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A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins [1]

['Mengwen Zhang', 'Department Of Chemistry', 'Yale University', 'New Haven', 'Connecticut', 'United States Of America', 'Jason M. Berk', 'Department Of Molecular Biophysics', 'Biochemistry', 'Adrian B. Mehrtash']

Date: 2022-07

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: M.H., M.Z. and J.M.B. are inventors on a U.S. Patent Application No. No. 17/061,347 filed on October 1, 2020 that covers methods of ubiquitin detection and enrichment using the OtUBD. The other authors declare no competing interests.

Funding: This work was supported by National Institutes of Health ( https://www.nih.gov/grants-funding ) grant GM136325 to M.H. The mass spectrometers at the Keck MS & Proteomics Resource at Yale University were funded in part by the Yale School of Medicine and by the Office of the Director, National Institutes of Health (S10OD02365101A1, S10OD019967, and S10OD018034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting Information files. All mass spectrometry proteomics data obtained in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD032294 (yeast results) and PXD032675 (HeLa cell results). The raw and fully uncropped images for all gels and blots have been deposited to Figshare ( https://figshare.com/articles/figure/S1_raw_images_pdf/19640367 ).

Copyright: © 2022 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

(A) Schematic showing the ubiquitin binding domain (OtUBD) within the O. tsutsugamushi DUB (OtDUB). OtUBD spans residues 170 to 264. (B) The different constructs of OtUBD and the control TUBE derived from the UBA domain of human Ubiquilin 1. His 6 tagged OtUBD and TUBE were used in the ubiquitylation protection experiment shown in Fig 1C and MBP-tagged OtUBD and 3xOtUBD were used in the ubiquitin pulldown experiment in Fig 1D. (C) IB analysis of bulk ubiquitylated proteins (top panel) and histone H2B (bottom panel) from yeast cell lysates prepared in the presence of different reagents. OtUBD prevents deubiquitylation of bulk ubiquitylated substrates (top panel) and monoubiquitylated histone H2B (bottom panel). (D) IB analysis of MBP pulldowns from yeast cell lysates using different bait proteins. MBP or MBP-tagged bait proteins bound to an amylose resin were incubated with yeast lysates, and bound proteins were eluted by incubation in SDS sample buffer. Both OtUBD and 3xOtUBD bound (B) ubiquitylated substrates in the lysates. Concentration of bait protein indicates the amount of bait protein per unit volume of amylose resin. B, bound fraction; IB, immunoblot; MBP, maltose-binding protein; OtDUB, O. tsutsugamushi DUB; TUBE, tandem ubiquitin-binding entity; U, unbound fraction; UBD, ubiquitin-binding domain.

Recently, our group discovered a novel UBD within a DUB effector protein, OtDUB, from the intracellular bacterium Orientia tsutsugamushi, the causative agent of the disease scrub typhus [ 41 ]. The UBD from OtDUB, which was referred to as OtDUB UBD (we will use OtUBD for the remainder of the paper for simplicity), spans residues 170 to 264 of the 1,369-residue OtDUB polypeptide ( Fig 1A ) and binds monomeric ubiquitin with very high affinity (K d , approximately 5 nM), which is more than 500-fold tighter than any other natural UBD described to date. Co-crystal structures of OtDUB and ubiquitin revealed that OtUBD binds ubiquitin at the isoleucine-44 hydrophobic patch, a ubiquitin feature commonly recognized by ubiquitin-binding proteins [ 42 ]. We reasoned that the small, well-folded OtUBD could serve as a facile enrichment reagent for ubiquitylated proteins. The advantages of such reagent include its low cost, lack of bias between monoubiquitylated and polyubiquitylated proteins, and ability to detect unconventional ubiquitin-substrate linkages.

Each of these methods has its advantages and limitations, which have been reviewed elsewhere [ 16 , 39 ]. For example, TUBEs are excellent tools to study polyubiquitylation, but in some mammalian cell types, over 50% of ubiquitylated proteins are only monoubiquitylated [ 17 ] and can easily be missed by TUBEs. Anti-diGly antibodies, while extremely effective in identifying ubiquitin–lysine linkages, are not capable of recognizing ubiquitylation sites on other nucleophilic side chains in proteins or other macromolecules [ 40 ]. Due to the importance and complexity of ubiquitylation, the development of sensitive and economical reagents to study the entire ubiquitylome is crucial.

