Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress

Ubp2 is a linkage-specific DUB regulated by ROS

We previously identified that the deletion of UBP2 impairs the cellular capacity to reverse K63-ub chains that accumulate in response to oxidative stress in yeast (5). However, it remained unknown whether Ubp2 is directly responsible for the cleavage of K63-ub chains as part of the RTU and whether Ubp2’s activity could be regulated by ROS. Expression of WT Ubp2 in ubp2Δ rescues the deubiquitination phenotype, which reverses K63-ub globally and from ribosomes after stress induction (Figs. 1A and S1). By mutating Ubp2’s catalytic cysteine residue to serine (C745S), we confirmed that Ubp2’s activity is required for the reversal of K63-ub from ribosomes that accumulate under H₂O₂ (Fig. 1A). To further test Ubp2’s role in directly cleaving these K63-ub chains, we purified recombinant Ubp2 and showed that Ubp2 acts preferentially on K63-ub in comparison to K48-ub chains (Figs. 1B and S2). We also showed that Ubp2 is able to generate degradation intermediates of varied lengths when tested against synthetic tetra- and hexa-K63-ub chain substrates (Fig. 1C). Combined, our results suggest that Ubp2 is able to specifically trim K63-ub directly from its targets. Given that Ubp2 is a cysteine protease, we hypothesized that the accumulation of K63-ub under oxidative stress occurs due to oxidative inactivation of Ubp2. To test this possibility, we performed in vitro activity assays, and we observed that the catalytic activity of Ubp2 against the fluorogenic substrate Ub-Rho can be inhibited by H₂O₂ in a dose-dependent manner (Figs. 1D and S3, A and B). Cleavage of the substrate by DUBs releases the product rhodamine 110, which can be determined fluorimetrically. As expected, the pan DUB inhibitor PR-619 and the cysteine alkylator iodoacetamide (IAA) abrogated Ubp2 enzymatic activity (Figs. 1D and S3C). Importantly, oxidative inhibition of Ubp2 activity can be rescued by the reducing agent DTT, which indicates a reversible regulatory process (Figs. 1D and S3D). These results suggest that H₂O₂ promotes the oxidation of Ubp2’s catalytic cysteine residue, which can be reversed by thiol reducing agents. This redox inhibition also occurs when Ubp2 is tested against synthetic K63-ub chains (Fig. 1E). While we observed a redox regulation of recombinant Ubp2 in vitro, it was unclear whether this process also occurred in the cellular context. To test this, we immunoprecipitated hemagglutinin (HA)-tagged Ubp2 from yeast cells subjected to oxidative stress and we showed that Ubp2’s activity is partially inhibited by H₂O₂, which can also be rescued by DTT (Fig. 1F). HA-immunoprecipitation from cells expressing the catalytic dead Ubp2^C745S did not display activity against Ub-7-amido-4-methylcoumarin (AMC), demonstrating that this deubiquitinating activity was produced by Ubp2 and not by other contaminant proteases (Fig. 1F). Therefore, our results indicate that Ubp2’s activity is redox-regulated by H₂O₂, and Ubp2 is responsible for the reversal of the K63-ub chains that accumulate on ribosomes under stress.

