Ubiquitin is a carbon dioxide–binding protein

Description


INTRODUCTION
Carbon dioxide is an absolute requirement for life. However, we know relatively little of the mechanisms that underpin direct interactions of CO 2 with the cell, despite the importance of the gas to biology. The only identified signaling molecules that respond directly to inorganic carbon [excluding a potential signaling role for carbonic anhydrases (1)] are the class III nucleotidyl cyclases of animals, fungi, and prokaryotes (2)(3)(4)(5); a subset of connexins (typified by Cx26) in mammals (6); and receptor protein tyrosine phosphatase  of mammals (7).
One hypothesis for how CO 2 regulates signaling is that it mediates a protein posttranslational modification (PTM); CO 2 might regulate the activity of multiple proteins and signaling pathways. There is direct evidence that CO 2 and protein can interact through carbamylation of neutral lysine -aminoand N-terminal -amino groups and affect the activities of RuBisCO (8) and hemoglobin (Hb) (9), respectively. Several proteins carry a stable carbamate required for catalysis, e.g., urease, alanine racemase, transcarboxylase 5S, class D -lactamase, and phosphotriesterase (10). Reversible carbamylation of neutral protein amino groups could yield responses to fluctuating pCO 2 (partial pressure of CO 2 ) that might, therefore, constitute a widespread mechanism for protein regulation (10,11). The hypothesis centers on the dissociation of cationic ammonium groups to neutral amines within structurally privileged environments within CO 2 -responsive proteins. Carbamate formation, mediated by nucleophilic attack of the neutral amines on CO 2 , leads to the formation of anionic groups, with the possibility for profound biological consequences as evidenced in Hb and RuBisCO. Our previous work developed the use of the triethyloxonium (TEO) ion as a tool to covalently trap the carbamate PTM on the protein (10). TEO is a water-soluble reagent that traps carbamates by selective alkylation. TEO has a t 1/2 of ~6 min at pH 7.4 under aqueous conditions. This t 1/2 permits its use as a trapping agent with mixing and pH control on a convenient laboratory time scale and we have used TEO as a tool to identify new CO 2 -binding proteins. Here, we have deployed TEO to identify mammalian CO 2 -binding proteins and identified ubiquitin (Ub) as a CO 2 -binding protein.
Ub is a highly conserved 8.5-kDa protein found in all eukaryotic cells, regulating protein activity and degradation through conjugation to target proteins. The identification of Ub as a CO 2 -binding protein can explain how CO 2 has these diverse effects in mammalian cells. The ubiquitination PTM involves Ub protein covalent conjugation to a lysine side chain on target proteins. Conjugation of Ub into poly-Ub chains can potentially occur at every Ub lysine side chain and the N-terminal  amino group producing eight functionally distinct chain formations. These varying linkages underpin different physiological processes (12). The well-characterized poly-Ub chains linked via lysine-48 result in protein targeting to the proteasome for degradation (12), while lysine-63-linked chains regulate proteasomeindependent reactions, including endocytosis (13). Poly-Ub conjugates at other lysine residues can affect the mammalian cell cycle (Lys 11 ) and 5′ AMP-activated protein kinase (AMPK)-related kinases to regulate enzymatic activity (Lys 29 and Lys 33 ) (14). CO 2 /HCO 3 at pH 7.4, and TEO was added. Trypsin was used to digest the trapping reaction mixture, and liquid chromatographytandem mass spectrometry (LC-MS-MS) was used to analyze samples, followed by data analysis using Peaks (Bioinformatics Solutions Inc.). The data were interrogated for modifications on the N terminus and lysine with masses of 72.0211 Da (trapped carbamate) and 28.0313 Da (O-ethylation on glutamate and aspartate side chains). Two lysine carbamylation sites were identified (MS-MS peptide amino acids 30 to 42 IQDKEGIPPDQQR, proposed carbamylation on K33; MS-MS peptide amino acids 43 to 54 LIFAGKQLEDGR, proposed carbamylation on K48) (Fig. 1, B and C). Within the datasets presented here, the carbamates were observed on both peptides on internal lysine residues that both exhibited a so-called missed cleavage. The missed cleavage is because carbamylation removes the cationic charge on the lysine essential for cleavage site recognition by trypsin. This supports the identification of carbamates on both Ub K33 and K48 as a missed cleavage is an otherwise rare event. The datasets also include peptides cleaved at K33 and K48, and these peptides do not carry a trapped carbamate. We performed the CO 2 -trapping experiments on Ub with 25 mM 13 CO 2 /H 13 CO 3 to corroborate the carbamate PTMs by interrogating the MS-MS data for a 73.0211-Da modification. The expected +1-Da mass/charge ratio (m/z) increase was observed for the carbamylation sites at both K33 (Fig. 1D) and K48 (Fig. 1E). MS-MS peptides encompassing the N-terminal -amino group, K6, K11, K27, K29, and K63, were observed, but no potential carbamylation sites were identified by this method.

