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A flexible new instrument derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins


Ubiquitin is a conserved posttranslational modifier that requires a cascade of enzymatic reactions for its attachment to proteins [1]. Every modification is catalyzed by a ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 [2]. The E1 enzyme prompts the carboxyl-terminal carboxylate of ubiquitin after which transfers the activated ubiquitin molecule to an E2. E3 ligases are answerable for the popularity of the substrate and catalyzing ubiquitin switch from the E2 to a nucleophilic residue on the substrate protein, usually the ε-amino group of a lysine residue, however probably additionally N-terminal amino teams, serine/threonine hydroxyl aspect chains, or the thiol group of cysteine [3]. Ubiquitin itself will be ubiquitylated via its N-terminal methionine (M1) or a number of of its 7 lysine residues (K6, K11, K27, K29, K33, K48, and K63) [4]. These various ubiquitin chain topologies and sizes can modulate the organic features of substrate ubiquitylation, typically described because the “ubiquitin code” [5]. For instance, monoubiquitylation has been reported to facilitate protein advanced formation in lots of circumstances [6,7]. Polyubiquitylation involving K48 linkages is a well-documented substrate marker for proteasomal degradation [8], whereas polyubiquitylation with K63 linkages is commonly a sign for membrane trafficking or DNA restore pathways [9,10]. Ubiquitylation will be reversed via hydrolysis by ubiquitin-specific proteases or deubiquitylases (DUBs) [11].

Defects in ubiquitylation have been linked to many human problems, together with cancers, viral infections, and neurodegenerative ailments [1214]. The broad biomedical influence of protein ubiquitylation has stimulated efforts to develop delicate strategies to check the ubiquitylated proteome [15,16]. As a result of the ubiquitylated fraction of a given protein substrate inhabitants is commonly very small at regular state [17], it’s typically essential to complement for the ubiquitylated proteins in organic samples of curiosity. Present strategies to complement ubiquitylated proteins will be roughly categorised into 3 classes: (1) ectopic (over)expression of epitope-tagged ubiquitin and affinity purification utilizing the tags; (2) immunoprecipitation with anti-ubiquitin antibodies; and (3) use of tandem ubiquitin-binding entities (TUBEs) [1820,2124].

The primary methodology was launched utilizing the budding yeast Saccharomyces cerevisiae [25]. In yeast, 4 completely different genes encode ubiquitin, both as fusions to ribosomal peptides or as tandem ubiquitin repeats [26]. It’s potential to create yeast strains the place the one supply of ubiquitin is a plasmid expressing epitope-tagged ubiquitin [27,28]; consequently, all ubiquitylated proteins on this particular yeast pressure bear the epitope tag, which may then be used for enrichment or detection of the ubiquitylated species. Plenty of earlier research have used this methodology to profile the ubiquitylated proteome [4]. One main concern with this methodology is that the (over)expression of tagged ubiquitin might lead to irregular ubiquitylation or intrude with endogenous ubiquitylation occasions.

To check endogenous ubiquitylated proteins, anti-ubiquitin antibodies—together with these towards all ubiquitylation sorts (equivalent to FK1 and FK2 monoclonal antibodies; [29]) or these particular for sure ubiquitin-chain linkages (equivalent to anti-K48 ubiquitin linkage antibodies)—have been used [30,31]. TUBEs, then again, are recombinant ubiquitin-affinity reagents constructed from a number of ubiquitin-binding domains (UBDs). UBDs have been characterised in a variety of ubiquitin-interacting proteins, and so they usually bind to ubiquitin with low affinity (Kd values within the micromolar vary) [32]. By fusing a number of copies of a UBD collectively to show it right into a TUBE, the avidity of the reagent towards polyubiquitin chain-modified proteins is tremendously elevated [21]. TUBEs are due to this fact helpful in defending polyubiquitylated proteins from DUB cleavages and enriching them in organic samples, and a few TUBEs are designed to acknowledge particular kinds of polyubiquitin chains [33]. On the whole, TUBE affinity towards monoubiquitylated proteins is low [21].

Along with the abovementioned strategies, ubiquitin remnant motif antibodies (diGly antibodies) are extensively utilized in bottom-up proteomics experiments to establish ubiquitylation websites on substrate proteins [34,35]. In bottom-up proteomics, proteins are digested by a protease (usually trypsin) into brief peptides, separated by liquid chromatography and recognized by tandem mass spectrometry (LC-MS/MS) [36]. Tryptic digestion of ubiquitylated proteins leaves a signature GlyGly (GG) remnant on ubiquitylated lysine aspect chains [18]. Anti-diGly-ε-Lys antibodies acknowledge this remnant motif and enrich such peptides for identification of ubiquitylation websites. The event of diGly antibodies has tremendously facilitated the systematic discovery and profiling of ubiquitylated proteins and their ubiquitylation websites and has enabled the institution of databases documenting ubiquitylation in people and different species [37,38].

Every of those strategies has its benefits and limitations, which have been reviewed elsewhere [16,39]. For instance, TUBEs are wonderful instruments to check polyubiquitylation, however in some mammalian cell sorts, over 50% of ubiquitylated proteins are solely monoubiquitylated [17] and might simply be missed by TUBEs. Anti-diGly antibodies, whereas extraordinarily efficient in figuring out ubiquitin–lysine linkages, will not be able to recognizing ubiquitylation websites on different nucleophilic aspect chains in proteins or different macromolecules [40]. As a result of significance and complexity of ubiquitylation, the event of delicate and economical reagents to check the complete ubiquitylome is essential.

Lately, our group found a novel UBD inside a DUB effector protein, OtDUB, from the intracellular bacterium Orientia tsutsugamushi, the causative agent of the illness scrub typhus [41]. The UBD from OtDUB, which was known as OtDUBUBD (we are going to use OtUBD for the rest 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 excessive affinity (Okayd, roughly 5 nM), which is greater than 500-fold tighter than another pure UBD described to this point. Co-crystal buildings of OtDUB and ubiquitin revealed that OtUBD binds ubiquitin on the isoleucine-44 hydrophobic patch, a ubiquitin function generally acknowledged by ubiquitin-binding proteins [42]. We reasoned that the small, well-folded OtUBD might function a facile enrichment reagent for ubiquitylated proteins. The benefits of such reagent embody its low price, lack of bias between monoubiquitylated and polyubiquitylated proteins, and skill to detect unconventional ubiquitin-substrate linkages.


Fig 1. The high-affinity UBD from OtDUB effectively protects and enriches for yeast ubiquitylated species.

(A) Schematic exhibiting the ubiquitin binding area (OtUBD) inside the O. tsutsugamushi DUB (OtDUB). OtUBD spans residues 170 to 264. (B) The completely different constructs of OtUBD and the management TUBE derived from the UBA area of human Ubiquilin 1. His6 tagged OtUBD and TUBE had been used within the ubiquitylation safety experiment proven in Fig 1C and MBP-tagged OtUBD and 3xOtUBD had been used within the ubiquitin pulldown experiment in Fig 1D. (C) IB evaluation of bulk ubiquitylated proteins (high panel) and histone H2B (backside panel) from yeast cell lysates ready within the presence of various reagents. OtUBD prevents deubiquitylation of bulk ubiquitylated substrates (high panel) and monoubiquitylated histone H2B (backside panel). (D) IB evaluation of MBP pulldowns from yeast cell lysates utilizing completely different bait proteins. MBP or MBP-tagged bait proteins certain to an amylose resin had been incubated with yeast lysates, and certain proteins had been eluted by incubation in SDS pattern buffer. Each OtUBD and 3xOtUBD certain (B) ubiquitylated substrates within the lysates. Focus of bait protein signifies the quantity of bait protein per unit quantity of amylose resin. B, certain fraction; IB, immunoblot; MBP, maltose-binding protein; OtDUB, O. tsutsugamushi DUB; TUBE, tandem ubiquitin-binding entity; U, unbound fraction; UBD, ubiquitin-binding area.


OtUBD can shield and enrich ubiquitylated species from complete cell lysates

We first expressed and purified recombinant OtUBD with an N-terminal His6 tag (Fig 1B). A beforehand reported TUBE primarily based on the UBA area of human Ubiquilin 1 (4xTR-TUBE) was used for comparability [23,43]. One use of TUBEs is to guard ubiquitylated proteins in vitro from being cleaved by endogenous DUBs or being degraded by the proteasome following cell lysis, which facilitates their evaluation [21]. We examined if OtUBD might do the identical. When yeast cells had been lysed within the presence of N-ethylmaleimide (NEM; a covalent cysteine modifier that inhibits most mobile DUBs), 3 μM OtUBD, or 3 μM TUBE, increased mass ubiquitylated species had been equally preserved by the two ubiquitin binders, with NEM having the strongest impact, as anticipated (Fig 1C).

We investigated whether or not this safety prolonged to monoubiquitylated proteins by inspecting Flag-tagged histone H2B (Htb2) in a ubp8Δ mutant [44]. Histone H2B is thought to be monoubiquitylated, and ranges 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 level akin to NEM (Fig 1C, backside). Against this, H2B-ubiquitin was utterly misplaced in extracts with none DUB inhibitor or when incubated with the TUBE protein.

We subsequent decided if OtUBD or tandem repeats of OtUBD might 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 had been first certain to an amylose resin after which incubated with yeast complete cell lysates. With decrease quantities of the resin-bound bait proteins, MBP-3xOtUBD enriched extra ubiquitylated proteins than MBP-OtUBD, doubtless on account of its increased capability for binding ubiquitin (3 ubiquitin binding websites versus 1 in OtUBD) (Fig 1D, left; examine certain (B) to unbound (U) lanes). After we elevated the quantity of the bait proteins, nevertheless, each MBP-OtUBD and MBP-3xOtUBD effectively depleted ubiquitylated proteins from the lysate (Fig 1D, proper). The detrimental management MBP didn’t detectably bind any ubiquitylated species at both focus. Notably, environment friendly enrichment was solely achieved when MBP-OtUBD was prebound to the amylose resin (S1A Fig). When free MBP-OtUBD was first incubated with the cell lysate after which certain to amylose resin, the enrichment effectivity was compromised (S1B Fig). MBP-OtUBD additionally effectively enriched ubiquitylated proteins from mammalian cell lysates, demonstrating its normal utility throughout species (S1C Fig).

