS analyses, below).

every 5 min. In parallel, beads were also incubated with 0.125 μg of Ub5+1 (ENZO Life Sciences) under identical conditions. Beads were thoroughly washed in binding buffer, and bound proteins were eluted with gel loading buffer. Eluted proteins were separated by SDS-PAGE and detected by Western blotting with anti-ubiquitin. Trypsin Digestion

Gel bands were manually excised and then subjected to trypsinolysis using standard automated robotic procedures. Briefly, bands were incubated with 1 mM DTT followed by 2 mM iodoacetamide in 0.1 M ammonium bicarbonate, 50% (v/ v) acetonitrile, with interim washing. Gel pieces were then washed with acetonitrile, dried, and rehydrated with appropriate volumes of sequencing grade porcine trypsin (Promega), at a ratio of approximately 1:10 enzyme/substrate in aqueous 0.1 M ammonium bicarbonate pH 8.0. Proteolytic peptides were extracted by recovering sequential washes with 0.1% formic acid(aq) and 0.1% formic acid in 70% acetonitrile(aq), dried, and resuspended in 4% acetonitrile, 0.1% formic acid(aq). NanoLC−MS and MS/MS Analyses

For the MS analyses, HPLC grade acetonitrile and formic acid were purchased from Fisher Scientific, and ultra pure water was generated using a Millipore Q water purification system. Monoubiquitin supplied by Sigma and lyophilized diubiquitin (K48-, K63-, K11-, and K27-linked; Boston Biochem) were prepared as a 1 μM samples in 0.1% formic acid in 95:5 water/ acetonitrile (v/v). For analysis of A20 Znf domain-purified ubiquitin dimers, samples were eluted from the USP2-treated beads (50 μL) using 0.1% formic acid, vortexed, and incubated at room temperature (∼20 °C) for 30 min followed by centrifugation (6800g for 10 min). An aliquot of the supernatant was removed and diluted 5-fold with 0.1% formic acid in 95:5 water/acetonitrile and transferred into polypropylene vials (Dionex) for analysis by capillary reversedphase-nanoLC (Ultimate 3000, Dionex) with subsequent MS and MS/MS analysis. Purified ubiquitin dimer samples and standards (K48-, K63-, K27-, and K11-linked) were concentrated on a (300 μm i.d. × 5 mm) trapping column packed with C4 PepMap300 (Dionex) using mobile phase A: water/ acetonitrile (95:5, v/v) with 0.1% formic acid delivered at 20 μL min−1. The trapping column was switched in-line with the analytical column after a 3 min loading time. Chromatographic separation was performed using either a reverse phase C4 column (length 15 cm × 75 μm i.d., manufactured in house) or a reverse-phase C18 column (manufactured in house, length 25 cm × 75 μm i.d., 3 μm Jupiter resin) and loading columns of appropriate phase. Samples were eluted from C4 and C18 media using a linear gradient of B, a mixture of acetonitrile/ water (95:5) with 0.1% formic acid, in buffer A, as follows: from 0% to 70% of buffer B in 30 min, switched to 90% buffer B for 5 min, followed by 15 min re-equilibration with buffer A at a constant flow rate of 0.2 μL min−1. The nanoLC was interfaced with an LTQ FT-Ultra mass spectrometer (ThermoFisher), which was operated in positive ion mode and equipped with a standard Thermo nanospray ion source. MS instrumental conditions were optimized using a diubiquitin standard. The heated capillary temperature was set at 200 °C, the spray voltage was set at 1.6 kV, and the capillary voltage was held at 43 V. Data were acquired for K48-, K63-, K27-, and K11-linked diubiquitin standards following LC separation using full scan mode (m/z 300−2000). Collision-induced dissociation (CID) of selected protein charge states (15+, 17+, and 19+) was

Znf UBP Domain Affinity Chromatography

Thrombin-cleaved Znf UBP domain was immobilized on CNBr Seph 4B (protein on beads at 10 mg/mL) to capture rat skeletal muscle unanchored polyubiquitin chains in a protocol identical to A20 Znf domain affinity chromatography, except beads were not USP2 treated. Proteins captured on beads were separated by SDS-PAGE using 5−20% gradient gels and again visualized by Coomassie staining (protein from 100 μL of beads) or immunoblotted with anti-ubiquitin and anti-K48polyubiquitin (Millipore) antibodies (protein from 10 μL of beads). Tryptically digested diubiquitin and tetraubiquitin gel bands were analyzed by MS/MS. When the sample heating step was included before affinity chromatography, muscle extract was heated (75 °C, 20 min) and centrifuged (35,000g for 30 min at 4 °C). The Znf UBP domain was confirmed to specifically bind ubiquitin with a free C-terminus using Ub5+1, a polymer in which a K48-Ub4 chain is conjugated to Ub1+, a frameshift mutant ubiquitin in which the C-terminal G76 of is replaced with a 20-residue (+1) nonsense sequence peptide extension so the free ubiquitin C-terminus is lost.28 CNBr Seph 4B-immobilized Znf UBP or A20 Znf domain or “control” CNBr Seph 4B beads with no protein coupled (all 50 μL) were incubated with 0.25 μg of commercial K48-linked polyubiquitin chains in binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% (v/v) NP-40, 0.1% (w/v) bovine serum albumin (Sigma), 1 mM DTT) for 30 min at 4 °C with gentle agitation 1971