Observation of carbamate formation on Ub by 13 C-NMR
We used 13 C-NMR (nuclear magnetic resonance) as an orthologous method to confirm Ub CO 2 -binding sites at K33 and K48, to investigate other carbamate formation sites not identified by MS-MS, and as a direct demonstration of the carbamate PTM on native protein. We initially mixed 1 mM 13 C/ 15 N-labeled Ub with 100 mM NaH 13 CO 3 and observed three peaks in 1D-13 C NMR spectra, which were not present in spectra for either Ub or NaH 13 CO 3 alone ( Fig. 2A). These new signals' chemical shifts-163. 25    consistent with the empirical range for carbamate PTMs (17). Unlabeled Ub (1 or 5 mM) was subsequently exchanged into buffers containing 20, 50, or 100 mM NaH 13 CO 3 , and we observed that the intensities of these carbamate signals increased with increasing [NaH 13 CO 3 ] or Ub concentration ( Fig. 2B and fig. S1), supporting the hypothesis that they are the product of reversible carbamate formation on Ub. The ratios of the peak intensities were unaltered at 20, 50, or 100 mM NaH 13 CO 3 . We therefore used 100 mM NaH 13  The only potential nucleophilic nitrogen atoms remaining in Ub K0 are the N-terminal amino group and, possibly, the imidazole of H68. N-acetyl-l-histidine cannot form adducts with CO 2 (18) but is identified in the binding pockets of proteins that can interact with CO 2 (19). An H68A mutation was introduced into Ub to examine whether H68 (the single histidine residue in Ub) was responsible for this remaining signal. This mutation altered the population of carbamylated K6, likely reflecting an altered pK aH of K6 due to the proximity of its -amino group to the imidazole of H68, but did not affect the resonance at 163.25 ppm ( fig. S3). We reacted Ub K0 with one equivalent of sodium cyanate, which ablated the carbamate resonance at 163.25 ppm, identifying it as originating from carbamylation of the N-terminal amine (residue M1; Fig. 2H). The pK aH value for the lysine -amino group is substantially above physiological pH. As carbamylation depends on the dissociation of the -amino moiety to a neutral group, those lysines with lower -amino pK aH values are potentially more likely to form carbamates. We therefore determined the pK aH for lysine -amino groups and the N-terminal amine in Ub. Lysine-specific N  or C  chemical shift data were obtained over the pH range from 8.6 to 12.3 and fit to a Henderson-Hasselbalch model to extract the pK aH for each -amino group (and the -amino group of M1) ( fig. S4). The data demonstrate an ordering of pK aH of K6 < K48 < K33 < K63 < K27 < K11 < K29 (Table 1), in agreement with a recent report (20). The observation of carbamylation on K6, K33, K48, and K63 using 13 C NMR, therefore, matches the ordering of pK aH for each -amino group. In conclusion, human Ub has been identified, through MS-MS and 13 C-NMR spectroscopy, to be capable of binding CO 2 through carbamate PTM formation.