In abstract, OtUBD can each shield ubiquitylated proteins from in vitro deubiquitylation and enrich for such proteins. Not like beforehand reported UBDs [21,46], OtUBD can effectively enrich ubiquitylated proteins even when used as a single entity as an alternative of tandem repeats.

A covalently linked OtUBD resin for ubiquitylated protein purification

We subsequent generated resins with covalently connected OtUBD to reduce the contamination by bait proteins seen with MBP-OtUBD and maltose elution (S1A–S1C Fig). Since OtUBD lacks cysteine residues, we launched a cysteine residue on the amino terminus of the OtUBD sequence as a useful deal with that may react with the commercially out there SulfoLink resin to kind a steady thioether linkage (Fig 2A). As a detrimental management, free cysteine was added to the SulfoLink resin to cap the reactive iodoacetyl teams. When incubated with yeast complete cell lysates ready in a buffer with 300 mM NaCl and 0.5% Triton-X100 detergent, the OtUBD resin certain a broad vary of ubiquitylated proteins and the certain proteins might be eluted with a low pH buffer (Figs 2B and S1D; see Supplies and strategies). No ubiquitylated species had been detected within the eluates from the management resin (Figs 2B and S1D).


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 response. An engineered cysteine on the amino terminus of OtUBD permits its covalent conjugation to the SulfoLink resin. (B) Ubiquitin blot of pulldowns from yeast cell lysate utilizing covalently linked OtUBD resin or management resin. Covalently linked OtUBD resin effectively pull down ubiquitylated species from yeast complete cell lysate. FT, flow-through; E1/E2/E3, eluted fractions utilizing a collection of stepwise, low pH elutions. (C, D) Extract pretreatment with M48 DUB cleaves ubiquitin from ubiquitylated species and tremendously reduces the overall protein pulled down by OtUBD resin. (C) Anti-ubiquitin blot of OtUBD pulldown of yeast lysate with or with out M48 DUB remedy. (D) Complete protein current within the eluted fractions of the OtUBD pulldowns visualized with SYPRO Ruby stain. (C and D are from 2 separate organic replicates.) IN, enter; FT, flowthrough; E, eluted fractions. (E, F) The V203D mutation in OtUBD, which tremendously impairs its binding of ubiquitin, prevents enrichment for ubiquitylated species from yeast lysate. (E) Anti-ubiquitin blot of pulldowns of yeast lysates utilizing OtUBD resin, Cys resin (detrimental management), and OtUBD(V203D) resin. (F) Complete protein current within the eluted fractions of the OtUBD pulldowns visualized with SYPRO Ruby stain. IN, enter; FT, flowthrough; E1/2/3, eluted fractions utilizing a collection of low pH elutions. MBP, maltose-binding protein; OtDUB, O. tsutsugamushi DUB.

By evaluating the anti-ubiquitin blot in Fig 2B to the final protein stain of the identical eluted fractions in S1D Fig, it was clear that many proteins eluted from the OtUBD resin weren’t themselves ubiquitylated. Pulldown experiments carried out below native or near-native situations are anticipated to copurify proteins that work together noncovalently with ubiquitylated polypeptides, e.g., complexes that harbor ubiquitylated subunits. To check whether or not the complete protein inhabitants eluted from OtUBD resin was nonetheless depending on substrate ubiquitylation, yeast lysates had been preincubated with the viral M48 DUB, which cleaves a broad vary of ubiquitylated proteins and reduces ubiquitin chains to free ubiquitin (Fig 2C) [47]. This remedy tremendously decreased the overall protein eluted from the OtUBD resin in comparison with the pulldown from untreated lysate (Fig 2D), indicating that almost all of proteins eluted from OtUBD resin had been both ubiquitylated themselves or interacted noncovalently with ubiquitin or ubiquitylated proteins.

To additional validate the specificity of the OtUBD resin towards ubiquitylated proteins, we made an OtUBD resin with a ubiquitin-binding poor mutation (V203D) [41] and examined its potential to purify ubiquitin and ubiquitylated proteins. This mutation tremendously diminished the resin’s potential to complement ubiquitylated species (Fig 2E) and likewise strongly decreased the overall protein eluate from the resin (Fig 2F). This means that the power of OtUBD resin to complement for ubiquitylated species relies on its binding affinity towards ubiquitin.

Taken collectively, these outcomes point out the OtUBD resin particularly enriches ubiquitin and ubiquitylated polypeptides in addition to proteins that work together with ubiquitin-containing proteins.

Purifications utilizing OtUBD with denatured extracts enrich ubiquitin–protein conjugates

To differentiate proteins covalently modified by ubiquitin from proteins co-purifying via noncovalent interplay with ubiquitin or ubiquitylated proteins, we optimized pulldown situations to incorporate a denaturation step (Fig 3A). Yeast lysates had been incubated with 8 M urea, a situation the place nearly all of proteins are unfolded, to dissociate protein complexes [48]. Denatured lysates had been then diluted with native lysis buffer (to a remaining urea focus of 4M) to facilitate the refolding of ubiquitin and utilized to the OtUBD resin. An analogous methodology was used beforehand in ubiquitin immunoprecipitation utilizing the FK2 monoclonal antibody [20]. Below such situations, the OtUBD resin concentrated ubiquitylated proteins with efficiencies much like these seen below native situations (Fig 3C). On the identical time, the denaturing remedy tremendously decreased the overall quantity of proteins eluted in comparison with native situations, and the spectrum of purified protein species additionally modified (Fig 3D). This implies that ubiquitylated proteins had been particularly enriched by the urea remedy.


Fig 3. OtUBD pulldown below denaturing situation particularly enriches for proteins covalently modified with ubiquitin.

(A) Workflow of OtUBD pulldowns following pattern denaturation (crimson arrows) or below native (blue arrows) situations. Within the first case, cell lysate is handled with 8 M urea to denature and dissociate proteins. The denatured lysate is then diluted 1:1 with native buffer to permit ubiquitin to refold and bind to OtUBD resin. Below such situations, solely ubiquitylated proteins are anticipated to be enriched. Within the second case, cell lysate comprises native ubiquitylated proteins in addition to proteins that work together with them. OtUBD pulldown below such situations is anticipated to yield each ubiquitylated substrates and ubiquitin-binding proteins. (B) Define for using tandem Co2+ resin pulldowns to validate OtUBD pulldown outcomes below completely different situations. Eluates from OtUBD after lysates had been incubated with denaturant (crimson arrows) or left untreated (blue arrows) are (re)handled with denaturant (8 M urea or 6 M guanidine•HCl) after which subjected to IMAC with a Co2+ resin in denaturing situations. Proteins covalently modified by His6-ubiquitin bind to the Co2+ resin whereas proteins that solely work together noncovalently with ubiquitin find yourself within the flowthrough. (C) Anti-ubiquitin blot of OtUBD pulldowns following native and urea denaturing therapies carried out as described in Fig 3A. FT, flowthrough; E, eluted fractions. The picture was spliced to take away irrelevant lanes. (D) Complete protein current in eluates of the OtUBD pulldowns in Fig 3C visualized by SYPRO Ruby stain. (E) Complete protein current in numerous fractions of the Co2+ IMAC (see Fig 3B; the outcomes proven right here used urea because the denaturant) visualized by SYPRO Ruby stain. IN, enter; FT, flowthrough; E, fraction eluted with 500 mM imidazole. (F) Anti-ubiquitin blot of fractions from Co2+ IMAC (see Fig 3B; the blot proven right here used urea because the denaturant) of eluates from native and denaturing OtUBD resin pulldowns. IN, enter; FT, flowthrough; E, fraction eluted with 500 mM imidazole. *The identification of the outstanding roughly 20 kDa species within the flowthrough from the native extract is unknown. (G) Anti-ubiquitin blot of OtUBD pulldowns from HeLa cell lysates carried out as described in Fig 3A following native or denaturing therapies. The picture was spliced to take away irrelevant lanes. IN, enter; FT, flowthrough; E, fraction eluted with low pH elution buffer. (H) Complete protein current in eluates in Fig 3G visualized by SYPRO Ruby stain. (I) Immunoblot evaluation of human proteasomal subunit Rpt6 in OtUBD pulldowns following native and urea denaturing therapies of lysates. Unmodified Rpt6 co-purified with OtUBD resin below native situations however not following denaturation of extract. N, native situation; D, denaturing situation. OtDUB, O. tsutsugamushi DUB.

To confirm that OtUBD pulldown following a denaturation step is particular for proteins covalently modified with ubiquitin, we utilized a yeast pressure whose endogenous ubiquitin-coding sequences had been all deleted and changed with a single plasmid-borne His6-tagged ubiquitin sequence [28]. The eluted fractions from OtUBD resin pulldowns carried out after both denaturing or nondenaturing therapies of lysates (Fig 3A) had been then denatured once more by incubation with urea or guanidine-HCl (Fig 3B). The denatured proteins had been utilized to a Co2+ (Talon) resin for immobilized metallic affinity chromatography (IMAC) through the His6-tagged ubiquitin. If the eluate from the OtUBD resin had contained solely (His6-)ubiquitylated proteins, most or the entire whole proteins ought to bind to the resin. We noticed that when OtUBD pulldowns had been accomplished following a denaturing lysate remedy, many of the eluted proteins had been certainly certain to the Co2+ resin (Fig 3E). Against this, a big portion of proteins from a “native” OtUBD pulldown remained within the flow-through of the Co2+ resin (Fig 3E). The general ranges of ubiquitylated species recovered, nevertheless, had been comparable between the two therapies (Fig 3F). In line with these findings with bulk ubiquitin conjugates, once we examined whether or not the proteasome, which binds noncovalently to many polyubiquitylated substrates [49], was within the OtUBD eluates, we readily detected unmodified proteasome subunits within the native pulldowns however not in pulldowns from denatured lysates (S2A Fig).