In addition to the abovementioned methods, ubiquitin remnant motif antibodies (diGly antibodies) are widely used in bottom-up proteomics experiments to identify ubiquitylation sites on substrate proteins [ 34 , 35 ]. In bottom-up proteomics, proteins are digested by a protease (typically trypsin) into short peptides, separated by liquid chromatography and identified by tandem mass spectrometry (LC-MS/MS) [ 36 ]. Tryptic digestion of ubiquitylated proteins leaves a signature GlyGly (GG) remnant on ubiquitylated lysine side chains [ 18 ]. Anti-diGly-ε-Lys antibodies recognize this remnant motif and enrich such peptides for identification of ubiquitylation sites. The development of diGly antibodies has greatly facilitated the systematic discovery and profiling of ubiquitylated proteins and their ubiquitylation sites and has enabled the establishment of databases documenting ubiquitylation in humans and other species [ 37 , 38 ].

To study endogenous ubiquitylated proteins, anti-ubiquitin antibodies—including those against all ubiquitylation types (such as FK1 and FK2 monoclonal antibodies; [ 29 ]) or those specific for certain ubiquitin-chain linkages (such as anti-K48 ubiquitin linkage antibodies)—have been used [ 30 , 31 ]. TUBEs, on the other hand, are recombinant ubiquitin-affinity reagents built from multiple ubiquitin-binding domains (UBDs). UBDs have been characterized in a range of ubiquitin-interacting proteins, and they typically bind to ubiquitin with low affinity (Kd values in the micromolar range) [ 32 ]. By fusing multiple copies of a UBD together to turn it into a TUBE, the avidity of the reagent toward polyubiquitin chain-modified proteins is greatly increased [ 21 ]. TUBEs are therefore useful in protecting polyubiquitylated proteins from DUB cleavages and enriching them in biological samples, and some TUBEs are designed to recognize specific types of polyubiquitin chains [ 33 ]. In general, TUBE affinity toward monoubiquitylated proteins is low [ 21 ].

The first method was introduced using the budding yeast Saccharomyces cerevisiae [ 25 ]. In yeast, 4 different genes encode ubiquitin, either as fusions to ribosomal peptides or as tandem ubiquitin repeats [ 26 ]. It is possible to create yeast strains where the only source of ubiquitin is a plasmid expressing epitope-tagged ubiquitin [ 27 , 28 ]; as a result, all ubiquitylated proteins in this specific yeast strain bear the epitope tag, which can then be used for enrichment or detection of the ubiquitylated species. A number of earlier studies have used this method to profile the ubiquitylated proteome [ 4 ]. One major concern with this method is that the (over)expression of tagged ubiquitin may result in abnormal ubiquitylation or interfere with endogenous ubiquitylation events.

Defects in ubiquitylation have been connected to many human disorders, including cancers, viral infections, and neurodegenerative diseases [ 12 – 14 ]. The broad biomedical impact of protein ubiquitylation has stimulated efforts to develop sensitive methods to study the ubiquitylated proteome [ 15 , 16 ]. Because the ubiquitylated fraction of a given protein substrate population is often very small at steady state [ 17 ], it is generally necessary to enrich for the ubiquitylated proteins in biological samples of interest. Current methods to enrich ubiquitylated proteins can be roughly classified into 3 categories: (1) ectopic (over)expression of epitope-tagged ubiquitin and affinity purification using the tags; (2) immunoprecipitation with anti-ubiquitin antibodies; and (3) use of tandem ubiquitin-binding entities (TUBEs) [ 18 – 20 , 21 – 24 ].

Ubiquitin is a conserved posttranslational modifier that requires a cascade of enzymatic reactions for its attachment to proteins [ 1 ]. Each modification is catalyzed by a ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 [ 2 ]. The E1 enzyme activates the carboxyl-terminal carboxylate of ubiquitin and then transfers the activated ubiquitin molecule to an E2. E3 ligases are responsible for the recognition of the substrate and catalyzing ubiquitin transfer from the E2 to a nucleophilic residue on the substrate protein, typically the ε-amino group of a lysine residue, but potentially also N-terminal amino groups, serine/threonine hydroxyl side chains, or the thiol group of cysteine [ 3 ]. Ubiquitin itself can be ubiquitylated through its N-terminal methionine (M1) or one or more of its 7 lysine residues (K6, K11, K27, K29, K33, K48, and K63) [ 4 ]. These diverse ubiquitin chain topologies and sizes can modulate the biological functions of substrate ubiquitylation, often described as the “ubiquitin code” [ 5 ]. For example, monoubiquitylation has been reported to facilitate protein complex formation in many cases [ 6 , 7 ]. Polyubiquitylation involving K48 linkages is a well-documented substrate marker for proteasomal degradation [ 8 ], while polyubiquitylation with K63 linkages is often a signal for membrane trafficking or DNA repair pathways [ 9 , 10 ]. Ubiquitylation can be reversed through hydrolysis by ubiquitin-specific proteases or deubiquitylases (DUBs) [ 11 ].