Carbamate formation down-regulates Ub conjugation at K48 in vitro
We hypothesized that Ub carbamylation would affect poly-Ub formation. Ubiquitination occurs through the sequential activity of a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3) (21). E1 forms a thioester between a catalytic cysteine and glycine-76 at the Ub C terminus. E1 transfers Ub via G76 to a catalytic cysteine on E2 and forms an E2-Ub thioester complex. E3s bind this complex and substrate and enable formation of an isopeptide bond between the Ub C-terminal carboxyl group and the -amino group of a substrate lysine or an N-terminal amino group. Successive reaction rounds can produce poly-Ub chains linked via the seven Ub lysine residues or the N terminus of M1. We hypothesized that carbamate formation on Ub would alter the charge/binding capacity of the modified lysine -amino group and therefore downregulate poly-Ub chain formation by blocking the transfer of free Ub onto a target Ub molecule. Ub conjugation at K48 is well characterized in vitro and in vivo, particularly concerning proteasomal function (22). We identified K48 as a site for carbamylation by TEO-trapping ( Fig. 1, C and E) and 13 C-NMR spectroscopy (Fig. 2C). Therefore, we selected Ub conjugation at K48 to investigate the biochemical relevance of Ub carbamylation. A carbamate was identified on Ub K63 by 13 C-NMR spectroscopy (Fig. 2F), but not TEO trapping. Therefore, we selected Ub conjugation at K63 as an additional site for analysis. Conjugation assays at both Ub K48 and Ub K63 used the mE1 protein as a Ub-activating enzyme (23). Ub conjugation at specific lysine side chains can be investigated using E2 and E3 enzymes specific for conjugation at that site. Conjugation at Ub K48 used the E2-25K protein, which functions as both an E2 and an E3 enzyme, while conjugation at Ub K63 used the UEV1-Ubc13 heterodimer. These assays were performed over a concentration range that incorporated physiologically relevant CO 2 concentrations (a reference range of 1.8 to 2.3 mM dissolved CO 2 corresponding to a pCO 2 of 4.6 to 6.0 kPa) as well as pathophysiological hypocapnic CO 2 (<1.8 mM dissolved CO 2 ) and up to severe pathophysiological hypercapnic CO 2 (3.0 mM dissolved CO 2 ) (24). pH was monitored before, during, and at the end of each assay and was within ±0.1 pH units. Any observations are therefore independent of pH.
We observed an approximate 12% decrease in di-Ub formation at K48 over 0.0 to 3.0 mM CO 2 (Fig. 3, A and C), consistent with an inhibition of E2-25K activity due to the carbamate on Ub K48. An increase in CO 2 from 1.8 to 3.0 mM CO 2 (corresponding to in vivo hypercapnia) revealed decreased di-Ub formation at K48. Reductions in CO 2 below 1.8 mM (corresponding to in vitro hypocapnia) showed increased di-Ub formation at K48. We observed no change in di-Ub formation at K63 over 0.0 to 3.0 mM CO 2 (Fig. 3, B and C). Analysis of the data for di-Ub formation at K48 by one-way analysis of variance (ANOVA) demonstrated a significant decrease in di-Ub formation between 1.8 and 3.0 mM CO 2 . Di-Ub formed represented ~30% of the input Ub, suggesting that the observations are robust and not reflective of a minor reaction. Thus, experiments in vitro demonstrate that pathophysiologically relevant changes in CO 2 significantly alter Ub conjugation at K48. It is formally possible that the influence of altered CO 2 on Ub conjugation at K48 is due to CO 2 binding to an alternative assay component to Ub. The Ub-activating enzyme, mE1, is common for conjugation at both K48 and K63 and is therefore unlikely to be a CO 2 target due to no observed influence of CO 2 in the K63 conjugation assay. We used TEO-based trapping in an attempt to identify a potential CO 2 -binding site on E2-25K to investigate the possibility that the E2/E3 enzyme for K48 conjugation is a CO 2 target. TEO trapping and subsequent MS-MS failed to identify any E2-25K peptides with trapped carbamates (174 peptides with 94% coverage of the E2-25K protein).
Note that CO 2 did not influence K63 conjugation under the assay's conditions. The carbamate on K48 may be more stable than that on K63, but this awaits further investigation. Therefore, while carbamates on both K48 and K63 are detectable by 13 C-NMR, we propose that only the carbamate at K48 has a sufficient residence time to influence Ub conjugation over the time scale of the in vitro assay.