OtUBD-based affinity purifications, below both native or denaturing situations, had been additionally efficient with human cell lysates. Each situations led to comparable enrichment of ubiquitin conjugates (Fig 3G), however the denaturing pretreatment tremendously decreased the quantities of co-purifying nonubiquitylated proteins (Fig 3H). Congruent with this, nonubiquitylated human proteasomal subunits had been solely current at substantial ranges in eluates from native lysates (Figs 3I and S2B). Curiously, low quantities of presumptive ubiquitylated proteasome subunits had been found in OtUBD pulldowns from each native and denatured lysates, and these species had been strongly enriched over the unmodified subunits below the latter situation (S2B Fig).

Total, these outcomes point out that OtUBD-based protein purification below denaturing situations can particularly enrich proteins which can be covalently modified by ubiquitin.

Comparability of OtUBD with different ubiquitin-enriching reagents

Having developed the native and denaturing protocols for OtUBD-based ubiquitin enrichment, we determined to match our new methodology aspect by aspect with current ubiquitin enrichment reagents primarily based on different UBDs or TUBEs. Dsk2 is a yeast protein that comprises a ubiquitin-associated area (UBA) at its carboxyl terminus [50]. Its potential to bind ubiquitylated proteins has been harnessed within the ubiquitin enrichment reagent GST-Dsk2, which has been efficiently used to check the DNA damage-induced ubiquitylation of RNA polymerase-II [51,52]. To immediately examine Dsk2 with OtUBD, we engineered an N-terminal cysteine residue as a deal with to conjugate Dsk2 to SulfoLink resin. As well as, the aforementioned TUBE TR-TUBE [23], in addition to the monomeric UBD it’s primarily based on (a trypsin resistant variant of the UBA area of human Ubiquilin 1; we are going to name it TR-UBA) had been equally engineered for resin conjugation and comparability with OtUBD. (Coincidentally and to our benefit, none of those proteins initially include a cysteine residue.)

After covalently attaching equimolar quantities of various ubiquitin affinity reagents to SulfoLink resin, we examined their respective skills to complement for ubiquitylated species from yeast complete cell lysates below each native and denaturing situations. Below native situations, Dsk2, TR-TUBE and OtUBD all effectively pulled down nearly all of ubiquitylated species from complete cell lysates when enough quantities of resin had been used; TR-UBA was a lot much less environment friendly even when a a lot increased quantity of resin was utilized (Fig 4A). OtUBD and TR-TUBE resins displayed equal effectivity on the lowest resin quantity examined. Though Dsk2 comprises solely a single UBA area, it was capable of effectively purify poly-ubiquitylated proteins when ample quantities of resin had been used, in keeping with its excessive affinity towards poly-ubiquitin [50]. We additionally examined an outdated batch of OtUBD resin that had been saved at 4°C for about one 12 months, and it carried out equally to the freshly ready OtUBD resin, demonstrating a wonderful shelf-life for this resin (Fig 4A, final 2 lanes). Strikingly, when the lysate denaturing pretreatments described earlier had been used with the completely different resins, solely OtUBD might effectively enrich ubiquitylated species (Fig 4B). TR-TUBE solely partially enriched ubiquitylated substrates in comparison with OtDUB below such situations, whereas Dsk2 and TR-UBA barely pulled down any ubiquitylated proteins.


Fig 4. Comparability of OtUBD with different ubiquitin enriching reagents.

(A) Anti-ubiquitin immunoblot of ubiquitin pulldowns from yeast complete cell lysate utilizing completely different UBD/TUBE-based reagents below native situations. Yeast complete cell lysate with 1mg of whole protein was utilized in every pulldown. Elution was achieved by incubating resins with SDS pattern buffer. (B) Anti-ubiquitin immunoblot of ubiquitin pulldowns from yeast complete cell lysate utilizing completely different UBD/TUBE-based reagents following lysate denaturation. Yeast complete cell lysate with 1 mg of whole protein was utilized in every pulldown. Elution was achieved by incubating resins with SDS pattern buffer. (C) Anti-Flag immunoblot of OtUBD and TUBE pulldowns from lysates of WT, bre1Δ and ubp8Δ yeasts expressing Flag-tagged histone H2B. OtUBD resin however not TUBE certain monoubiquitylated histone H2B from complete cell lysate of WT and ubp8Δ yeasts. IN, enter; UBD, eluted fraction from OtUBD pulldown; TUBE, eluted fraction from TUBE pulldown. Elution was achieved by incubating resins with SDS pattern buffer. (D) Anti-Flag immunoblot of pulldowns utilizing completely different UBD/TUBE-based reagents from lysate of ubp8Δ yeast expressing Flag-tagged histone H2B. Solely the OtUBD resin was capable of bind detectably to monoubiquitylated histone H2B. (E) Western blot of yeast Rpt5 from pulldowns of rpt2-P103A rpt5-P76A (rpt2,5PA) mutant yeast lysates utilizing completely different ubiquitin affinity resins below completely different situations. N, pulldown carried out below native situations; D1, pulldown carried out after 8 M urea remedy of lysate as described in Fig 3A; D2, pulldown carried out with lysate immediately extracted in buffer containing 8 M urea. (F) Western blot evaluation of RNAPII subunit Rpb1 in OtUBD and Dsk2 pulldowns of native HeLa cell lysates. Cells had been handled with 4-NQO to induce RNAPII ubiquitylation. (G) Western blot evaluation of RNAPII subunit Rpb1 in OtUBD and Dsk2 pulldowns of denatured HeLa cell lysates. Cells had been handled with 4-NQO to induce RNAPII ubiquitylation. OtDUB, O. tsutsugamushi DUB; TUBE, tandem ubiquitin-binding entity; UBD, ubiquitin-binding area; WT, wild-type; 4-NQO, 4-nitroquinoline-1-oxide.

We then in contrast the completely different resins for his or her skills to detect ubiquitylated types of particular proteins of curiosity. Histone H2B is monoubiquitylated by the ubiquitin E3 ligase Bre1 and deubiquitylated by the DUB Ubp8 in yeast [45,53]. The monoubiquitylated spinoff of histone H2B is troublesome to detect immediately in the entire cell lysate on account of its low abundance compared to unmodified H2B (Fig 4C). To find out if the OtUBD resin might assist within the detection of monoubiquitylated H2B, we used OtUBD resin to purify whole ubiquitylated proteins from cell lysates of wild-type (WT), bre1Δ and ubp8Δ yeast strains expressing Flag-tagged histone H2B after which analyzed the proteins by immunoblotting. A slower migrating band within the anti-Flag immunoblot, which represents the monoubiquitylated H2B, was detected within the WT and ubp8Δ yeasts however not within the bre1Δ yeast (Fig 4C). Against this, the TR-TUBE-resin did not seize the monoubiquitylated H2B in any of the yeast samples, together with these with elevated ranges of H2B monoubiquitylation on account of deletion of UBP8 (Fig 4C). We additionally examined whether or not any of the opposite resins we made had been capable of seize mono-ubiquitylated histone H2B in ubp8Δ yeast lysate and located that neither Dsk2 nor TR-UBA might (Fig 4D). Thus, though each Dsk2 and TR-TUBE-resin can effectively enrich bulk ubiquitylated species from yeast lysates (Fig 4A), these reagents will be restricted of their skills to detect sure monoubiquitylated proteins. These outcomes once more spotlight a possible benefit of OtUBD relative to different UBDs or TUBEs in finding out monoubiquitylated substrates.

Our group beforehand recognized Professional-to-Ala mutations in yeast proteasomal subunits Rpt2 and Rpt5 (pressure “rpt2,5PA”) that result in their misfolding and ubiquitylation below regular development situations [54]. In that examine, ubiquitylation of the Rpt subunits was confirmed by overexpressing His-tagged ubiquitin within the proteasome mutant yeast pressure and performing IMAC below denaturing situations to seize ubiquitylated species. We carried out OtUBD and TUBE pulldowns with rpt2,5PA yeast lysates with out ubiquitin overexpression. Primarily based on anti-Rpt5 immunoblotting, each resins captured a smear of upper mass Rpt5PA species, that are doubtless endogenous polyubiquitylated Rpt5PA species (Fig 4E). Unmodified Rpt5PA co-purified with each OtUBD and TUBE below native situations however was largely eradicated from each pulldowns following lysate denaturation. Below native situations, each OtUBD and TUBE purified comparable quantity of ubiquitylated Rpt5PA (Fig 4E, lane 4 and 5) whereas below denaturing situations, OtUBD captured extra ubiquitylated species particularly on the decrease molecular weight vary (Fig 4E, lane 6 to 9). In comparison with the TUBE pulldown, OtUBD pulldown captured an extra slower-migrating Rpt5PA species which, primarily based on the obvious molecular mass, is probably going monoubiquitylated Rpt5PA. On this experiment, we additionally discovered that denatured lysate extracted immediately with urea buffer (situation D2) gave extra ubiquitylated Rpt5PA in comparison with when the lysate was extracted with a local buffer first after which denatured with added urea (situation D1). The previous methodology is anticipated to solubilize ubiquitylated species that might usually precipitate.