Ubp2 has different sensitivity to peroxides

To further understand the redox regulation of Ubp2, we tested whether Ubp2 activity was responsive to other peroxides beyond H₂O₂. To different extents, we observed that organic peroxides are also able to induce the accumulation of K63-ub chains in yeast (Fig. 2A). As DUBs are a diverse group of enzymes, their sensitivity to ROS can vary substantially. To understand the impact of ROS on DUBs, we determined cellular DUB activity by incubating the whole cell extract or purified proteins with the DUB fluorescent substrate Ub-Rho. We first showed that cellular DUB activity was more affected by the organic peroxides cumene (CHP) and tert-butyl hydroperoxide (t-BHP) than H₂O₂ at our standard concentration of 0.6 mM, with CHP promoting the strongest effect (Fig. 2B, left). At 2.5 mM, organic and inorganic peroxides had a similar and acute effect on global DUB activity (Fig. 2B, right). These results disagreed with our previous findings that showed higher levels of K63-ub under H₂O₂ than organic peroxides (Fig. 2A). Therefore, we tested whether Ubp2 itself would be more sensitive to H₂O₂, while global DUB activity would be more affected by organic peroxides. Using purified Ubp2, we confirmed that H₂O₂ promoted a stronger reduction of Ubp2 activity (∼60%), while organic peroxide treatments showed only a small reduction at the same concentration (Fig. 2C, ∼5–20%). Upon increasing concentrations, organic peroxides promoted a stronger inhibition of Ubp2 activity (Fig. S4A). Thus, our findings show that Ubp2 can be redox-regulated by organic and inorganic peroxides, and its sensitivity correlates to the levels of K63-ub chains observed in yeast cells (Figs. 2, A and C and S4A). Finally, because several DUBs are cysteine proteases (28), this redox regulation could affect many Ub-mediated pathways in eukaryotic cells at once. Thus, we tested whether Cezanne, a human K11 linkage-specific DUB involved in cell cycle regulation (29), could also be redox-regulated by peroxides. Indeed, we observed an increased sensitivity of the catalytic domain of Cezanne (Cezanne^CAT) to H₂O₂, in comparison to t-BHP and CHP (Figs. 2D and S4B). Cezanne^CAT activity can also be rescued by DTT (Fig. S4C). Therefore, the redox mechanisms that we are exploring for Ubp2 could have further implications for Ub dynamics in a plethora of eukaryotic pathways that are responsive to ROS.

Ubp2 is reactivated during stress recovery

Although we observed that Ubp2 activity can be reversibly regulated by ROS (Fig. 1, D and F), it remained unclear whether this process is required to reverse the K63-ub chains that accumulate under stress. As the intracellular fate of oxidized Ubp2 is unknown, the reversal of K63-ub chains during stress recovery could be mediated either by Ubp2 reactivation or by de novo Ubp2 synthesis. Using the translation inhibitor cycloheximide (CHX), we first showed that the degradation of Ubp2 is not enhanced upon stress induction, and Ubp2 remains present and abundant during the time required for deubiquitination (Fig. 3A). Moreover, yeast cells were able to cleave K63-ub chains even in the presence of CHX, suggesting that de novo synthesis of Ubp2 is not required for K63-ub reversal (Fig. 3A). Accordingly, we showed that cells treated with CHX can regain their DUB activity during stress recovery, further indicating that redox reactivation of DUBs occurs in the cellular context (Fig. 3B). Thus, our data suggest that inactivation and reactivation of Ubp2 are critical to controlling the levels of K63-ub chains following H₂O₂ stress. Although cysteine oxidation is largely a chemical process (30), intracellular reduction of these residues commonly relies on the glutathione or thioredoxin system as the main thiol-based antioxidant pathways in eukaryotes (31, 32). Therefore, we tested whether alterations of the cellular redox balance could also affect the dynamics of K63-ub chains during stress. Double deletion of thioredoxins (TRX1/TRX2), glutaredoxins (GRX1/GRX2), and single deletion of thioredoxin peroxidases (TSA1 and TSA2) did not show sustained high levels of K63-ub chains during recovery (Figs. 3C and S5). However, deletion of glutathione reductase consistently impaired the cell’s ability to reverse K63-ub chains during stress recovery (Fig. 3C). Our findings show that Ubp2 is reactivated during the recovery phase of stress in a redox-dependent manner and suggest that cell’s reductive capacity, mediated by NADPH-dependent redox systems, participates in this process.