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performed in the linear ion trap, and fragments were subsequently transferred and measured in the ICR cell. An isolation width of m/z = 8 was used for selected protein charge states, which were subsequently activated for 30 ms using 18% normalized collision energy and an activation q of 0.25. A mass resolving power of 200,000 at m/z 400 was used, and Automatic Gain Control (AGC) was applied in all data acquisition modes. Identical parameters were used for the LC− MS and LC−MS/MS analyses of the intact ubiquitin dimers purified from rat skeletal muscle. For bottom-up analyses, peptides generated from in gel digestion of diubiquitin standards and purified polyubiquitin were subjected to LC− MS/MS. CID (conditions as above) and ECD using 6% electron energy and a 70 ms duration time with no delay were carried out. Data was processed using ThermoFisher Xcalibur software.

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RESULTS

A20 Znf Domain Allows Isolation of a Ubiquitin Dimer from Skeletal Muscle

Figure 1. Purification of an endogenous ubiquitin dimer from skeletal muscle using the A20 Znf domain. (A) Purification of polyubiquitinmodified proteins from rat skeletal muscle using the A20 Znf domain captured a complex mixture of polyubiquitinated proteins (P) detected by anti-ubiquitin Western blotting. Sample analyzed is equivalent to 1.25% (10 μL of beads) of the total material purified from 15 g of muscle. Load (Lo) is equivalent to 0.5% of clarified muscle lysate applied to 10 μL of beads. Note the high molecular weight smear of ubiquitinated proteins in the purified fractions and discrete lower molecular weight ubiquitin-positive bands, several of which co-migrate with components of commercial unanchored K48 and K63 linked polyubiquitin chain mixtures (denoted K48, K63; 0.25 μg per lane, numbers of ubiquitin moieties are indicated). A band with mobility similar to that of diubiquitin standards is marked with an arrow. (B) On-bead deubiquitination of A20 Znf domain-immobilized polyubiquitinated proteins as in panel A. Beads (400 μL) were incubated with (+) or without (−) USP2 (10 μg in 400 μL of buffer) for 1 h at 37 °C, and then beads (P, 10 μL) or elution (E, 30 μL/2.5%) was analyzed by anti-ubiquitin Western blotting. While the higher molecular weight ubiquitin-immunoreactive smear was effectively removed by USP2 treatment for 1 h, with a concomitant increase in monoubiquitin in the eluted fraction, the presumed ubiquitin dimer (arrow) was spared from disassembly.

We initially utilized the A20 Znf (A20-like zinc finger) UBD of the ubiquitin receptor ZNF216, which we have recently characterized in structural detail,26 to affinity purify ubiquitinated proteins. This domain is specific for the acidic D58 interaction surface on ubiquitin, binding to individual ubiquitin moieties with a Kd of ∼12 μM at 298 K,26 making it one of the higher affinity UBDs.29 We confirmed that pull-downs using covalently immobilized recombinant A20 Znf peptide allowed the capture of in vitro generated (commercial) unanchored K48-linked polyubiquitin chains containing two or more ubiquitin moieties, but not monoubiquitin (for example, see Figure 3A). As ZNF216 is functionally relevant in muscle, acting as a shuttle delivering ubiquitinated substrates to the proteasome,30 we attempted the purification of ubiquitinmodified proteins from rat skeletal muscle. Immobilized A20 Znf domain captured a complex mixture of polyubiquitinated proteins from muscle extracts, as assessed by anti-ubiquitin Western blotting (Figure 1A). A double mutant of the A20 Znf domain (C30A/C33A) previously shown to be defective in ubiquitin-binding30 was unable to capture polyubiquitin chains (not shown). The blots revealed a characteristic high molecular weight smear of ubiquitinated proteins in purified fractions, as well as several discrete lower molecular weight bands, which comigrated with components of commercial unanchored K48and K63-linked polyubiquitin chain mixtures. A prominent ubiquitin-immunoreactive band with mobility similar to that of diubiquitin standards (both K48- and K63-linked) was evident in the A20 Znf-purified fractions (Figure 1A, arrow), and we devised a strategy that allowed us to further analyze this protein, based on deubiquitination on beads using the catalytic core of the USP2 DUB.8,31 Specifically, we found that although the higher molecular weight ubiquitin-immunoreactive smear was effectively removed by USP2 treatment for 1 h, with a concomitant increase in monoubiquitin in the eluted fraction, the presumed ubiquitin dimer was spared from disassembly (Figure 1B) even upon overnight incubation. There was no apparent change in the intensity of the ubiquitin dimer band upon USP2 treatment, indicating the polyubiquitin processed by the DUB was converted to ubiquitin monomers. No monoubiquitin was generated by incubation of beads in the absence of USP2, confirming the absence of co-purifying DUB activity (Figure