As a remaining instance of single protein evaluation, we used OtUBD to detect ubiquitylated RNA polymerase II (RNAPII) in cultured human cells. RNAPII turns into ubiquitylated upon UV-induced DNA harm [55]. Rpb1, the most important subunit of RNAPII, is closely ubiquitylated below such situations [56]. We handled HeLa cells with the chemical 4-nitroquinoline-1-oxide (4-NQO), which mimics the organic results of UV on DNA (57), and carried out OtUBD and Dsk2 pulldowns of each native (Fig 4F) and denatured (Fig 4G) lysates with saturating quantities of every resin (as decided in Fig 4A). In each circumstances, OtUBD pulldown captured comparable slower migrating bands when analyzed by anti-Rpb1 immunoblotting. As a result of Rpb1 is a big protein of over 200 kDa and exists in numerous phosphorylation states, it’s troublesome to differentiate nonubiquitylated and monoubiquitylated species primarily based on migration via an SDS-PAGE gel [58]. OtUBD pulldown below denaturing situations, on this case, supplies confidence that Rpb1 is ubiquitylated even below basal situations and turns into closely ubiquitylated upon remedy with 4-NQO (Fig 4G). Dsk2 pulldown captured a lot much less ubiquitylated Rpb1 below each situations in comparison with OtUBD. The two reagents might probably seize completely different populations of ubiquitylated Rpb1 primarily based on the looks of the bands noticed (Fig 4F).

These examples illustrate how OtUBD resin can facilitate the detection of monoubiquitylated and polyubiquitylated proteins in each yeast and human cells. Importantly, in a number of completely different examples, OtUBD demonstrated its benefits over different UBD/TUBE-based reagents examined, notably in its potential to seize monoubiquitylated proteins and to bind ubiquitylated species following protein denaturation.

OtUBD-pulldown proteomic profiling of the yeast and human ubiquitylome and ubiquitin interactome

By evaluating OtUBD pulldowns of native and denatured cell lysates, we will probably differentiate completely different ubiquitin-related proteomes in a organic pattern. The “ubiquitylome,” i.e., the gathering of covalently ubiquitylated proteins, will be outlined because the protein inhabitants eluted from an OtUBD affinity resin used with denatured cell extracts. The “ubiquitin interactome” will be roughly outlined as these proteins which can be particularly enriched following OtUBD pulldowns from native extracts however not pulldowns from denatured lysates (Fig 3A). Notably, the latter definition will exclude circumstances the place a subpopulation of a protein is ubiquitylated whereas the nonubiquitylated inhabitants of the identical protein interacts noncovalently with ubiquitin or different ubiquitylated proteins. For instance, some proteasomal subunits are identified to be ubiquitylated [59], however proteasome particles the place these subunits are unmodified nonetheless work together noncovalently with ubiquitylated proteins. Proteins equivalent to these proteasomal subunits can be excluded from the ubiquitin interactome as outlined right here. Nonetheless, these definitions present a normal image of the ubiquitylome and ubiquitin interactome.

We carried out OtUBD pulldowns of complete yeast lysates with and with out prior denaturation (S3A–S3C Fig) and profiled the eluates utilizing shotgun proteomics. The quantity of resins used was predetermined empirically to keep away from saturation of binding capability. For every situation, we included 2 organic replicates and for every organic replicate, 2 technical repeats of the LC-MS/MS run. Management pulldowns by SulfoLink resin with out OtUBD had been carried out in parallel to get rid of proteins that nonspecifically bind to the resin. As was seen in earlier experiments, the management pulldowns yielded no detectable ubiquitylated species and solely hint quantities of proteins (S3A–S3C Fig). Some proteins had been recognized in a subset of the management pulldown replicates (S3D Fig), due partially to carryover of excessive abundance peptides from earlier runs, however the total portions of proteins in these management samples, as demonstrated by TIC (whole ion present), had been a lot decrease in comparison with the OtUBD pulldown samples (S3E Fig). Therefore, for every organic replicate, solely proteins current at considerably increased ranges (>20-fold) within the OtUBD pulldown samples over the corresponding management pulldown samples had been thought-about actual hits (S3F Fig and S2 Knowledge).

The two pulldown situations yielded comparable whole numbers of proteins (Fig 5A) with a significant overlap of protein identities. Over 400 proteins had been found completely below native situations, suggesting they don’t seem to be ubiquitylated themselves however co-purify with ubiquitin or ubiquitylated proteins. Curiously, over 600 proteins had been recognized solely below denaturing situations. As a result of OtUBD pulldowns following lysate denaturation yield a lot much less whole protein than below native situations (S3C Fig), the potential of figuring out low abundance proteins within the LC-MS/MS evaluation is probably going elevated.


Fig 5. OtUBD pulldown-proteomics permits profiling of the ubiquitylome and ubiquitin interactome of yeast and human cells.

(A) Venn diagram of yeast proteins recognized by OtUBD pulldown-proteomics with the pulldowns carried out below following both nondenaturing or urea denaturing therapies. The gathering of proteins recognized in OtUBD pulldowns below denaturing situation is outlined because the ubiquitylome (blue define). The gathering of proteins recognized solely in native OtUBD pulldown is outlined because the ubiquitin interactome (purple define). (B) Venn diagram evaluating the yeast ubiquitylome outlined by OtUBD pulldown-proteomics with 3 earlier research utilizing the di-Gly antibody IP methodology [6062]. (C, D) Prime organic pathways concerned in OtUBD pulldown-defined yeast ubiquitylome (C) and ubiquitin interactome (D) primarily based on GO evaluation. The numeric values supporting these panels will be present in S3 Knowledge. (E) Venn diagram evaluating the human ubiquitylome outlined by OtUBD pulldown and FK2 antibody IP carried out within the present examine. (F) Venn diagram evaluating the printed human ubiquitylomes outlined by diGly antibody [63] and TUBE-based enrichments [64] and the OtUBD-defined ubiquitylome obtained on this examine. GO, Gene Ontology; OtDUB, O. tsutsugamushi DUB; TUBE, tandem ubiquitin-binding entity.

We in contrast the OtUBD-defined yeast ubiquitylome with information from beforehand printed research utilizing di-Gly remnant antibody-based strategies (Fig 5B) [6062]. Our examine recognized 1,811 ubiquitylated yeast proteins, the second highest quantity among the many 4 research in contrast right here. About two-thirds of proteins recognized in our examine have been reported to be ubiquitylated by no less than considered one of these di-Gly antibody-based research. Round 600 ubiquitylated proteins had been uniquely recognized on this examine. A few of these may contain noncanonical ubiquitylation, the place the ubiquitin modifier is covalently connected to a nucleophilic residue on the substrate aside from a lysine [3].

GO evaluation indicated that the yeast ubiquitylome outlined by OtDUB binding spans proteins from all kinds of mobile processes, together with a number of biosynthesis pathways, protein localization, vesicle-mediated transport, and protein high quality management pathways (Fig 5C). Against this, the ubiquitin interactome, as outlined above, appeared to yield larger illustration in nucleic acid-related processes such DNA replication, RNA transcription, ribosome biogenesis, and noncoding RNA processing (Fig 5D).

We additionally carried out OtUBD pulldowns with denatured HeLa cell lysates alongside immunoprecipitations utilizing the FK2 anti-ubiquitin monoclonal antibody, which has been used efficiently with the same denaturation protocol [20]. Three organic replicates, every with 2 technical repeats of LC-MS/MS runs, had been analyzed. OtUBD resins effectively and constantly enriched ubiquitylated proteins from HeLa cell lysates. Against this, we noticed that completely different batches of FK2 antibodies gave completely different efficiencies of enrichment; that is evident within the quantity of ubiquitylated species current within the flowthrough fractions of FK2 immunoprecipitations in 2 of the organic replicates utilizing completely different batches of antibodies (S4A and S4B Fig) in addition to the overall variety of proteins recognized in every organic replicate. This might be a results of inconsistency in antibody high quality throughout batches or in effectivity of antibody immobilization. Nearly all of ubiquitylated proteins recognized by the FK2 antibody resin had been additionally discovered within the ubiquitylated proteome recognized by OtUBD resin (Fig 5E). In comparison with the FK2 immunoprecipitations, OtUBD pulldowns recognized about 1,000 extra ubiquitylated proteins, nearly all of which have no less than one reported ubiquitylation web site in a earlier examine utilizing diGly antibodies [63].

We additionally in contrast our outcomes with printed information primarily based on TUBE or diGly antibody-based enrichment strategies. For human ubiquitylome discovery, completely different research utilizing the identical enrichment methodology yielded a variety within the whole variety of detected proteins [35]. Because the human ubiquitylome is way more advanced than the yeast ubiquitylome, proteomics strategies and practices that may have an effect on the depth of protein identifications will extra strongly affect the general outcomes with human cells (see instance in Dialogue). Subsequently, evaluating the variety of recognized proteins throughout completely different research will mirror not solely the relative efficiencies of the completely different ubiquitin enrichment strategies but additionally variations within the proteomics methodology and cell tradition situations.

With this caveat in thoughts, we in contrast our outcomes by way of whole variety of identifications and the identities of ubiquitylated proteins with printed information units describing the human ubiquitylome utilizing the diGly antibody enrichment [63] and TUBE strategies [64], respectively (Fig 5F). The diGly-based outcome from Elia and colleagues, which found the most important variety of ubiquitylated proteins amongst completely different diGly literatures we examined, encompassed a a lot increased whole variety of ubiquitylated proteins in comparison with our examine and the TUBE-based examine, which yielded comparable numbers of whole proteins. In comparison with the TUBE-based identifications, our information total confirmed larger overlap with the diGly antibody-based outcome—77% of proteins detected in our evaluation and 58% of these within the TUBE examine had been supported by no less than one diGly-remnant-containing peptide reported within the diGly antibody-based examine. One clarification for the decrease congruence for the TUBE-based enrichment was that it was accomplished below native situations and due to this fact doubtless included many nonubiquitylated polypeptides that co-purified with ubiquitin or ubiquitylated proteins.