Carbamate formation down-regulates Ub conjugation at Ub K48 in cellulo
We hypothesized that exposure of cells to elevated CO 2 would affect Ub-dependent processes in the cell. Ubiquitination of proteins regulates nuclear factor B (NF-B) signaling (17). Two NF-B activation pathways have been described: the canonical (classical) and noncanonical (alternative) pathways. Various ligands, including tumor necrosis factor- (TNF-), associated with local inflammatory and immune responses, induce the activation of the canonical NF-B pathway. NF-B is maintained in the nonactivated state in the cytoplasm through binding to the inhibitor of NF-B (IB) proteins. Phosphorylation of IB proteins results in ubiquitination with K48linked poly-Ub and subsequent degradation of IB by the proteasome. NF-B is subsequently transported to the nucleus, where it activates a transcriptional response.
Elevated CO 2 suppresses NF-B-mediated transcription (25)(26)(27)(28). This suppression is proposed to have therapeutic potential (29) but remains controversial (30). Regardless of the controversy, the mechanism(s) by which CO 2 influences NF-B-mediated transcription is unknown. We investigated whether Ub was able to determine responses of NF-B-mediated transcription to CO 2 . Experiments used human embryonic kidney (HEK) 293 cells (NF-B/293/GFP-Luc) transduced with HIV-based pseudoviral particles packaged with a lentivector that coexpressed destabilized copGFP [but whose stability is not altered by pH (31)] driven by the minimal cytomegalovirus promoter (mCMV) in conjunction with four copies of the NF-B consensus transcriptional response element upstream of mCMV. We exposed NF-B/293/GFP-Luc cells to increasing concentrations of TNF- under culture media equilibrated to normocapnic [5% (v/v) CO 2 ] or hypercapnic [10% (v/v) CO 2 ] conditions (endpoint pH 7.5) (Fig. 4A). Physiological hypercapnia occurs above 45 mmHg pCO 2 . We selected cell culture conditions of 10% (v/v) CO 2 as representative of CO 2 levels encountered in disease (32). Extracellular pH was monitored before, during, and after assays and was constant across all conditions. Cells were permitted to undergo intracellular pH (pHi) homeostasis using our previously established methodology to ensure that changes in pHi did not influence the results (33). Resting pHi for HEK 293 cells (~7.4) is consistent with our in vitro assay conditions (34). NF-B-dependent green fluorescent protein (GFP) reporter activity was suppressed at 10% (v/v) compared to 5% (v/v) CO 2 as hypothesized.
We transfected NF-B/293/GFP-Luc cells with plasmids encoding wild-type (WT) Ub, a mutant K48R Ub, a mutant K63R Ub, or an empty vector. We hypothesized that overexpression of K48R Ub would alter the relative response of the NF-B pathway to elevated CO 2 . In contrast, for the cases of WT Ub and Ub K63R, we expected the NF-B response to be insensitive to CO 2 in vitro (Fig. 3, B and C).
We observed a ratio of fluorescence reporter activity at 5% (v/v) compared to 10% (v/v) >1 in vector-transfected cells, consistent with a reduction in NF-B-dependent transcription (Fig. 4B). A similar observation was made in cells transfected with WT or a K63R Ub. However, we observed a ratio of fluorescence reporter activity at 5% (v/v) compared to 10% (v/v) not significantly different from 1.0 in K48Rtransfected cells, consistent with no change in NF-B-dependent transcription. This finding suggests Ub K48 to be the target for CO 2 in the NF-B-dependent transcriptional response to hypercapnia.