1B). The ability to remove the majority of the polymeric ubiquitin from the sample, while retaining the presumed ubiquitin dimer, permitted its subsequent detailed MS characterization. MS Characterization of A20 Znf-Purified Diubiquitin

Following 1 h USP2 treatment of A20 Znf-immobilized rat muscle ubiquitinated proteins (as in Figure 1B), the remaining proteins bound to the beads were eluted with formic acid and subjected to accurate intact mass determination. NanoLC−MS showed the eluted sample consisted of predominately diubiquitin with some monoubiquitin (the latter being USP2derived and retained on beads) consistent with the Western blot data (Figure 1B). Further, the observed mass of a major component equivalent to diubiquitin (m/z 17102) was consistent with it being unmodified and unanchored, i.e., non-substrate-linked. A typical mass spectrum obtained under denaturing conditions is shown in Figure 2A. Diubiquitin was observed with charge states in the range 10+ to 21+. The peaks at m/z 714 and 779 correspond to the 12+ and 11+ charge states of monoubiquitin also present in the sample. For this reason the charge states selected for isolation and subsequent 1972



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Figure 2. MS analyses of the A20 Znf-purified ubiquitin dimer. (A) LC−ESI−MS spectrum of diubiquitin purified from rat skeletal muscle showing diubiquitin charge states from 10+ to 21+ and the presence of a small amount of monoubiquitin (11+ and 12+). (B) MS/MS spectrum of tryptic peptide 43−54 from the ubiquitin dimer resulting from CID of the [M + 2H]2+ at m/z 731.0. C. MS/MS spectrum of tryptic peptide 43−54 from the ubiquitin dimer resulting from ECD of the [M + 3H]3+ at m/z 487.7 unequivocally locating the linkage site to K48.

top-down MS/MS (see later) were those that correspond only to the diubiquitin. To investigate the linkage(s) present within the purified diubiquitin, we used a combination of MS/MS approaches. LC−MS/MS analysis of tryptic peptides derived from SDS-

PAGE-purified diubiquitin, using both CID and electroncapture dissociation (ECD) fragmentation, detected only the signature peptide corresponding to K48-linked ubiquitin (Figure 2B,C). We failed to detect peptides characteristic of any other isopeptide linkages (or N-terminally linked linear 1973



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polyubiquitin) despite detecting unbranched ubiquitin peptides (e.g., residues 12−17 and 64−72), nor did we detect peptide sequences corresponding to other proteins. These observations suggest the purified dimer to be free, unattached K48-linked diubiquitin. Further, digest MS/MS analyses of the diubiquitin band from purified samples that had not been treated with USP2 did not provide any evidence of other linkages (not shown), discounting the possibility that the ubiquitin dimer with this linkage was selectively protected from USP2-mediated deubiquitination. Purification of Longer Unanchored Polyubiquitin Chains Using the Znf UBP Domain