These experiments show that the OtUBD affinity resin can be utilized to profile the ubiquitylated proteome of each yeast and human cells. Furthermore, all 7 lysine ubiquitin–ubiquitin linkages had been recognized within the yeast proteomics (S4C Fig), and their ratios roughly agreed with earlier quantitative research of relative linkage frequencies [4], indicating comparatively unbiased enrichment of those completely different lysine linkages. The molecular foundation of this obvious indifference to chain linkage is unsure. Earlier evaluation by isothermal calorimetry of OtUBD binding to monomeric ubiquitin revealed a 1:1 advanced with a Okayd of roughly 5 nM; ubiquitin dimers linked by both K48 or K63 certain extraordinarily tightly, stopping correct willpower of their dissociation constants by this methodology [41]. OtUBD may bind to the distal ubiquitin in all chain sorts with excessive affinity, resulting in unbiased enrichment of various polyubiquitin chains, in addition to detection of noncanonical ubiquitylation. All besides the K33 ubiquitin–ubiquitin linkage had been additionally recognized within the HeLa cell proteomics. We observe that K33 is a low abundance linkage [4], and employment of methods that may enhance the depth of identifications, equivalent to an orthogonal fractionation step previous to LC-MS/MS, would doubtless enable its detection.

OtUBD and label-free quantitation allow identification of potential E3 ligase substrates

Lastly, we sought to use OtUBD-pulldown proteomics for figuring out substrates of particular E3 ubiquitin ligases. Identification of substrates for specific E3 ligases will be difficult as a result of transient nature of E3-substrate interactions and the low abundance and instability of many ubiquitylated proteins [65]. One option to display screen for potential substrates is to match the ubiquitylome of cells with and with out (or with decreased degree/perform of) the E3 of curiosity [66]. Proteins with increased ubiquitylation ranges within the cells expressing the E3 in comparison with cells missing it could be candidate substrates. We used OtUBD-pulldown proteomics to match the ubiquitylomes of wildtype BY4741 yeast and a couple of congenic E3 deletion strains, bre1Δ and pib1Δ, obtained from a yeast gene knockout library [67]. Bre1 is a comparatively well-characterized E3 ligase that monoubiquitylates histone H2B [53]. This ubiquitylation doesn’t result in H2B proteolysis however is concerned in essential chromosomal processes, together with transcription and DNA harm restore [68]. Different substrates of Bre1 are largely unknown [69]. Pib1 is a a lot much less studied E3 ligase that localizes to endosomes and the vacuole and participates in endosomal sorting [70].

We harvested WT, bre1Δ, and pib1Δ yeast cells and following lysate denaturation, carried out OtUBD pulldowns. Proteins eluted from the OtUBD resin had been topic to label-free quantitative proteomics (Figs 6A, S5A and S5B). Three organic replicates had been examined for every group, and every replicate was analyzed by 2 separate LC-MS/MS runs. Quantitation was carried out utilizing whole TIC after normalization among the many analyzed samples. As anticipated, histone H2B (recognized as Htb2) offered at a a lot increased degree within the ubiquitylome of WT cells in comparison with that of bre1Δ cells (Fig 6B). Curiously, we recognized 2 completely different ubiquitylation websites on histone H2B (Htb2) in numerous samples (Figs 6C, S6A and S6B). The K123 ubiquitylation web site, which is the key reported ubiquitylation web site of Bre1 on histone H2B [71,72], was detected in WT cells however not in bre1Δ cells. Against this, the opposite ubiquitylation web site, K111, confirmed up in each WT and bre1Δ cells. This implies there may be an E3 ligase(s) aside from Bre1 that ubiquitylates histone H2B on K111. Though this ubiquitylation web site had been reported in a diGly antibody-based proteomics examine [61], its perform stays to be studied. Apart from histone H2B, we additionally recognized 16 different proteins current in considerably increased ranges within the WT cell ubiquitylome in comparison with bre1Δ cells (Fig 6D). As well as, 35 proteins had been completely detected within the ubiquitylome of WT cells (Fig 6E). Taken collectively, these proteins are thought-about potential Bre1 substrates. Curiously, a few of these proteins (Fig 6D and 6E, inexperienced) had been proven to be metabolically stabilized in bre1Δ cells in an earlier examine [73], which indicated that they might be direct or oblique proteolytic ubiquitylation substrates of Bre1.


Fig 6. Identification of potential E3 substrates by OtUBD pulldown and label-free quantitation.

(A) Scheme for E3 substrate identification utilizing OtUBD pulldown and quantitative proteomics. WT and E3 deletion (bre1Δ and pib1Δ) yeast strains had been subjected to OtUBD pulldowns following extract denaturation. The eluted proteins had been then analyzed by label-free quantitation. (B) Volcano plot evaluating WT and bre1Δ samples. Orange dots symbolize proteins that had been considerably enriched in WT samples in comparison with bre1Δ samples. Horizontal dashed line signifies p = 0.05. Vertical sprint strains point out relative change of +/− 1.5-fold. The numeric values supporting this determine will be present in S2 Knowledge. (C) Two completely different ubiquitylation websites recognized on histone H2B (Htb2) in numerous samples. (D) Listing of proteins that had been considerably enriched in WT samples in comparison with bre1Δ samples (orange dots in B). Inexperienced coloration signifies proteins that had been beforehand reported to be stabilized in bre1Δ yeast. (E) Ubiquitylated proteins detected completely in WT however not bre1Δ samples. Inexperienced coloration signifies proteins beforehand reported to be stabilized in bre1Δ cells. (F) Volcano plot evaluating WT and pib1Δ samples. Orange dots symbolize proteins that had been considerably enriched in WT samples in comparison with pib1Δ samples. Horizontal sprint line signifies p = 0.05. Vertical sprint strains point out relative change of +/− 1.5-fold. The numeric values supporting this determine will be present in S2 Knowledge. (G) Listing of proteins that had been considerably enriched in WT samples in comparison with pib1Δ samples (orange dots in F). Inexperienced coloration signifies proteins that had been beforehand reported to be stabilized in pib1Δ yeast. (H) Ubiquitylated proteins detected completely in WT however not pib1Δ samples. Inexperienced coloration signifies proteins beforehand reported to be stabilized in pib1Δ yeast. OtDUB, O. tsutsugamushi DUB; WT, wild-type.

Analogous to the Bre1 information, we recognized 3 proteins whose ubiquitylated kinds had been discovered at considerably increased ranges in WT cells versus pib1Δ cells (Fig 6F and 6G) and 38 proteins that had been detectably ubiquitylated in WT cells however not pib1Δ cells (Fig 6H). Of those proteins, 6 have been proven beforehand to be stabilized in pib1Δ cells (Fig 6G and 6H, inexperienced) [73].

Whether or not these potential E3 substrates are direct or oblique ubiquitylation substrates of the examined E3s will have to be validated by biochemical assays. Nonetheless, our outcomes demonstrated that OtUBD can be utilized to profile ubiquitylomes quantitatively, which can be helpful within the identification of novel substrates for E3 ligases and different ubiquitin-related enzymes equivalent to E2s and DUBs.

Among the many numerous proteomics information obtained from our OtUBD pulldowns, we noticed quite a few potential nonlysine ubiquitylation websites assigned by the Mascot search algorithm (S2 Knowledge), substantiating the concept that OtUBD doubtless can enrich proteins with nonlysine ubiquitylation websites. (Please observe that on this checklist, the GG-peptide assignments by the search algorithm haven’t been validated and can embody some inaccurate assignments. Additional validation is required to substantiate any particular person task.) We confirmed considered one of these websites by guide validation of the spectrum task (S6C Fig).


Protein ubiquitylation continues to be of nice curiosity due its very important contributions to many basic mobile processes and for its essential roles in human illness. Many enzymes concerned in ubiquitylation are being pursued as targets for therapeutics [74,75]. For instance, quite a few drug candidates concentrating on E3 ligases equivalent to MDM2 and XIAP have entered scientific trials for remedy of a number of kinds of most cancers [76]. Quite a lot of reagents and strategies to check ubiquitylation or ubiquitylation-related processes have been developed, however these strategies all have limitations [16,39]. For instance, TUBEs are efficient at detecting polyubiquitin chains, however this creates a bias towards polyubiquitylated substrates; they typically fail to detect protein monoubiquitylation indicators (e.g., Fig 4C and 4D), which may dominate the ubiquitylome in no less than some mammalian cell sorts [17]. In consequence, new and economical reagents and strategies to research the various kinds of ubiquitin modification are nonetheless wanted, notably when these modifications are current at very low ranges.

The versatile high-affinity UBD area of O. tsutsugamushi DUB supplies a ubiquitin affinity reagent with a number of benefits over current instruments. First, it’s simple and comparatively cheap to generate the affinity resin utilizing the small recombinant OtUBD protein expressed and purified from Escherichia coli. Second, ubiquitin enrichment utilizing sOtUBD is relevant to each monoubiquitylation and polyubiquitylation, in distinction to the bias of TUBEs and different UBD-based reagents equivalent to those we examined (Fig 4D). Third, OtUBD pulldowns will be carried out below native situations for the examine of each ubiquitylated substrates and proteins that affiliate noncovalently with them; alternatively, by subjecting extracts to denaturing situations previous to pulldown, OtUBD pulldowns will be tuned towards proteins covalently modified by ubiquitin. We demonstrated that OtUBD pulldowns, coupled with proteomics, can be utilized to profile the ubiquitylated proteomes of yeast and mammalian cells; it could possibly undoubtedly be used to characterize the ubiquitylomes of different eukaryotic cells as effectively. Fourth, comparative OtUBD pulldown-proteomics can be utilized to establish substrates of ubiquitylating enzymes (E2s or E3s), as proven right here, or DUBs. Lastly, not like the anti-diGly immunoaffinity instrument that’s particular for diGly remnants on Lys aspect chains, OtUBD-based purifications can probably assist establish noncanonical ubiquitin–protein linkages equivalent to these via Cys, Ser, or Thr aspect chains, the N-terminal amino group, or chemical bonds that don’t contain the ubiquitin carboxyl terminus, as in ubiquitylation mediated by Legionella SidE proteins [3,77,78]. It also needs to be potential to complement ubiquitin linkages to macromolecules aside from proteins, such because the just lately found ubiquitin-lipopolysaccharide adducts fashioned throughout Salmonella infections [40].