Next, we investigated the role of Ubp2 cysteine residues (Cys) on its redox regulation. Beyond their catalytic cysteine, thiol-dependent enzymes can use additional Cys to aid in their catalytic cycle and redox regulation (30, 31). Ubp2 has 13 cysteine residues, and analysis of its 3D model from AlphaFold2 (33, 34) predicted that the cysteine residues C821 and C944 are in proximity to the catalytic site, which could foster the formation of disulfide bonds upon conformational changes (Fig. 4A). By running nonreducing gels, we observed that Ubp2 forms H₂O₂-dependent disulfide bonds (Fig. S6, A–C) that are fully reduced by DTT (Fig. S6C). Mutational analysis revealed that the disulfide bond formation was abrogated in the Ubp2^C754S and Ubp2^C944S mutants following H₂O₂ treatment, indicating that these residues are involved in disulfide bond formation (Fig. 4B). To determine the effect of these cysteine residues on Ubp2’s activity, we evaluated the cellular growth of strains carrying selective Ubp2 mutations through their cellular sensitivity to the proteotoxic agent L-azetidine-2-carboxylic acid (ADCB). Functional Ubp2 provides resistance to ADCB by deubiquitinating and recycling selective ADCB transporters (13, 35). First, we showed that cells expressing the catalytic dead Ubp2^C745S mutant are highly sensitive, while cells expressing WT Ubp2 show tolerance to ADCB (Fig. 4C). Next, we tested whether mutation to other cysteine residues affects cell’s sensitivity to ADCB. While we observed a mild effect for the double mutation C821S/C944S, mutations to cysteine C621 and C645 show no change in ADCB sensitivity (Fig. 4C). Importantly, no growth defects were observed in the absence of ADCB (Fig. S7). Although the expression of the Ubp2 C821S/C944S mutant blocks the formation of Ubp2 disulfide bond and presents sensitivity to ADCB, this strain was still able to remove K63-ub chains in a timely fashion (Fig. 4D). As our experiments are not able to capture all Ubp2 states of cysteine oxidation, which might vary under different phases and magnitudes of stress, the functional role of Cys944 and its oxidation remain to be elucidated. Under the conditions tested, our data show that Ubp2 is reactivated upon stress cessation and is able to form disulfides bonds in the presence of ROS, which correlates to the levels of K63-ub chains that accumulate under stress.

Ubp2 activity is regulated by a series of repeated domains

In addition to the involvement of key cysteine residues, additional functional elements are necessary to drive Ubp2’s deubiquitinating function. Ubp2 is comprised of a nonconserved N-terminus extension, three repeated domains (RDs), and a conserved C-terminus UBP/USP catalytic domain (Figs. 4A and 5A). While it is known that Ubp2 plays a role in several pathways mediated by K63-ub (5, 36), the protein domains and residues that are critical for its activity have only been broadly characterized in the context of its interaction with the E3 Rsp5 and the cofactor Rup1 in vesicle trafficking (37). As the reversal of K63-ub chains that accumulates under stress works independently of Rup1 (Fig. S8A), we sought to characterize the role of Ubp2’s domains in the RTU. To determine the functional role of Ubp2 domains, we turned again to ADCB, which allowed us to test several experimental conditions in parallel. We observed that expression of the N terminus only (Ubp2^Nterm) or the catalytic domain only construct (Ubp2^CAT) led to cellular sensitivity toward ADCB (Fig. 5B). Ubp2^CAT still retained partial activity against the fluorogenic substrate Ub-AMC (Fig. S8B), suggesting that additional domains are important for Ubp2 cellular function. By producing additional truncated constructs, we showed that cells expressing Ubp2 lacking its N-term but expressing its three RDs and C terminus (Ubp2^RD1-3+CAT) are sensitive to ADCB (Fig. 5C). Surprisingly, upon deletion of RD1 (Ubp2^RD2-3+CAT), cellular resistance to ADCB was rescued similarly to levels observed in WT cells (Fig. 5C). We then removed Ubp2 RD2 (Ubp2^RD3+CAT), which also showed an increased sensitivity to ADCB (Fig. 5C), suggesting that RD2 and the catalytic domain are required to promote resistance against ADCB. To test whether RD2 alone would be sufficient to support Ubp2 function, we created new constructs and showed that cells expressing RD2 with the catalytic domain (Ubp2^RD2+CAT) remain sensitive to ADCB (Fig. 5D). Thus, our results indicate that RD2 is required but not sufficient to regulate Ubp2 activity. Addition of RD1 (Ubp2^RD1-2+CAT) made cells more sensitive to ADCB again (Fig. 5D). The presence of RD1 consistently reduces Ubp2 activity, suggesting that it might serve as an inhibitory domain that could reduce the exposure of key catalytic residues to the substrate.

Following our initial functional screen of Ubp2’s RDs, we asked whether these regulatory domains also affected the reversal of K63-ub chains that accumulate in response to stress. We have not identified any relationship of these domains with Ubp2’s capacity to bind or associate with ribosomes (Fig. S8, C and D), but they indeed affected the cell’s ability to reverse K63-ub chains. In agreement with the ADCB experiments, cells expressing Ubp2 containing all repeated domains (Ubp2^RD1-3+CAT) were inefficient in reversing K63-ub chains, while deletion of RD1 (Ubp2^RD2-3) also rescued the phenotype and contributed to reversal of these Ub chains (Fig. 5E). Supporting the notion that RD1 might play an inhibitory effect, we observed that Ubp2^RD2-3+CAT forms high levels of disulfides after H₂O₂ (Figs. 5E and S6A), which are also prevented by the presence of RD1. Here, we showed that Ubp2 activity is regulated by a series of repeated domains that controls cellular resistance to ADCB and the levels of K63-ub chains in the RTU.