The ability to isolate unanchored K48-linked diubiquitin from rat skeletal muscle using the A20 Znf domain, combined with the recent descriptions of physiological roles for unanchored polyubiquitin chains,16,18−20 prompted us to develop a UBDbased affinity purification that would select only unanchored (poly)ubiquitin. We reasoned that this approach would also allow us to experimentally determine if the K48 linkage is predominantly used in ubiquitin dimers in vivo or whether the A20 Znf domain displays a linkage-selective bias in pull-downs from tissue extracts. In this case we made use of a different UBD, the Znf UBP (BUZ) domain of human isoT, which exhibits high-affinity binding to the free C-terminal tail of ubiquitin but does not bind to substrate-linked ubiquitin/polyubiquitin17 and, based on the structure of its complex with ubiquitin, is highly unlikely to exhibit any polyubiquitin linkage or chain length bias. Indeed, in vitro quantitative MS binding studies (to be reported elsewhere) revealed this to be the case. This UBD was utilized in the recently developed ubiquitin-PSAQ approach to selectively capture and allow quantification of free ubiquitin.31 We first confirmed that, when covalently immobilized on Sepharose beads, the recombinant Znf UBP peptide binds in vitro generated unanchored K48-linked polyubiquitin chains that contain a free ubiquitin C-terminus, as well as free monoubiquitin (Figure 3); for comparison binding of these chains to the Znf A20 domain is shown. In addition we showed that Ub5+1, a ubiquitin polymer in which the free C-terminus of the proximal ubiquitin is lost,28 readily binds to the A20 Znf beads but not the Znf UBP beads (Figure 3B). We then applied the immobilized Znf UBP domain to the affinity purification of rat skeletal muscle. In this case analysis of purified fractions by anti-ubiquitin Western blotting revealed discrete ubiquitin-positive bands, including a prominent ubiquitin dimer, and the complete absence of a high molecular weight smear equivalent to ubiquitinated proteins. These bands co-migrated with commercial polyubiquitin chain mixtures (in particular K48-linked standards) (Figure 4A). Unanchored polyubiquitin chains containing as many as 10 ubiquitin moieties were readily detected by ubiquitin antibodies, and chains were completely disassembled by incubation with isoT (not shown). For clarity, the abundant free monoubiquitin band (which is also purified by the Znf UBP domain) was omitted from the blots, and for comparison A20 Znf-purified ubiquitinated proteins prepared in parallel are also shown (Figure 4A). Ubiquitin-reactive bands similar to those purified from muscle were readily detected in fractions purified from different cultured human cells (e.g., U20S, HEK293; not shown) using the Znf UBP domain, suggesting that ubiquitin dimers and longer unanchored polyubiquitin chains are widely expressed.

Figure 3. Znf UBP domain specifically binds polyubiquitin chains with a free ubiquitin C-terminus. Znf UBP (UBP), A20 Znf (A20), or control-Sepharose (Con) (50 μL each) were incubated with (A) 0.25 μg of K48-linked commercial polyubiquitin chains or (B) 0.125 μg of Ub5+1, and bound proteins were detected by anti-ubiquitin Western blotting. Control Sepharose failed to bind any ubiquitin species; A20 Znf-Sepharose bound K48-linked unanchored polyubiquitin and Ub5+1. The Znf UBP domain bound only unanchored K48-linked polyubiquitin (and monoubiquitin) but not Ub5+1, which lacks a free ubiquitin C-terminus. K48 ‘Lo’ represents 100% of the input, Ub5+1 ‘Lo’ represents 50% of input.

Linkages within the Unanchored Polyubiquitin Chains

As the Znf UBP domain is predicted to be capable of capturing polyubiquitin irrespective of linkages,17 MS analyses of the ubiquitin dimer(s) purified in this case better inform as to which linkages occur in vivo. LC−MS/MS analysis of tryptic peptides derived from the gel-purified diubiquitin, again using both CID and ECD fragmentation, detected signature peptides from both K48 and K11 linkages (see Supplementary Figure 2). From a muscle-derived gel-purified band co-migrating with K48-tetraubiquitin standard we detected only the K48 signature by LC−MS/MS (not shown). Western blotting with K48specific polyubiquitin antibodies (which we found to preferentially bind to epitopes in the longer unanchored chains) confirmed the presence of the K48 linkage within the presumed tetraubiquitin band (on longer blot exposures) and also allowed us to detect chains containing as many as 15 ubiquitin moieties within the purified fractions (Figure 4B). To enable analyses of these longer unanchored chains, we noted that inclusion of a sample heating (75 °C, 20 min) and centrifugation step18 immediately before affinity purification, which presumably denatures endogenous ubiquitin receptors that compete for binding to the free chains while sparing the heat-stable polyubiquitin chains themselves, significantly increased recovery of the chains as assessed by Western blotting (Figure 4C). Notably, however, little or no immunoreactivity was detected in samples of purified chains from either untreated or heat-treated samples using K63specific polyubiquitin antibodies under conditions where we readily detected K63-linked standards (not shown). This suggests K63-linked unanchored chains to be absent or at a concentration too low to detect in these muscle-derived fractions. Top-Down MS of A20 Znf-Purified K48-Linked Diubiquitin

Although top-down MS offers the potential to provide insights into the connectivity within polyubiquitin modifications, such 1974