Results

OtUBD can protect and enrich ubiquitylated species from whole cell lysates We first expressed and purified recombinant OtUBD with an N-terminal His 6 tag (Fig 1B). A previously reported TUBE based on the UBA domain of human Ubiquilin 1 (4xTR-TUBE) was used for comparison [23,43]. One use of TUBEs is to protect ubiquitylated proteins in vitro from being cleaved by endogenous DUBs or being degraded by the proteasome following cell lysis, which facilitates their analysis [21]. We tested if OtUBD could do the same. When yeast cells were lysed in the presence of N-ethylmaleimide (NEM; a covalent cysteine modifier that inhibits most cellular DUBs), 3 μM OtUBD, or 3 μM TUBE, higher mass ubiquitylated species were similarly preserved by the 2 ubiquitin binders, with NEM having the strongest effect, as expected (Fig 1C). We investigated whether this protection extended to monoubiquitylated proteins by examining Flag-tagged histone H2B (Htb2) in a ubp8Δ mutant [44]. Histone H2B is known to be monoubiquitylated, and levels of this species are enhanced by deleting Ubp8, the DUB that reverses the modification [45]. Strikingly, OtUBD added to the cell lysate preserved the monoubiquitylated H2B to a degree comparable to NEM (Fig 1C, bottom). By contrast, H2B-ubiquitin was completely lost in extracts without any DUB inhibitor or when incubated with the TUBE protein. We next determined if OtUBD or tandem repeats of OtUBD could be used for affinity enrichment of ubiquitylated proteins. We fused maltose-binding protein (MBP) to the amino terminus of OtUBD or 3 tandem OtUBD repeats (Fig 1B). Purified MBP or the MBP fusion proteins were first bound to an amylose resin and then incubated with yeast whole cell lysates. With lower amounts of the resin-bound bait proteins, MBP-3xOtUBD enriched more ubiquitylated proteins than MBP-OtUBD, likely due to its higher capacity for binding ubiquitin (3 ubiquitin binding sites versus 1 in OtUBD) (Fig 1D, left; compare bound (B) to unbound (U) lanes). When we increased the amount of the bait proteins, however, both MBP-OtUBD and MBP-3xOtUBD efficiently depleted ubiquitylated proteins from the lysate (Fig 1D, right). The negative control MBP did not detectably bind any ubiquitylated species at either concentration. Notably, efficient enrichment was only achieved when MBP-OtUBD was prebound to the amylose resin (S1A Fig). When free MBP-OtUBD was first incubated with the cell lysate and then bound to amylose resin, the enrichment efficiency was compromised (S1B Fig). MBP-OtUBD also efficiently enriched ubiquitylated proteins from mammalian cell lysates, demonstrating its general utility across species (S1C Fig). In summary, OtUBD can both protect ubiquitylated proteins from in vitro deubiquitylation and enrich for such proteins. Unlike previously reported UBDs [21,46], OtUBD can efficiently enrich ubiquitylated proteins even when used as a single entity instead of tandem repeats.

A covalently linked OtUBD resin for ubiquitylated protein purification We next generated resins with covalently attached OtUBD to minimize the contamination by bait proteins seen with MBP-OtUBD and maltose elution (S1A–S1C Fig). Since OtUBD lacks cysteine residues, we introduced a cysteine residue at the amino terminus of the OtUBD sequence as a functional handle that can react with the commercially available SulfoLink resin to form a stable thioether linkage (Fig 2A). As a negative control, free cysteine was added to the SulfoLink resin to cap the reactive iodoacetyl groups. When incubated with yeast whole cell lysates prepared in a buffer with 300 mM NaCl and 0.5% Triton-X100 detergent, the OtUBD resin bound a broad range of ubiquitylated proteins and the bound proteins could be eluted with a low pH buffer (Figs 2B and S1D; see Materials and methods). No ubiquitylated species were detected in the eluates from the control resin (Figs 2B and S1D). PPT PowerPoint slide