Ubp2 supports the re-establishment of translation upon stress cessation

We have previously shown that K63-ub chains that accumulate in response to oxidative stress modifies ribosomes and pauses translation at the elongation stage (5, 7, 10). Following oxidative stress induction, Ubp2 is required for Ub reversal, however, it remains unclear how Ubp2 regulates translation through its deubiquitinating activity. To start to elucidate the role of Ubp2 in translation, we first showed that purified Ubp2 is able to deubiquitinate isolated ribosomes in vitro, and the deubiquitination of ribosomes by Ubp2 can also be impaired by H₂O₂ (Figs. 6A and S9, A and B). Using polysome profiling, we showed that Ubp2 remains associated to the ribosome fraction in the presence or absence of stress (Fig. 6B). Additionally, we observed that cells lacking Ubp2 retained high levels of K63-ub chains in the monosome and polysome fractions during stress recovery (Fig. 6C), further suggesting that Ubp2 is responsible for removing K63-ub chains from elongating ribosomes. Because of the role of K63-ub chains in ribosome pausing (7, 10), we hypothesized that cells lacking Ubp2 would show impaired resumption of protein synthesis during stress recovery. Therefore, we expressed an episomal inducible GFP-based reporter in the WT and ubp2Δ strains to evaluate whether the absence of UBP2 would lead to a decrease in GFP protein synthesis during recovery. As a stable protein (38), accumulation of GFP is largely driven by synthesis. While both strains have decreased GFP accumulation due to translation shut down after H₂O₂ treatment, GFP accumulation is rapidly restored after stress in the WT, but remains largely unaltered in ubp2Δ (Fig. 6D). Our findings suggest that Ubp2 plays a role in translation resumption following stress; however, it was unclear whether the results observed for this GFP reporter could be extrapolated to the proteome level.

To further investigate Ubp2’s role in restoring protein synthesis, we performed quantitative proteomics to understand how Ubp2 contributes to a global mechanism of translation regulation during different phases of the oxidative stress response. For this purpose, we cultivated WT and ubp2Δ cells and measured protein abundance in cells untreated or incubated with H₂O₂ for 30 and 120 min using label-free mass spectrometry. We chose 120 min after stress induction because at this time, cells generally start to reverse the K63-ub chains that accumulated under stress and resume translation that had been inhibited following H₂O₂ treatment (Figs. 5E and 6D). Our processed dataset is comprised of 4422 proteins representing ∼75% of the yeast proteome (39, 40). For a small number of proteins (179 proteins, 4.0% of the dataset) that presented three or fewer missing values across the 18 samples (i.e., 2 strains, 3 time points, 3 replicates), intensity values were imputed using the k-nearest neighbor method (41). To confirm that cells were responding to H₂O₂, we showed that both strains significantly enhanced the expression of key antioxidant enzymes such as thioredoxin Tsa2 and catalase Ctt1 after stress induction (Fig. S10A). When accounting for basal differences between strains, we observed that after 30 min of stress, 23 proteins were significantly downregulated (<1.5 fold) in ubp2Δ compared to the WT (Fig. S10B). Gene ontology (GO) analysis revealed that proteins involved in mitotic spindle and membrane proteins are significantly downregulated in the ubp2Δ background (Fig. S10D). When focusing on the recovery phase (30–120 min), and accounting for basal differences among strains, we observed that 8 and 27 proteins are significantly downregulated or upregulated, respectively, in the ubp2Δ strain (Fig. S10C). GO analysis confirmed that molecular functions related to exocytosis, vacuolar membrane, and mitosis remain up even 120 min after H₂O₂ addition to media (Fig. S10E).