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Figure 4. Purification of unanchored polyubiquitin chains from skeletal muscle using the Znf UBP domain. Znf UBP (UBP) or A20 Znf (A20) Sepharose was incubated with rat skeletal muscle extract, and samples equivalent to 1.25% (10 μL of beads) of the total material purified from 15 g of muscle were probed with (A) anti-ubiquitin and (B) anti-K48-polyubiquitin antibodies. The Znf UBP domain captures a discrete ladder of ubiquitinated species, which co-migrate with bands within commercial K48- and K63-linked polyubiquitin chain mixtures (denoted K48, K63; 0.25 μg each). Purified polyubiquitin chains co-migrating with K48-linked polyubiquitin2−10 were detected with anti-ubiquitin; the ubiquitin dimer is indicated with an arrow. K48-linked polyubiquitin chains as long as Ub15 were detected in the purified fractions using K48-polyubiquitin-specific antibodies. (C) Optimized affinity capture with Znf UBP Sepharose was achieved by including an additional step, in which muscle extract was heated at 75 °C for 20 min prior to purification. Load (Lo) equivalent to 0.67% of muscle extract applied to 10 μL of beads. Purified proteins from 10 μL of beads were Western blotted. This step increased the recovery of heat-stable unanchored K48-linked polyubiquitin chains (as detected by anti-K48polyubiquitin antibodies).

Table 1. Predicted m/z Values of Diagnostic b′ and y Series Ions (Intact Distal, Fragmented Proximal Ubiquitin; see Figure 5) Produced by a Single CID-Induced Fragmentation of Diubiquitin with the Indicated Lysine Linkagesa

a

A shaded box indicates the ion is predicted to be produced by fragmentation of the given ubiquitin dimer. Diubiquitin standards (in vitro generated) with the underlined linkages were analysed in this study, and diagnostic ions that were detected are indicated ‘x’.

presence/absence of diagnostic ions produced by single fragmentation events (see Table 1). Fragmentation of the 15+, 17+, and 19+ charge states of in vitro generated K11, K27, K48, and K63 diubiquitin standards resulted in the formation of various b/b′and y ions (for simplicity the b series, in which the proximal ubiquitin has an intact N-terminus, i.e., the distal ubiquitin is fragmented, is not considered here). High resolution mass analysis allowed the determination of fragment masses via deconvolution using the integrated Xcalibur Extract software, with peak assignment subsequently conducted manually (see Table 1). Figure 5A and B shows the LC−MS/MS deconvoluted spectra obtained for the 15+ precursor ion (m/z 1141) for both K48- and K63linked diubiquitin standards. Although the resultant MS/MS spectra for all three charge states (17+ and 19+ not shown) were dominated by the y58 ion, they also consisted of product ions that are capable of distinguishing between K48 and K63 polyubiquitin linkages; most notably, the b52′ and y24 ions cannot be generated from diubiquitin with a K63-linked topology. CID of selected charge states (19+, 17+, and 15+) was then performed on the A20 Znf-purified rat muscle diubiquitin. Figure 5C shows the deconvoluted mass spectrum obtained for the 15+ precursor ion. The presence of the b52′ (m/z 14375.7) and y24 (m/z 2726.5) ions confirms that the dimer is not K63-linked and, combined with the absence of any

an approach has to date not been applied to topography mapping of endogenous ubiquitin polymers. Given our ability to isolate unanchored K48-linked diubiquitin from rat skeletal muscle using the A20 Znf domain (with the use of USP2), we investigated whether the approach could be used to provide information related to isopeptide linkage within the purified ubiquitin dimer. Top-down protein MS relies upon CID where the target ion undergoes multiple collisions with an inert buffer gas (in this case helium). Upon collision the kinetic energy is converted into internal energy, and fragmentation will ensue if sufficient energy is deposited in the precursor ion. When proteins/peptides are subjected to CID at low energy, fragmentation occurs along the backbone. The charge may be retained on either the N-terminus, resulting in formation of fragment ions designated as an, bn, and cn, or at the C-terminus, when xn, yn, and zn type ions are observed. Examination of the MS/MS spectrum of monoubiquitin, resulting from low energy CID of m/z 779.2 ([M + 11H]11+) in a linear ion-trap reveals relatively few species, namely, b18 = m/z 2032.13, b39 = m/z 4307.30, b52 = m/z 5834.12, y24 = m/z 2725.50, y58 = m/z 6527.50, and y74 = m/z 8300.52 (Supplementary Figure 1). Comparison with the sites of polyubiquitin isopeptide linkage shows that it should theoretically be possible to distinguish between K6/11-, K27/29/33-, K48-, and K63-linked ubiquitin dimers using these primary fragmentation modes and the 1975



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Figure 5. Top-down MS of ubiquitin dimers. LC−MS/MS deconvoluted spectra obtained for the 15+ precursor ion (m/z 1141.8) for (A) commercial K48-diubiquitin, (B) commercial K63-diubiquitin, and (C) diubiquitin purified from rat skeletal muscle. Top, schematic representation of the b52′ and y24 ions derived from K48-linked diubiquitin. These data are consistent with the presence of K48-linked diubiquitin in the rat skeletal muscle sample.