We’ve got demonstrated that OtUBD is particular towards ubiquitin and ubiquitylated proteins. Nonetheless, a number of caveats ought to be famous. Though OtUBD pulldowns following extract denaturation considerably reduces the quantity of interacting proteins co-purifying with ubiquitylated proteins, a small variety of noncovalently interacting proteins should still be co-purified in some circumstances (e.g., S2B Fig). Further stringent wash steps might assist mitigate this drawback. OtUBD additionally binds to the carefully associated ubiquitin-like modifier Nedd8, though with a a lot decrease affinity than for ubiquitin [41]. Like ubiquitin, Nedd8 is used for protein posttranslational modification [79] and since they go away the identical -GG remnant after trypsin digestion, it’s arduous to distinguish the two modifiers utilizing the traditional diGly antibody methodology [16]. We regarded for potential neddylation substrate(s) in our proteomics research. Rub1 (yeast Nedd8), Cdc53, Rtt101, and Cul3 (3 yeast cullin proteins reported to endure neddylation [80]) had been detected within the OtUBD-defined ubiquitylome, which can have been enriched on the OtUBD resin via Nedd8 binding. Nonetheless, neddylation happens at a lot decrease ranges in comparison with ubiquitylation [37] and primarily based on the specificity evaluation we carried out (Figs 2D and 3E), neddylated proteins (if any) ought to account for less than a small fraction of the OtUBD-enriched proteome.

In our OtUBD pulldown-proteomics experiments, the overall variety of ubiquitylated yeast proteins recognized was akin to earlier research utilizing the di-Gly antibody enrichment methodology [6062]. For the evaluation of human cells, OtUBD pulldowns carried out higher than FK2 antibody immunoprecipitations in our arms, each by way of total effectivity and repeatability. Inconsistency within the efficiency of antibodies from completely different batches has been a widely known concern [81]. Recombinant OtUBD, against this, seemed to be constant throughout completely different batches in addition to steady throughout long-term storage. Because the human ubiquitylome is way more advanced in comparison with the yeast ubiquitylome, proteomics identification depth tremendously influences the overall variety of ubiquitylated proteins recognized in every examine [35]. Subsequently, it’s troublesome to interpret outcomes evaluating our human ubiquitylome information with printed information from different research.

Optimization of our proteomics pipeline would doubtless enhance the variety of recognized ubiquitylated proteins and ubiquitylation websites, particularly for low-abundance proteins. In a paper benchmarking a TUBE-based enrichment methodology referred to as ThUBD with human cell lysates [46], the authors initially fractionated the eluted proteins by SDS-PAGE previous to trypsin digestion and LC-MS/MS; they recognized 1,663 ubiquitylated proteins of their TUBE enriched samples. Switching to an orthogonal LC-based fractionation of digested peptides previous to the LC-MS/MS run, the authors had been capable of enhance their total identification to over 7,000 proteins, highlighting the robust affect of proteomics methodologies in ubiquitylome characterization. Within the samples we analyzed, peptides derived from ubiquitin accounted for a major share of the overall variety of recognized peptides. This doubtless suppressed detection of low-abundance peptides, particularly these with comparable retention instances. Preclearing of free ubiquitin from the eluted samples earlier than LC-MS/MS, for instance, by gel separation or an affinity depletion particular without cost ubiquitin [82], would doubtless cut back this drawback. Alternatively, fractionation of the protein or peptide samples previous to LC-MS/MS also needs to improve the general discovery price.

OtUBD-based ubiquitin purification might be used along with strategies that effectively enrich sure ubiquitylated species. For instance, OtUBD pulldowns might be carried out earlier than di-Gly antibody immunoprecipitation. When carried out towards the large pool of peptides derived from the complete cell proteome, di-Gly antibody IP typically must be accomplished in a number of batches or for a number of rounds to make sure environment friendly enrichment [63]. A preliminary OtUBD pulldown step would considerably enrich for ubiquitylated substrates within the pattern with out creating any bias towards polyubiquitylated species. It will tremendously enhance the share of di-Gly-linked peptides current within the digested pattern. Since OtUBD has exceptionally excessive affinity towards free ubiquitin, it may be used with the Ub-Clipping method [83]. In Ub-Clipping, ubiquitylated proteins are cleaved on the ubiquitylation websites by the protease Lb-Professional to generate diGly-linked monoubiquitin species and free ubiquitin1-74. These species carry info on ubiquitin chain topology and posttranslational modifications of ubiquitin that may be deciphered by MS evaluation. Deployment of OtUBD for different purposes will be readily envisioned.

With the characterization of OtUBD-ubiquitin binding and crystal buildings of OtUBD out there [41], one can think about additional modifications that might adapt or improve OtUBD for different makes use of. For instance, directed evolution or structure-based rational mutagenesis could also be carried out to alter OtUBD binding specificity towards ubiquitin, particular ubiquitin chains or ubiquitin-like proteins. OtUBD might be used to make different ubiquitin detection instruments by attaching a fluorophore or different useful handles to it. As a recombinant protein reagent that’s versatile and simple to arrange, OtUBD can be a cost-effective addition to the ubiquitin analysis toolbox.

Supplies and strategies

Plasmids and DNA cloning

The coding sequence for 3xOtUBD was synthesized by Genscript USA. pRSET-4xTR-TUBE was a present from Yasushi Saeki (Addgene plasmid # 110312) [43]. The pRT498 vector, a bacterial expression plasmid modified from pET42b to incorporate an N-terminal His6-MBP with a cleavable TEV web site, was used for expression of MBP and MBP-fusion proteins made in our lab. pET21a and pET42b vectors had been used to specific His6-tagged proteins in micro organism. Plasmids and primers used on this examine in addition to insert sequences are described intimately in S1 Knowledge. All PCR reactions had been accomplished utilizing Phusion Excessive-Constancy DNA Polymerase (New England Biolabs).

Yeast strains and development

Yeast strains used are listed in S1 Knowledge. Yeast cultures had been grown in a single day in yeast extract-peptone-dextrose (YPD) medium to saturation. The following day, the tradition was diluted in contemporary YPD to an OD600 of 0.1 to 0.2 and cultured at 30°C with shaking till reaching mid-exponential part (OD600 0.8 to 1.2). Cells had been pelleted, washed with water, and flash frozen in liquid nitrogen and saved at −80°C till used.

Expression and purification of recombinant proteins

Recombinant His6-tagged proteins had been purified from Rosetta (DE3) competent E. coli cells (Novagen) remodeled with the suitable plasmids. Bacterial cells had been grown in a single day in Luria-Bertani (LB) broth supplemented with both 100 μg/mL ampicillin (for pET21a-based plasmids) or 50 μg/mL kanamycin (for pRT498- and pET42b-based plasmids) and diluted 1/100 the following morning in contemporary LB broth supplemented with the corresponding antibiotics. When cell density had reached 0.5 to 0.8 OD600, protein manufacturing was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a remaining focus of 0.3 mM, and cells had been cultured at 16 to 18°C in a single day with shaking. Micro organism had been pelleted and resuspended in micro organism lysis buffer (50 mM Tris•HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 2 mM freshly added phenylmethylsulfonyl fluoride (PMSF)) supplemented with lysozyme and DNaseI, incubated on ice for half-hour and lysed utilizing a French press. For purification of His6-tagged proteins and His6-MBP-tagged proteins, the lysates had been clarified by centrifugation for 1 hour at 4°C at 10,000 rcf earlier than being subjected to Ni-NTA (QIAGEN) affinity purification following the producer’s protocol. For additional purification of His6-tagged OtUBD, Dsk2, TR-UBA, or 4xTR-TUBE, the protein eluted from the Ni-NTA matrix was supplemented with 5 mM tris(2-carboxyethyl)phosphine) (TCEP) (from a 1M TCEP inventory neutralized with NaOH to pH 7), concentrated, after which fractionated by Quick Protein Liquid Chromatography (FPLC) on a Superdex 75 gel filtration column (Cytiva) utilizing FPLC buffer (50 mM Tris•HCl, pH 7.5, 150 mM NaCl, 1 mM TCEP). For additional purification of His6-MBP-tagged proteins, the protein eluate from the Ni-NTA resin was concentrated and fractionated by FPLC on a Superdex 75 column utilizing 50 mM Tris•HCl, pH 7.5, 150 mM NaCl buffer supplemented with 1 mM dithiothreitol (DTT).

For purification of OtUBD variants utilizing pRT498-based plasmids, the His6-MBP-tagged proteins eluted from the Ni-NTA resin had been topic to buffer change in a 50 mM Tris•HCl, pH 7.5, 150 mM NaCl buffer supplemented with 10 mM TCEP utilizing a centrifugal filter system (Amicon, 3000 MWCO) following producer’s protocol. His-tagged TEV protease was added to take away the His6-MBP tag, and the combination was incubated on ice in a single day. The cleavage combination was then allowed to move via a column of unpolluted Ni-NTA resin to seize the cleaved His6-MBP tag. The flow-through was concentrated and purified by FPLC with a Superdex 75 column utilizing FPLC buffer.

The M48 DUB protein was ready as described earlier [47].

All proteins had been flash-frozen in liquid nitrogen and saved at −80°C till use. Protein concentrations had been decided by both SDS-PAGE and GelCode Blue (Thermo) staining or a BCA assay (Thermo) utilizing bovine serum albumin (BSA) as the usual.

Immunoblotting and antibodies

Proteins resolved via SDS-PAGE gels had been transferred to Immobilon-P PVDF membranes (Millipore) and blocked with 3% nonfat milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH adjusted to 7.5 with HCl) with 0.1% Tween-20 (TBST). Membranes had been incubated first with the specified main antibody diluted in TBST containing 1% nonfat milk for 1 hour at room temperature or in a single day at 4°C, washed extensively, after which incubated with an HRP-linked secondary antibody diluted in TBST with 1% milk for 1 hour at room temperature or in a single day at 4°C.