Our results using GFP as a reporter protein indicated that Ubp2 is important for the re-establishment of protein synthesis during the recovery phase of stress (Fig. 6D). When comparing 120 min to our initial time point, we observed that 341 proteins are still differentially expressed in ubp2Δ in contrast to the 186 in the WT strain (Fig. S10, F and G). Of that, 90 proteins were shared among strains (Fig. S10G). Here, we show that only a fraction of the proteome is substantially different across strains and that deletion of UBP2 affects specific cellular pathways. However, other meaningful physiological changes might be present below our fold change cut-off. Our findings related to mitosis and mitotic spindle, motivated us to inspect the distribution of functional protein groups that are directly associated with cellular growth/division and stress response. We first observed that proteins from the GCN4 regulon, which are expressed as part of the integrated stress response (42), are significantly lower in ubp2Δ (Fig. 7B). This suggests that translation dysregulation might also affect cells capacity to mount a proper antioxidant defense. We also observed that ribosomal proteins are largely enriched among the upregulated proteins in WT cells (Fig. 7A). However, these proteins are mostly represented among the downregulated proteins and are significantly lower in ubp2Δ (Fig. 7, B and C). As the expression of these proteins is controlled by the target of rapamycin complex pathway (43), their levels are highly correlated with cellular growth and fitness (44), suggesting that different physiological states are present in these strains. Interestingly, we observed that only 16.7% and 9.1% of the proteins that are upregulated or downregulated, respectively, overlap when comparing WT and ubp2Δ strains, which suggests that these cells are mounting different cellular responses following stress induction (Fig. 7D). Taken together, our results support a model where Ubp2 is an integral part of the RTU, in which its redox regulation determines the dynamics of ribosome ubiquitination that is required to achieve proper stress recovery.

Yeast strains, plasmids, culture, and protein extraction

All S. cerevisiae strains and plasmids used in this study are described in Table S1. Proteomics processed data, analyses, and metadata are present in Table S2. Unless specified, yeast cells were cultivated into synthetic dextrose minimal medium (SD: 0.67% yeast nitrogen base, 2% dextrose, and required amino acids) at 30 °C at 200 rpm agitation. Cells were harvested at exponential phase A₆₀₀ 0.3 to 0.5. Protein extraction, ribosome isolation, polysome profile, GFP expression, and preparation for immunoblotting assays were performed as described previously (11).

Protein expression and purification

Escherichia coli BL21 and BL21-CodonPlus (DE3)-recombinant inbred line were transformed with pCS (Cold-Shock induced bacterial vector)/UBP2-HA-TEV-His and pGEX/Cezanne^CAT (catalytic domain)-TEV-glutathione-S-transferase, respectively. Bacterial cells were grown until A₆₀₀ reached 0.6 to 0.8. Ubp2 expression was induced overnight at 16 °C in the presence of 1 mM IPTG and Cezanne^CAT was expressed with 0.6 mM IPTG for 4 h at 37 °C. Ubp2’s cell lysis was carried out by incubation with 1 mg/ml lysozyme for 1 h at 4 °C in buffer containing 50 ml Tris–HCl pH 7.5, 500 mM NaCl, 1 mM of DTT, and protease inhibitors (1 mM PMSF and 10 μM leupeptin) during 4× rounds of 2 min of sonication on ice followed by 1 min of rest. E. coli cells expressing Cezanne^CAT were lysed by sonication in 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5% octylphenoxypolyethoxyethanol, 1 mM DTT, 1 mM PMSF, 1 mM leupeptin, and 1 mg/ml lysozyme. The extract was cleared by centrifugation at 12,000 rpm for 30 min prior to 2 h incubation with nickel affinity beads for Ubp2 purification (GOLDBIO catalog H-355) or glutathione-containing beads (GOLDBIO G-250-5) for 2 h at 4 °C for Cezanne^CAT. The elution of Ubp2 was carried out with 250 mM imidazole, followed by size-exclusion chromatography column (SEC 200 – Cytiva HiLoad 26/600 Superdex 200 pg, cat. #28989336) in buffer containing 50 mM Tris–HCl pH 7.5, 150 mM and 1 mM DTT. Cezanne^CAT was eluted by cleavage of the glutathione-S-transferase tag with 15 units/ml tobacco etch virus (TEV) protease (SigmaT4455-1KU) in PBS buffer overnight at 4 °C and desalted using a PD-10 column (GE Healthcare, cat. #17085101) in 50 mM Tris–HCl pH 7.5, 100 mM NaCl buffer. Fractions were combined and concentrated using an Amicon centrifugal filter with 100 kDa or 30 kDa cut-off (Sigma), for Ubp2 and Cezanne^CAT, respectively.