mammalian cells indicating ubiquitin dimers are widely expressed. That the former was from biopsies sampled from healthy volunteers and immediately snap-frozen in liquid nitrogen, with direct homogenization in a buffer containing protease (proteasome and DUB) inhibitors, strongly indicates that free ubiquitin dimers represent biologically relevant species rather than arising artifactually from proteolysis of polyubiquitinated proteins during sample preparation. With regards to possible in vivo role(s) of endogenous ubiquitin dimers, it is noteworthy that diubiquitin chains appear to be the fundamental units recognized by linkage-selective UBDs,4 indicating they may themselves have biological activity. It is feasible that ubiquitin dimers could represent preformed “building blocks” for the assembly of longer polyubiquitin chains.33 Ubiquitin dimers may also be generated by the incomplete but regulated deubiquitination of longer polyubiquitin chains. Indeed isoT- and CYLD-mediated disassembly of longer endogenous free polyubiquitin chains results in the formation of ubiquitin dimers,16 as does disassembly of in vitro generated polyubiquitin chains by various other DUBs.35 With respect to biological activity, although the ability of longer unanchored K48-linked polyubiquitin chains to competitively inhibit the 26S proteasome in vitro has been demonstrated,24 K48-diubiquitin was found to be only a weak inhibitor of

specific ions associated with other non-K48 linkages, is consistent with the purified ubiquitin dimer being predominantly K48-linked.

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DISCUSSION Although structures of in vitro generated ubiquitin dimers with the majority of the possible polyubiquitin linkages have been determined, to date the physiological relevance of ubiquitin dimers has been largely overlooked. We show that an unmodified endogenous ubiquitin dimer, linked via K48, can be readily purified from rat skeletal muscle with a strategy based on UBD affinity purification using an A20 Znf domain. Further, purification with an alternate UBD, the Znf UBP domain, also allowed purification of endogenous K11-linked diubiquitin. Our observations are in accordance with a limited number of previous studies showing the existence of cellular diubiquitin,32 including a report of the purification by column chromatography of a ubiquitin dimer from wheat germ shown by Edman degradation to contain the K48 linkage.33 A more recent study also detected a ubiquitin dimer(s) of undefined linkage in rat skeletal muscle extracts but did not draw attention to its presence.34 Using our affinity chromatography approach, we have also been able to purify ubiquitin dimers from human skeletal muscle biopsies (Supplementary Figure 3) and cultured 1976



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possibility that our failure to find evidence (signature peptides) for non-K48/K11 linkages in any of our MS analyses is related to the relative sensitivity of their detection. Ubiquitin peptide standards could readily be detected at levels between 20 fmol to 1 pmol for all but the K29 linkage. Signal intensities for the K48 tryptic peptide in the Znf UBP and A20 Znf capture experiments were found to be similar, but the K11 peptide was detectable only in the Znf UBP samples, demonstrating the specificity of A20 Znf for the K48 polyubiquitin linkage. Further, in the longer unanchored polyubiquitin chains we purified we could readily detect K48 but saw no evidence for K63 linkages by Western blotting; this is especially salient given the emerging roles for K63-linked unanchored polyubiquitin chains in cellular signaling.16,18,19 As noted earlier, our purification strategies make it highly unlikely that the unanchored dimers or polyubiquitin chains we purified arose as a result of adventitious proteolysis. Were this the case it would require that ubiquitin dimers and polyubiquitin chains containing the K48/K11 linkages were preferentially protected from proteolysis. Although detailed studies of the relative stability of different polyubiquitin linkages in the cellular milieu have not been performed, we previously noted that K48-linked chains were rapidly disassembled in cell extracts.28 We favor the notion that K48-linked unanchored polyubiquitin chains may arise as intermediates in the degradation of polyubiquitinated substrates and/or represent de novo assembled post-translational modifiers (see below). Even so, this still does not explain the dominance of the K48 and K11 linkages nor our failure to detect K63-linked chains or other linkages in Znf UBP-purified fractions. Future studies are needed to determine the source of free K48/K11-diubiquitin and longer K48-linked chains and also characterize unanchored polyubiquitin chains purified under “stimulated” conditions (e.g., where there is activation of NF-κB signaling16) as opposed to under “basal” conditions (i.e., resting muscle) in the current study. So what could be the physiological significance of the longer unanchored polyubiquitin chains revealed using the Znf UBP domain? The role of free polyubiquitin chains in activation of protein kinases related to cellular signaling remains topical,16,18,19 leading to the concept that unanchored polyubiquitin chains of certain topologies may act like second messengers that are rapidly assembled in response to various stimuli. Other studies have suggested that kinases involved in energy metabolism can be activated by unanchored K48-linked polyubiquitin chains, suggesting roles for non-K63-linked unanchored chains in enzyme regulation. 38 Direct E3 ubiquitin-ligase-mediated transfer of preformed unanchored polyubiquitin chains to substrates is also plausible,39 indicating certain unanchored chains might act as a preassembled source of polyubiquitin that can be “extracted” from the cytoplasm for transfer en bloc to substrates, rapidly promoting ubiquitinmediated processes.40 In this regard, some chains may act as reservoirs of intermediates and bioactive ubiquitin polymers that directly and indirectly regulate all ubiquitin-mediated processes. It is tempting to speculate unanchored chains may act as, for example, endogenous natural regulators of 26S proteasomal degradation21 and more broadly ‘”fine-tune” all ubiquitin-mediated interactions. Interestingly, some proteasome-associated DUBs have ubiquitin chain amputating activities, or activities that deconjugate multiple ubiquitins in a single cleavage event, potentially generating unanchored polyubiquitin when ubiquitinated substrates are degraded41,42