Major antibodies used on this examine had been rabbit polyclonal anti-ubiquitin antibody (Dako, discontinued, 1:2,000 dilution), monoclonal mouse anti-ubiquitin antibody P4D1 (Enzo, 1:1,000), monoclonal mouse anti-Flag M2 (Sigma, 1:5,000 or 1:10,000), monoclonal mouse anti-human Rpt6 (PSMC5) (Invitrogen, 2SU-1B8, 1:10,000), monoclonal mouse anti-human Rpt4 (Enzo, p42-23, 1:1,000), mouse monoclonal anti-yeast Rpt4 (reward from W. Tansey, 1:2,500), rabbit polyclonal anti-Rpt5 (Enzo Life Sciences), rabbit polyclonal anti-Pre6 (reward from D. Wolf, 1:5,000) and anti-Rpb1 (RNA Pol II) monoclonal mouse antibody (Lively Motif, 4H8, 1:2,000). For rabbit main antibodies, the HRP-linked anti-rabbit IgG secondary antibody (GE Healthcare, NA934) was used at a dilution of 1:5,000 or 1:10,000. For mouse main antibodies, the HRP-linked anti-mouse secondary antibody (GE Healthcare, NXA931V) was used at a dilution of 1:10,000.

Blots had been visualized by enhanced chemiluminescence on a G:Field imaging system with GeneSnap software program (Syngene). Photographs had been processed with ImageJ software program.

Safety of ubiquitylated species in complete cell yeast lysates

Yeast ubp8Δ cells expressing Flag-tagged histone H2B [44] had been grown in YPD medium and harvested throughout exponential part development. The cell pellet was washed with water, flash-frozen, and lysed by grinding below liquid nitrogen in a mortar. Proteins had been extracted by addition of lysis buffer (50 mM Tris•HCl, pH 7.5, 150 mL NaCl, 1 mM EDTA, 10% glycerol, cOmplete EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF) within the presence of 20 mM NEM, 3 μM OtUBD, 3 μM 4xTR-TUBE (all remaining concentrations), or nothing. The ensuing lysates had been cleared by centrifugation at 21,000 x g for 12 minutes at 4°C and incubated at room temperature for 1 to 4 hours. Flag-tagged H2B was purified by anti-Flag immunoprecipitation with ANTI-FLAG M2 Affinity Gel (Millipore) following the producer’s protocol. Complete cell lysates had been analyzed by anti-ubiquitin immunoblotting. The anti-Flag precipitates had been analyzed by anti-Flag immunoblotting.

Pulldown with MBP-tagged bait proteins

Pulldowns of MBP-tagged fusion proteins had been carried out utilizing an amylose resin (New England Biolabs). Applicable quantities of (see Figs 1D, S1A and S1B) MBP or MBP fusion proteins diluted in 300 μL amylose column buffer (20 mM Tris•HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA) had been incubated with 50 μL amylose resin for 1 hour at 4°C with rotation. The resin was pelleted by centrifugation at 5,000 x g for 30 seconds, and the supernatant was eliminated. One mL of yeast lysate (1 to 2 mg/mL) ready in column buffer freshly supplemented with protease and DUB inhibitors (cOmplete mini EDTA-free (Roche), 10 mM NEM, 1 mM PMSF) was added to the beads. (For detailed strategies of lysate preparation, see part “Ubiquitin pulldown with protein-linked resins” beneath.) The combination was incubated with rotation at 4°C for two hours. The resin was washed 5 instances with 1 mL column buffer after which eluted by incubating with column buffer containing 50 mg/mL maltose for two hours at 4°C with rotation. Alternatively, certain proteins might be eluted by incubating with SDS pattern buffer for quarter-hour at room temperature.

Within the different incubation methodology described in S1B Fig, MBP-OtUBD was first incubated with yeast lysate for 4 hours at 4°C with rotation. The combination was then added to the amylose resin and incubated with rotation at 4°C for one more 2 hours, adopted by the identical washing and elution steps described above.

Era of covalently linked affinity purification resins

OtUBD resin.

Covalently linked OtUBD resin was made by conjugating Cys-OtUBD or Cys-His6-OtUBD to SulfoLink coupling resin (Thermo Scientific) based on the producer’s protocol. Briefly, 2 mL (mattress quantity) of SulfoLink resin was positioned in a gravity column and equilibrated with 4 mattress volumes of SulfoLink coupling buffer (50 mM Tris•HCl, 5 mM EDTA, pH 8.5). 4 mg of Cys-OtUBD was diluted in 4 mL coupling buffer supplemented with 20 mM TCEP and incubated at room temperature with rotation for half-hour. The diluted Cys-OtUBD was loaded onto the SulfoLink resin, and the combination was incubated at room temperature for 30–60 minutes with rotation. (Cys-His6-OtUBD required an extended incubation time (60 minutes) than Cys-OtUBD (half-hour).) The resin was allowed to accept one other half-hour earlier than being drained and washed as soon as with 6 mL coupling buffer. Furthermore, 4 mL freshly ready 50 mM L-cysteine dissolved in coupling buffer (pH adjusted to eight.5 with NaOH) was added to the resin and the combination was incubated at room temperature for half-hour with rotation. The resin was allowed to accept one other half-hour earlier than being drained and washed with 12 mL 1 M NaCl adopted by 4 mL OtUBD column buffer (50 mM Tris•HCl, 150 mM NaCl, 1 mM EDTA, 0.5% Triton-X, 10% glycerol, pH 7.5). For long-term storage (greater than 2 days), the resin was saved in column buffer containing 0.05% NaN3 and stored at 4°C. The resin might be saved at 4°C for no less than 1 12 months with out important lack of efficacy.

FK2 resin.

FK2 anti-ubiquitin antibodies had been covalently linked to a Protein-G resin following a earlier protocol with modifications [20]. Briefly, 500 μg of FK2 mouse monoclonal IgG1 antibody (Cayman Chemical) was diluted in 500 μL DPBS. The answer was added to 250 μL (mattress quantity) Protein G Sepharose 4 Quick Movement resin (GE Healthcare) prewashed with DPBS. The combination was incubated at 4°C for two hours with rotation. The resin was washed twice with 100 mM triethanolamine•HCl (pH 8.3), and the antibody was then crossed-linked to the resin by incubation with 500 μL 50 mM dimethyl pimelimidate (DMP) freshly dissolved in 100 mM triethanolamine•HCl buffer (pH 8.3) for 4 hours at 4°C with rotation. The response was terminated by incubating with 1.5 mL 100 mM Tris•HCl buffer (pH 7.5) for two hours at room temperature. Unconjugated antibody was faraway from the resin by washing with 500 μL of 100 mM glycine•HCl buffer (pH 2.5). The resin was equilibrated with DPBS and saved at 4°C earlier than used.

Ubiquitin-conjugate purifications with protein-linked resins

Native situations

Preparation of yeast lysate.

Most often, the frozen yeast pellet was lysed by grinding with mortar and pestle in liquid nitrogen. To 1 quantity of the resultant yeast powder, 1 quantity of chilly native lysis buffer (50 mM Tris•HCl, 300 mM NaCl, 1 mM EDTA, 0.5% Triton-X100, with freshly added 20 mM NEM, cOmplete mini EDTA-free protease inhibitor cocktail (Roche) and 1 mM PMSF, pH 7.5) was added to extract proteins. The combination was vortexed completely and incubated on ice for 10 minutes with intermittent vortexing. The crude extract was centrifuged at 21,000 x g for 12 minutes, and the supernatant was rigorously transferred to a clear tube.

Alternatively, yeast might be lysed by glass bead beating. Cell pellets had been resuspended in 1 mL chilly native lysis buffer containing 10% glycerol and 0.6 mL acid-washed glass beads (Sigma) and lysed in a FastPrep homogenizer (MP Bio) at 4°C (5.0 m/s, 3×(30 seconds, 1 minute relaxation on ice), 4 minutes relaxation on ice, 3×(30 seconds, 1 minute relaxation on ice)). The ensuing combination was left on ice for five extra minutes after which centrifuged at 8,000 x g for five minutes at 4°C. The supernatant was transferred to a brand new tube whereas 0.5 mL extra lysis buffer was added to the beads and pelleted cell particles. The pellet was resuspended and handled as above. The supernatants had been mixed and centrifuged at 21,000 x g for 12 minutes at 4°C. The cleared lysate was transferred to a clear tube.

For mammalian cells, the frozen cell pellets had been resuspended in chilly native lysis buffer and incubated on ice for 30 to40 minutes with occasional vortexing. After centrifugation at 21,000 x g for 20 minutes, clarified lysates had been transferred to a contemporary tube.

Protein focus within the lysates was measured by the BCA assay, and lysates had been adjusted to 2 to 4 mg/mL remaining protein focus utilizing native lysis buffer.


If the downstream software was solely western blotting, the certain proteins might be eluted by incubating the resin with 2 to three resin volumes of 1x SDS pattern buffer (50 mM Tris•HCl, pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.005% bromophenol blue) for quarter-hour at room temperature with rotation. In our arms, the TUBE resin might solely be effectively eluted utilizing this methodology.

If the purified proteins had been to be analyzed by LC-MS/MS, 2 resin volumes of pure water had been handed via the resin to push off residue buffers. Then, certain proteins had been eluted by incubation in 2 resin volumes of elution buffer (100 mM glycine•HCl, pH 2.5) for five minutes at 4°C with rotation. The eluate was collected and instantly neutralized with 0.2 resin quantity of 1M Tris•HCl pH 9 buffer. The elution course of was repeated to make sure full elution (the two eluates, E1 and E2, had been generally mixed to offer eluate E). In some experiments, the primary elution step was accomplished with 100 mM glycine•HCl, pH 3.0 and a 3rd elution step, additionally with the pH 2.5 buffer, was included to make sure full elution.

Ubiquitin conjugates within the enter, flow-through and eluate for every pattern had been analyzed by anti-ubiquitin western blotting. The amount loaded onto the SDS-PAGE gel for every pattern was normalized to mirror a 1:1:1 scaling of enter, flow-through and eluate (e.g., if the overall quantity of the eluate is 1/10 that of the enter, we load 1 quantity of the enter and 0.1 quantity of the eluate on the identical SDS-PAGE gel) except in any other case specified. Complete protein from the pulldowns was analyzed by SYPRO Ruby staining of the gels following producer’s protocol. SYPRO Ruby stained gels had been imaged on a Bio-Rad ChemiDoc imager and quantified utilizing ImageJ software program.