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TIFF original image Download: Fig 2. A covalently linked OtUBD resin purifies ubiquitin and ubiquitylated proteins from yeast lysates. (A) OtUBD constructs used for covalent coupling to resin and mechanism of the coupling reaction. An engineered cysteine at the amino terminus of OtUBD enables its covalent conjugation to the SulfoLink resin. (B) Ubiquitin blot of pulldowns from yeast cell lysate using covalently linked OtUBD resin or control resin. Covalently linked OtUBD resin efficiently pull down ubiquitylated species from yeast whole cell lysate. FT, flow-through; E1/E2/E3, eluted fractions using a series of stepwise, low pH elutions. (C, D) Extract pretreatment with M48 DUB cleaves ubiquitin from ubiquitylated species and greatly reduces the total protein pulled down by OtUBD resin. (C) Anti-ubiquitin blot of OtUBD pulldown of yeast lysate with or without M48 DUB treatment. (D) Total protein present in the eluted fractions of the OtUBD pulldowns visualized with SYPRO Ruby stain. (C and D are from 2 separate biological replicates.) IN, input; FT, flowthrough; E, eluted fractions. (E, F) The V203D mutation in OtUBD, which greatly impairs its binding of ubiquitin, prevents enrichment for ubiquitylated species from yeast lysate. (E) Anti-ubiquitin blot of pulldowns of yeast lysates using OtUBD resin, Cys resin (negative control), and OtUBD(V203D) resin. (F) Total protein present in the eluted fractions of the OtUBD pulldowns visualized with SYPRO Ruby stain. IN, input; FT, flowthrough; E1/2/3, eluted fractions using a series of low pH elutions. MBP, maltose-binding protein; OtDUB, O. tsutsugamushi DUB. https://doi.org/10.1371/journal.pbio.3001501.g002 By comparing the anti-ubiquitin blot in Fig 2B to the general protein stain of the same eluted fractions in S1D Fig, it was clear that many proteins eluted from the OtUBD resin were not themselves ubiquitylated. Pulldown experiments performed under native or near-native conditions are expected to copurify proteins that interact noncovalently with ubiquitylated polypeptides, e.g., complexes that harbor ubiquitylated subunits. To test whether the entire protein population eluted from OtUBD resin was nevertheless dependent on substrate ubiquitylation, yeast lysates were preincubated with the viral M48 DUB, which cleaves a broad range of ubiquitylated proteins and reduces ubiquitin chains to free ubiquitin (Fig 2C) [47]. This treatment greatly reduced the total protein eluted from the OtUBD resin compared to the pulldown from untreated lysate (Fig 2D), indicating that the majority of proteins eluted from OtUBD resin were either ubiquitylated themselves or interacted noncovalently with ubiquitin or ubiquitylated proteins. To further validate the specificity of the OtUBD resin toward ubiquitylated proteins, we made an OtUBD resin with a ubiquitin-binding deficient mutation (V203D) [41] and tested its ability to purify ubiquitin and ubiquitylated proteins. This mutation greatly diminished the resin’s ability to enrich ubiquitylated species (Fig 2E) and also strongly reduced the total protein eluate from the resin (Fig 2F). This indicates that the ability of OtUBD resin to enrich for ubiquitylated species is based on its binding affinity toward ubiquitin. Taken together, these results indicate the OtUBD resin specifically enriches ubiquitin and ubiquitylated polypeptides as well as proteins that interact with ubiquitin-containing proteins.

Purifications using OtUBD with denatured extracts enrich ubiquitin–protein conjugates To distinguish proteins covalently modified by ubiquitin from proteins co-purifying through noncovalent interaction with ubiquitin or ubiquitylated proteins, we optimized pulldown conditions to include a denaturation step (Fig 3A). Yeast lysates were incubated with 8 M urea, a condition where the majority of proteins are unfolded, to dissociate protein complexes [48]. Denatured lysates were then diluted with native lysis buffer (to a final urea concentration of 4M) to facilitate the refolding of ubiquitin and applied to the OtUBD resin. A similar method was used previously in ubiquitin immunoprecipitation using the FK2 monoclonal antibody [20]. Under such conditions, the OtUBD resin concentrated ubiquitylated proteins with efficiencies similar to those seen under native conditions (Fig 3C). At the same time, the denaturing treatment greatly reduced the total amount of proteins eluted compared to native conditions, and the spectrum of purified protein species also changed (Fig 3D). This suggests that ubiquitylated proteins were specifically enriched by the urea treatment. PPT PowerPoint slide