proteasomal proteolysis and probably does not exert such an effect in vivo. In contrast, unanchored K63-linked polyubiquitin chains containing at least three ubiquitins were found to be potent and specific activators of RIG-I,18 indicating that shorter chains with different linkages may be physiologically relevant, consistent with observations in yeast.25 We speculate that ubiquitin dimers could also play a role in the regulation of ubiquitin receptor dimerization or function.32 Indeed our observations that the immobilized A20 Znf domain does not capture free monoubiquitin in pull-downs but is capable of binding K48-diubiquitin (Figure 3A) indicates that binding to the dimer is likely achieved via avidity effects associated with multiple immobilized UBDs interacting simultaneously, with enhanced affinity. While we have no direct evidence that K48diubiquitin may act to promote dimerization of ZNF216 in vivo, this represents a testable concept. It is noteworthy that (when bound to immobilized A20 Znf) K48-diubiquitin purified from muscle appears to be completely spared from USP2-mediated deubiquitination, even upon prolonged incubation, unlike other ubiquitin conjugates that are readily disassembled. Indeed the USP2 used for deubiquitination represents the catalytic core of the enzyme and has been shown to be a versatile tool for the in vitro removal of ubiquitin from ubiquitin conjugates,31,36 although a recent study suggested the K27 linkage may be selectively spared from deubiquitination.37 We speculate that other cobinding ubiquitin receptors from the muscle sample, which are able to form ternary complexes with individual A20 Znf/ ubiquitin units in the dimer through non-D58 interaction surfaces on ubiquitin,26 may provide a steric block that protects the dimer from deubiquitination. Consistent with this proposal we noted that purified in vitro generated K48-diubiquitin was not spared from processing by USP2 when bound to immobilized A20 Znf domain (not shown), and we have shown it is possible to coprecipitate the I44-binding ubiquitin receptor p62 with ubiquitinated proteins from cultured cells using immobilized ZNF216 protein.26 Competitive binding to non-K48-linked ubiquitin dimers by other linkage-selective ubiquitin receptors in the muscle extract (which does not preclude binding by the Znf UBP domain) may also explain selectivity seen for K48-linked dimers in pull-downs with the A20 Znf domain. Indeed, selectivity of the A20 Znf domain for K48-linked polyubiquitin was not apparent in in vitro quantitative MS binding studies with K48- and K63-linked diubiquitin, where binding affinities to these two linkage types were very similar within the limits of uncertainty.26 However, in structural models, both the A20 Znf and, for example, I44binding UBA domain (of p62) appear to be co-located in reasonably close proximity to the conjugated K48 side chain, suggesting that there could be occlusion around this linkage site that affects deubiquitination by USP2 of a K48-diubiquitin ternary complex with several different bound UBDs (Supplementary Figure 4). Given that all seven of ubiquitin’s lysine residues can participate in polyubiquitin chain formation and that K48 or K11 linkages do not predominate in ubiquitin conjugates per se (see discussion of ref 14 for a comprehensive review of ubiquitin linkage abundance in different studies), it is curious that our purifications using the Znf UBP domain suggest ubiquitin dimers with the K48/K11 linkages may be most abundant in normal skeletal muscle. However, it has been noted that many targets of K11-linked polyubiquitin may be modified by short chains.14 We cannot completely exclude the 1977