Denaturing situations

Preparation of lysates.

Yeast or human cells had been lysed as described above for the native situation protocol. After the measurement of protein focus, the lysates had been adjusted to as much as 12.58 mg/mL protein with native lysis buffer. The lysate was stored on ice for the entire length till applicable quantities of stable urea had been added on to the native lysate to achieve a remaining focus of 8 M (1 g of urea was added per 0.763 mL of lysate; calculations had been primarily based on [48]), and the lysate was vortexed and agitated till the urea had totally dissolved. The urea lysate was incubated at 25°C for half-hour, chilled on ice and diluted 1:1 with native lysis buffer (remaining focus of urea, 4 M).

Alternatively, yeast lysate was immediately extracted in a urea-containing buffer (Fig 4E, D2 situation). This methodology might assist solubilize precipitated ubiquitylated species. Floor-up yeast powders had been resuspended immediately in urea lysis buffer (50 mM Tris•HCl, 300 mM NaCl, 8 M urea, 1 mM EDTA, 0.5% Triton-X100, 20 mM NEM, cOmplete mini EDTA-free protease inhibitor cocktail (Roche), 1 mM PSMF, pH 7.5) or the yeast cells might be lysed immediately in urea lysis buffer by bead-beating.

The focus of the cleared lysate was decided by BCA assay and the focus was adjusted to match different samples. The cleared lysate was incubated at 25°C for quarter-hour, chilled on ice, and diluted 1:1 with native lysis buffer. This methodology might in principle embody insoluble ubiquitylated proteins and could also be helpful in particular purposes.

M48 DUB remedy of yeast cell lysates

Yeast powder ensuing from grinding the BY4741 pressure in liquid nitrogen was reconstituted in M48 lysis buffer (50 mM Tris•HCl, 300 mM NaCl, 1 mM EDTA, 0.5% TritonX, 10% glycerol, pH 7.5, supplemented with 7.6 μM pepstatin A, 5 mM aminocaproic acid (ACA), 5 mM benzamidine, 260 μM AEBSF, 1 mM PMSF, and 1 mM DTT), incubated for 10 minutes on ice and clarified by centrifugation at 21,000 x g at 4°C. Inhibitors of cysteine proteases had been averted to forestall inhibition of the M48 cysteine protease [84]. Protein concentrations within the lysates had been decided by BCA assay, and the lysates had been adjusted to 2 to 4 mg/mL protein with M48 lysis buffer. M48 DUB was added to the lysate to offer a remaining enzyme focus of 100 nM. The combination was incubated at 37°C with rotation for 1 hour earlier than subjecting to pulldown evaluation. Within the management samples the place M48 was not added, 10 mM NEM and 20 μM MG132 (a proteasome inhibitor) had been additionally included within the lysis buffer.

IMAC below denaturing situations

Eluates from the OtUBD pulldowns had been denatured by including a stable denaturant, both urea to a remaining focus of 8 M (used for the outcomes proven in Fig 3E and 3F) or guadinine•HCl to a remaining focus of 6 M. (Quantities of denaturants had been calculated primarily based on [48].) After the denaturant had totally dissolved, the answer was incubated at 25°C for half-hour earlier than making use of to a prewashed HisPur Cobalt resin (Thermo Scientific). The combination was incubated at room temperature with rotation for 1.5 hours, washed with 8 M urea wash buffer (50 mM Tris•HCl, pH 7.5, 8M urea), and eluted twice, every time by boiling for five minutes in 2 resin volumes of 500 mM imidazole in 2x SDS pattern buffer (100 mM Tris•HCl, pH 6.8, 4% SDS, 10% glycerol, 200 mM DTT, 0.01% bromophenol blue). Samples had been resolved by SDS-PAGE and analyzed by anti-ubiquitin immunoblotting and SYPRO Ruby staining. Particularly, samples containing guanidine was first diluted with 3 parts of pure H2O after which rigorously blended with 4x SDS pattern buffer earlier than loaded onto an SDS-PAGE gel to keep away from precipitation of SDS.


In resolution protein digestion.

Protein pellets had been dissolved and denatured in 8M urea, 0.4M ammonium bicarbonate, pH 8. The proteins had been decreased by the addition of 1/10 quantity of 45mM DTT (Pierce Thermo Scientific #20290) and incubation at 37°C for half-hour, then alkylated with the addition of 1/20 quantity of 200 mM methyl methanethiosulfonate (MMTS, Pierce Thermo Scientific #23011) with incubation at midnight at room temperature for half-hour. Utilizing MMTS avoids the potential false optimistic identification of GG modification arising from iodoacetamide (IAA) alkylation [86]. The urea focus was adjusted to 2M by the addition of water previous to enzymatic digestion at 37°C with trypsin (Promega Seq. Grade Mod. Trypsin, # V5113) for 16 hours. Protease:protein ratios had been estimated at 1:50. Samples had been acidified by the addition of 1/40 quantity of 20% trifluoroacetic acid, then desalted utilizing BioPureSPN PROTO 300 C18 columns (The Nest Group, # HMM S18V or # HUM S18V) following the producer’s instructions with peptides eluted with 0.1% TFA, 80% acetonitrile. Eluted peptides had been speedvaced dry and dissolved in MS loading buffer (2% acetonitrile, 0.2% trifluoroacetic acid). A nanodrop measurement (Thermo Scientific Nanodrop 2000 UV-Vis Spectrophotometer) decided protein concentrations (A260/A280). Every pattern was then additional diluted with MS loading buffer to 0.08μg/μl, with 0.4ug (5μl) injected for many LC-MS/MS evaluation, aside from the detrimental management samples, which had been diluted to and injected the identical quantity because the corresponding OtUBD pulldown samples.

Peptide identification.

Knowledge had been analyzed utilizing Proteome Discoverer software program v2.2 (Thermo Scientific). Knowledge looking out is carried out utilizing the Mascot algorithm (model 2.6.1) (Matrix Science) towards a customized database containing protein sequences for OtUBD in addition to the SwissProt database with taxonomy restricted to S. cerevisiae (7,907 sequences) or Homo sapiens (20,387 sequences). The search parameters included tryptic digestion with as much as 2 missed cleavages, 10 ppm precursor mass tolerance, and 0.02 Da fragment mass tolerance, and variable (dynamic) modifications of methionine oxidation; NEM, NEM+water, carbamidomethyl, or methylthio on cysteine; and GG adduct on lysine, protein amino terminus, serine, threonine or cysteine. Regular and decoy database searches had been run, with the boldness degree set to 95% (p < 0.05). Scaffold (model Scaffold_5.0, Proteome Software program, Portland, Oregon, USA) was used to validate MS/MS primarily based peptide and protein identifications. Peptide identifications had been accepted in the event that they might be established at larger than 95.0% chance by the Scaffold Native FDR algorithm. Protein identifications had been accepted in the event that they might be established at larger than 99.0% chance and contained no less than 2 recognized peptides. Protein and peptide FDR calculated by Scaffold for every experiment had been additionally reported in S2 Knowledge. The mass spectrometry proteomics information have been deposited to the ProteomeXchange Consortium through the PRIDE [87] companion repository with the dataset identifier PXD032294 (yeast outcomes) and PXD032675 (HeLa cell outcomes).

Quantitative evaluation was accomplished by Scaffold 5 (Proteome Software program) primarily based on normalized whole TIC (MS/MS whole ion present). Pearson correlation coefficients had been calculated utilizing GraphPad Prism 9 software program. Volcano plots had been generated utilizing GraphPad Prism 9 software program. Proteins are chosen as a possible E3 substrate in the event that they meet one of many following standards: (1) Its common quantitative worth (normalized whole TIC) is no less than 1.5 instances increased within the WT samples in comparison with the E3 deletion samples and p-value < 0.05. (2) It appeared in no less than 3 of the 6 technical replicates of the WT samples however not in any of the 6 technical replicates of the E3 deletion samples.

GO enrichment evaluation on particular protein populations was carried out utilizing the web Gene Ontology engine [8890] accessible at

Supporting info

S3 Fig. Further figures for OtUBD pulldown proteomics experiment in yeast.

(A) Consultant anti-ubiquitin western blot of OtUBD pulldowns (below native situations) used for proteomics evaluation. IN, enter; FT, flowthrough; E1/E2/E3, eluted fractions from a collection of low pH elutions. (B) Consultant anti-ubiquitin blot of OtUBD pulldown samples following extract denaturation (urea) and used for proteomics evaluation. (C) Consultant SYPRO Ruby protein stain of OtUBD eluates resolved by SDS-PAGE. (D) Variety of proteins detected in every organic replicate of OtUBD pulldown-MS and detrimental management. Error bar represents distinction amongst technical replicates. (E) Overlay of TIC chromatographs of consultant OtUBD pulldowns and detrimental management samples. The detrimental controls total have a lot much less peptide spectra in comparison with the OtUBD pulldown samples. This determine is generated with Thermo Xcalibur Qual Browser (v3.0.63) utilizing.uncooked recordsdata from the corresponding runs (QEp21-2054_Zhang_A1_Native_UBD_pos, QEp21-2050_zhang_a2_native_ubd_neg, QEp21-2036_zhang_a3_denatured_ubd_pos, QEp21-2032_zhang_a4_denatured_ubd_neg), which have been deposited to the ProteomeXchange Consortium and made out there to the general public (see the Strategies part for particulars). (F) Adjusted variety of proteins detected in every organic replicate of OtUBD pulldowns. Solely proteins whose TIC worth are no less than 20 instances increased within the OtUBD pulldown samples in comparison with the corresponding detrimental management samples are included. OtDUB, O. tsutsugamushi DUB.



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