We speculate that K48R Ub might be introduced into endogenous Ub chains at a rate sufficient to permit eventual fluorescence reporter activation and ablate the impact of CO 2 on poly-Ub formation. Analysis of the ratio of production of the transfected Ub protein at 5% (v/v) versus 10% (v/v) CO 2 demonstrated no significant difference between WT Ub, K48R Ub, and K63R Ub; thus, differences in protein production do not explain these results (Fig. 4C). A faint band was visible above the predominant signal in the WT Ub-transfected sample. The identity of the protein in this band is not known, but its density does not alter the experimental findings. Data were reported as ratios of fluorescence reporter activity at 5% (v/v) compared to 10% (v/v) CO 2 , as the variation in raw values for fluorescence reporter activity was greater than the change in ratio. All values were normalized to total loaded protein. This variation was likely due to variation in Ub plasmid transfection efficiency and Ub protein production. We cannot, therefore, rule out whether the influence of CO 2 on reporter activity occurs specifically at 5% (v/v) versus 10% (v/v) CO 2 (or both). We investigated whether Ub conjugation on IB was sensitive to elevated CO 2 . IB is conjugated with Ub under basal cell conditions that form a high-molecular weight complex, and activation of the NF-B pathway can enhance this conjugation (35). We treated HEK 293 cells with or without TNF- at 5% (v/v) versus 10% (v/v) CO 2 . TNF- treatment was optimized such that bulk IB was not degraded and thus able to be analyzed for Ub conjugation. Western blot analysis demonstrated approximately equivalent amounts of endogenous Ub and IB under the varying conditions (Fig. 5A, -IB and -Ub Input, and fig. S5). A faint band was visible below the predominant signal in the 10% (v/v) CO 2 input sample analyzed with an -Ub antibody. The identity of the protein in this band is not known, but its density does not alter the experimental findings. We analyzed IB by Western blot after immunoprecipitation with -Ub antibody to identify an IB-Ub conjugate. A high-molecular weight IB-Ub conjugate was observed in the absence of TNF- at 5% (v/v) CO 2 as previously observed (35) (Fig. 5A, arrow, top, and fig. S5). The IB-Ub conjugate was not observed in the presence of TNF- at 5% (v/v) CO 2 , consistent with its degradation in the proteasome. Significantly, no highmolecular weight IB-Ub conjugate was observed in the absence of TNF- at 10% (v/v) CO 2 . This observation is consistent with a decrease in Ub conjugation to IB at elevated CO 2 and the in vitro data of Fig. 3A. The experimental observations were independent of small variations in input Ub and IB evident across the biological replicates. We permitted the higher Ub input signal level at 10% (v/v) CO 2 to be certain that the loss of the high-molecular weight IB-Ub conjugate was not an artefact of a lower input Ub. Note that under the conditions of this experiment, the IB-Ub conjugate was observed to degrade in the presence of TNF- at 10% (v/v) CO 2 . Thus, the observation of a change in the formation of an IB-Ub complex was, by necessity, made in the absence of TNF-. Future developments will be required to observe changes in IB-Ub conjugation in the presence of TNF-.
We assessed the impact of 10% (v/v) CO 2 on IB degradation in response to TNF-. A comparison was made to ambient CO 2 to increase the likelihood of observing a difference (25). HEK 293 cells transfected with Ub WT or Ub K48R were compared for their sensitivity to elevated CO 2 after stimulation with TNF- (0.26 ng ml −1 ) and harvesting cells at 30 min (Fig. 5B). We observed that the ratio of the impact of elevated CO 2 for Ub K48R:Ub WT was consistently <1, indicating that the CO 2 effect was more significant for Ub WT than Ub K48R.  We further assessed the impact of 10% (v/v) CO 2 on p65 nuclear localization in response to TNF-. HEK 293 cells transfected with Ub WT or Ub K48R were compared for their sensitivity to elevated CO 2 after stimulation with TNF- (10 ng ml −1 ) and harvesting cells at 30 min (Fig. 5B). We observed that the ratio of the impact of elevated CO 2 for Ub K48R:Ub WT was also consistently <1, indicating that Ub K48R altered the sensitivity of the experiment to CO 2 . In this case, the result indicates that Ub K48R overexpression increased the sensitivity of p65 nuclear localization to CO 2 . Therefore, it is interesting to note that CO 2 , through Ub K48, might have varying effects at different parts of the NF-B pathway depending on its local roles. For example, Ub K48 is linked to protein nuclear export (36), and these processes might influence p65 nuclear localization and downstream effects at chromatin. However, whatever the impact of CO 2 on varying parts of the TNF- response, all with their characteristic response and dynamics, the result is the down-regulation of NF-B expression.
Considering the observations that Ub binds CO 2 , has its biochemistry altered by CO 2 in vitro and in vivo and overexpression of a mutant Ub that cannot bind CO 2 in cellulo, ablates a response to hypercapnia, and alters the CO 2 response as varying parts of the response pathway, we conclude that Ub is a CO 2 -binding protein.