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biological samples, now affording a route to their future detailed characterization, and established that free chains containing as many as 15 ubiquitins can be recovered from biological samples. Even if some unanchored polyubiquitin chains are found to represent cellular “garbage”, for example, representing byproduct of ubiquitin-dependent processes, comprehensive molecular analyses of pools of these chains and how they change under different conditions will still provide indirect insights in to polyubiquitin topologies and their responses to physiological stimuli. The ability to purify polyubiquitin modifications free of substrate proteins also now allows further detailed analyses of additional post-translational modifications of polyubiquitin.

and raising the possibility of negative feedback that may in turn be antagonized by other ubiquitin receptors.42 Quite why free polyubiquitin chains as long as 15 ubiquitins, the maximum chain length we have so far detected, would be required by the cell remains a mystery, especially considering that (K48-linked) tetraubiquitin is the minimum targeting signaling for the proteasome and increasing chain length beyond eight ubiquitin moieties does not increase proteasomebinding affinity.2 Further, are unanchored polyubiquitin chains sufficiently abundant in cells to be biologically relevant? It was estimated previously by comparisons of gel band intensities that a typical mammalian (cultured) cell may contain as many as 6000 molecules of free K63-linked Ub6,18 which is comparable with the median protein copy number.43 Further, recent work in yeast suggest unanchored polyubiquitin chains represent a prominent fraction of the total ubiquitin conjugates, at least during conditions of vigorous growth.22 K63-linked Ub6 can activate RIG-I with a EC50 of about 50 pM (equivalent to only 15 chains per cell), showing polyubiquitin chains are potent regulators of RIG-I activity. The ubiquitin-PSAQ approach did recover low levels of unanchored polyubiquitin from different biological samples, although not optimized for the bulk purification of free chains.31 Although future studies will be required to provide precise information on the abundance of free polyubiquitin chains in different cell types, as well as to assess the overall efficiency of our own capture methods, on the basis of comparisons of Western blot band intensities of our free polyubiquitin chain fractions (purified using the Znf UBP domain following heat-treating of samples) with polyubiquitin chain standards of known protein concentration, we estimate that from 1 mg of soluble muscle protein (extracted from 62.5 mg wet weight of muscle) we could recover as much as 500 ng of free polyubiquitin, although the ubiquitin dimers predominate. Although the potential of top-down protein MS to study ubiquitin modifications has been considered, to date the characterization of polymeric ubiquitin species purified from an in vivo source using this approach has not been achieved. Such an approach, which avoids the need for an enzymatic digest step that destroys polyubiquitin connectivity, potentially represents an analytical method for probing absolute polyubiquitin chain topologies. Our results show for the first time that it is possible to gain information related to the linkage connectivity within unanchored polyubiquitin chains using a top-down proteomics approach, albeit with the simplest ubiquitin polymer, diubiquitin. In our study we subjected K11-, K27-, K48-, and K63-linked diubiquitin standards to LC− MS and CID MS/MS and gained insights into chain topology. A top-down type of approach therefore offers great promise for polyubiquitin topology mapping, including cases of heterogeneous chain linkage. Where it is not possible to distinguish linkage through examination of primary MS/MS spectra, e.g., K6- versus K11-linked chains, MS3 (either CID or ECD) could be employed to provide an additional level of information. Although top-down MS alone does not allow the absolute topology of ubiquitin dimers to be determined and top-down analyses of longer polyubiquitin chains are more challenging, our observations provide the first proof of concept that this approach, in combination with other MS techniques, is directly applicable to determining the connectivity within polyubiquitin chains. In summary, our studies have demonstrated that endogenous unanchored polyubiquitin chains can be directly purified from

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. LC−MS/MS (CID) deconvoluted spectrum obtained for the [M + 11H]11+ precursor ion (m/z 779.2) of monoubiquitin. Figure S2. ECD MS/MS spectra obtained for diubiquitin captured from rat skeletal muscle by the ZnF UBP domain. Figure S3. Western blot (anti-ubiquitin) showing capture of ubiquitin dimers from human skeletal muscle biopsies from two normal volunteers collected essentially according to published protocols.44 Figure S4. Space filling representation of the complex of K48-linked diubiquitin with A20 Znf domains (of ZNF216) bound at the two D58 binding sites on each half of the dimer, and a UBA domain (of p62) at the I44 site on the proximal ubiquitin; ribbon representation of the same structure showing the position of key residues at the binding interfaces and the side chain of K48 conjugating the two ubiquitin molecules. This information is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44-115-8230107. Fax: +44-115-9709259. E-mail: robert. [email protected]. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by BBSRC grants BB/I006052/1 and BB/F019297/1 to R.L. and N.J.O. We wish to thank Dr. P. A. Atherton for the provision of human muscle samples.

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Insights into the Molecular Composition of Endogenous Unanchored

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