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TIFF original image Download: Fig 3. OtUBD pulldown under denaturing condition specifically enriches for proteins covalently modified with ubiquitin. (A) Workflow of OtUBD pulldowns following sample denaturation (red arrows) or under native (blue arrows) conditions. In the first case, cell lysate is treated with 8 M urea to denature and dissociate proteins. The denatured lysate is then diluted 1:1 with native buffer to allow ubiquitin to refold and bind to OtUBD resin. Under such conditions, only ubiquitylated proteins are expected to be enriched. In the second case, cell lysate contains native ubiquitylated proteins as well as proteins that interact with them. OtUBD pulldown under such conditions is expected to yield both ubiquitylated substrates and ubiquitin-binding proteins. (B) Outline for the use of tandem Co2+ resin pulldowns to validate OtUBD pulldown results under different conditions. Eluates from OtUBD after lysates were incubated with denaturant (red arrows) or left untreated (blue arrows) are (re)treated with denaturant (8 M urea or 6 M guanidine•HCl) and then subjected to IMAC with a Co2+ resin in denaturing conditions. Proteins covalently modified by His 6 -ubiquitin bind to the Co2+ resin while proteins that only interact noncovalently with ubiquitin end up in the flowthrough. (C) Anti-ubiquitin blot of OtUBD pulldowns following native and urea denaturing treatments performed as described in Fig 3A. FT, flowthrough; E, eluted fractions. The image was spliced to remove irrelevant lanes. (D) Total protein present in eluates of the OtUBD pulldowns in Fig 3C visualized by SYPRO Ruby stain. (E) Total protein present in different fractions of the Co2+ IMAC (see Fig 3B; the results shown here used urea as the denaturant) visualized by SYPRO Ruby stain. IN, input; FT, flowthrough; E, fraction eluted with 500 mM imidazole. (F) Anti-ubiquitin blot of fractions from Co2+ IMAC (see Fig 3B; the blot shown here used urea as the denaturant) of eluates from native and denaturing OtUBD resin pulldowns. IN, input; FT, flowthrough; E, fraction eluted with 500 mM imidazole. *The identity of the prominent approximately 20 kDa species in the flowthrough from the native extract is unknown. (G) Anti-ubiquitin blot of OtUBD pulldowns from HeLa cell lysates performed as described in Fig 3A following native or denaturing treatments. The image was spliced to remove irrelevant lanes. IN, input; FT, flowthrough; E, fraction eluted with low pH elution buffer. (H) Total protein present in eluates in Fig 3G visualized by SYPRO Ruby stain. (I) Immunoblot analysis of human proteasomal subunit Rpt6 in OtUBD pulldowns following native and urea denaturing treatments of lysates. Unmodified Rpt6 co-purified with OtUBD resin under native conditions but not following denaturation of extract. N, native condition; D, denaturing condition. OtDUB, O. tsutsugamushi DUB. https://doi.org/10.1371/journal.pbio.3001501.g003 To verify that OtUBD pulldown following a denaturation step is specific for proteins covalently modified with ubiquitin, we utilized a yeast strain whose endogenous ubiquitin-coding sequences were all deleted and replaced with a single plasmid-borne His 6 -tagged ubiquitin sequence [28]. The eluted fractions from OtUBD resin pulldowns performed after either denaturing or nondenaturing treatments of lysates (Fig 3A) were then denatured again by incubation with urea or guanidine-HCl (Fig 3B). The denatured proteins were applied to a Co2+ (Talon) resin for immobilized metal affinity chromatography (IMAC) via the His 6 -tagged ubiquitin. If the eluate from the OtUBD resin had contained only (His 6 -)ubiquitylated proteins, most or all of the total proteins should bind to the resin. We observed that when OtUBD pulldowns were done following a denaturing lysate treatment, most of the eluted proteins were indeed bound to the Co2+ resin (Fig 3E). By contrast, a large portion of proteins from a “native” OtUBD pulldown remained in the flow-through of the Co2+ resin (Fig 3E). The overall levels of ubiquitylated species recovered, however, were comparable between the 2 treatments (Fig 3F). Consistent with these findings with bulk ubiquitin conjugates, when we tested whether the proteasome, which binds noncovalently to many polyubiquitylated substrates [49], was in the OtUBD eluates, we readily detected unmodified proteasome subunits in the native pulldowns but not in pulldowns from denatured lysates (S2A Fig). OtUBD-based affinity purifications, under either native or denaturing conditions, were also effective with human cell lysates. Both conditions led to similar enrichment of ubiquitin conjugates (Fig 3G), but the denaturing pretreatment greatly reduced the amounts of co-purifying nonubiquitylated proteins (Fig 3H). Congruent with this, nonubiquitylated human proteasomal subunits were only present at substantial levels in eluates from native lysates (Figs 3I and S2B). Interestingly, low amounts of presumptive ubiquitylated proteasome subunits were discovered in OtUBD pulldowns from both native and denatured lysates, and these species were strongly enriched over the unmodified subunits under the latter condition (S2B Fig). Overall, these results indicate that OtUBD-based protein purification under denaturing conditions can specifically enrich proteins that are covalently modified by ubiquitin.

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