In vitro ribosome deubiquitinating assay

The in vitro deubiquitinating assay was performed in the presence of 5 μg Ubp2, 10 mM DTT, and 40 μg of isolated ribosomes. All components were pre-incubated in reaction buffer (50 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgCl₂) for 10 min at room temperature, before the addition of ribosomes. When specified, 7 mM H₂O₂ and/or 10 mM IAA was added to the reaction before the addition of ribosomes. The reaction was incubated for 1 h at 30 °C at 300 rpm, stopped by the addition of 4× Laemmli sample buffer, and subjected to SDS-PAGE gel prior to immunoblotting.

K63-ub chain cleavage assay

The cleavage of K63-ub chains by Ubp2 was assessed through an in vitro reaction containing 3 μg Ubp2, 10 mM DTT, 150 ng of tetra (LifeSensors) or 250 ng hexa K63-ub chains (R&D Systems), and reaction buffer (50 mM Tris pH 7.5 and 100 mM NaCl). The reaction was incubated for 1 h at 30 °C at 300 rpm, stopped by the addition of 4× Laemmli sample buffer, and subjected to SDS-PAGE followed by immunoblotting.

Immunoprecipitation assay

Yeast cells expressing Ubp2-HA and Ubp2^C745S-HA were grown until A₆₀₀ 0.3 to 0.4 and challenged with 0.6 mM H₂O₂. Lysis was carried out in 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 20 mM KCl as described before (11). Twenty five microliters of the normalized lysate was added to pierce protein A/G magnetic beads and incubated for 1 h under mild rotation. Unbound sample was removed and beads were washed twice with lysis buffer. Bound protein was eluted by incubation with 50 ng/μl of HA peptide for 30 min. Protein concentration was normalized via Bradford assay prior to the DUB activity assay.

DUB activity assay

The DUB activity was measured using the fluorogenic substrates Ub-AMC (R&D Systems, ex 345 nm, em 445 nm) or Ub-Rho 110 (LifeSensors, ex 485 nm, em 535 nm) in reaction buffer containing 50 mM Tris–HCl pH 7.5 and 150 mM NaCl at 30 °C. When specified, 5 min preincubation with peroxides was carried out before the start of the reaction. For measuring DUB activity in the total cell extract, cell lysis was performed in the absence of IAA. Enzymatic activity was determined and normalized by standard curves of free AMC and Rho 110.

Immunoblotting

Proteins were separated by standard 10 to 15% SDS-PAGE and transferred to polyvinylidene fluoride membrane (Thermo Fisher Scientific) for immunoblotting. The antibodies used in this work were as follows: anti-K63 Ub (1:8000, EMD Millipore, cat. #05-1308, clone apu3), anti-Ub (1:10000; Cell Signaling Technology, cat. #3936S), anti-uL5/Rpl11 (1:6000; Cell Signaling, cat. #18163), anti-uS3/Rsp3 (1:6000; Cell Signaling, cat. #9538), anti-PGK1 (1:8000; Invitrogen, cat. #22C5D8), anti-GAPDH (1:3000, Abcam, cat. #ab9485), anti-HA (1:3000, Thermo Fisher Scientific, cat. #71-5500), anti-actin (1:5000, Cell Signaling, cat. #4967), and anti-Rabbit IgG (1:400–10000; Cytiva, cat. #NA934). Immunoblots were developed by chemiluminescence using the Amersham ECL Prime (Cytiva, cat. #RPN2232).

ADCB sensitivity assays

Yeast cell cultures were grown into SD-Ura until log phase and diluted back to A₆₀₀ 0.1. Cells were mixed 1:1 with fresh medium or medium containing 200 μg/ml of ADCB (100 μg/ml final concentration). Cells were grown into 96-well plates in triplicate and incubated for up to 96 h at 30 °C under agitation. Absorbance was measured at 600 nm every 15 min in a Tecan Sunrise microplate reader.

3D structural analysis

3D graphical images of Ubp2 structure were generated in Chimera X (33, 34, 62) using the predicted models deposited in Alphafold2 protein database and UniProt (ID: Q01476).

Mass spectrometry analysis