DISCUSSION
The sensing of bioactive gases is of fundamental importance to mammalian physiology. Soluble guanylate cyclase is the nitric oxide receptor (37), while oxygen sensing is achieved by PHD1-3 catalyzed prolyl hydroxylation of hypoxia-inducible factors (HIF-1, HIF-2, and HIF-3), which facilitates HIF- regulation in the PHD1-3-HIF--pVHL signaling axis (38,39). Analysis of transcriptional responses to elevated CO 2 in Drosophila identified upregulated gene ontology (GO) families relating to metabolic functions. In contrast, most of the down-regulated GO families had either immune-or fertility-related annotations (40). Similar experiments in Caenorhabditis elegans identified 488 up-or down-regulated genes after 1-hour exposure to elevated CO 2 (41). In addition to transcription responses, physiological responses to hypercapnia in Drosophila included altered embryo morphogenesis, egg laying, egg hatching, and innate immune responses. C. elegans responses included altered body muscle organization, slowed development, reduced fertility, and increased life span. No candidate CO 2 -binding protein that might explain its various physiological effects has been identified to date. Ub fulfils the criteria of a CO 2 -binding protein and might explain the diverse physiological impact of hypercapnia.
Elevated CO 2 is reasonably well tolerated in mammals, while Ub conjugation is an essential cellular function. Therefore, it is significant that carbamate formation on Ub across the range of 0 to 3 mM CO 2 reduces Ub conjugation at K48 by just over 10%. Such a reduction in Ub conjugation may be sufficient to have a physiological impact, but not too significant an effect of being lethal. The specific carbamate formation sites on Ub also guide future avenues of investigation for molecular responses to CO 2 . Carbamate formation at Ub K48 suggests an influence on signaling pathways regulated by proteasomal degradation, as evidenced here for NF-B-dependent transcription. We cannot exclude the possibility that carbamylation at K48 (or another site) acts to expand the Ub code in the presence of conjugates at other sites (42). Carbamate formation at Ub K6 and K33 suggests a mechanism by which hypercapnia might influence mitophagy/xenophagy and post-Golgi membrane protein trafficking, respectively (16,43). Also, of note, Ub N-terminal carbamylation might be physiologically important because the head-to-tail linked (also known as "linear") Ub chains also regulate immune signaling (44). The impacts of carbamylation upon Ub polymerization in vitro and in vivo at these sites await future investigation. No motif identifying the propensity of a site to carbamylation is evident, most likely because carbamylation depends on a structurally privileged local environment that lowers local pK aH , rather than a defined primary motif. Carbamylation in Ub-like molecules (e.g., prokaryotic Ub-like protein) will therefore require future experiments and is not currently amenable to prediction.
The identification of Ub as a CO 2 -binding protein might also decipher conflicting data surrounding the identity(ies) of the site(s) of action of CO 2 along the NF-B pathway. Several studies suggest that CO 2 affects the canonical NF-B pathway components, including IB- (45) and the noncanonical pathway (25,46,47). In the canonical pathway, in addition to IB, the E3 ligase cIAP1 and RIPK1 serine-threonine kinase at the TNF receptor complex require Ub K48-linked poly-Ub for pathway activation (48). In the noncanonical pathway, ubiquitination with K48-linked poly-Ub of TRAF2 by cIAP1 and of p100 for its processing to p52 is required for activation (49). Ub is the common entity that could underpin explanations as to why both pathways are sensitive to CO 2 . The future selection of other ligands will allow this to be selectively probed. HEK 293 cells transfected with Ub K48R compared to Ub WT on the ratio of the response for IB degradation and p65 nuclear localization (mean ± 95% CI; *P < 0.05, one-sample t test, theoretical mean = 1.000, t > 3.447, df = 2 to 3).

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In summary, Ub is a CO 2 -binding protein through carbamate formation. Physiological levels of CO 2 regulate Ub conjugation at K48 in vitro, and overexpression of a Ub mutant that is unable to conjugate at the K48 CO 2 -dependent site ablates a CO 2 -dependent phenotype in cellulo. On the basis of our findings, we postulate that the regulation of ubiquitination by CO 2 explains one of hypercapnia's broad physiological effects.

CO 2 trapping
All CO 2 -trapping experiments were carried out with recombinant protein (500 g) in phosphate buffer (3 ml, 50 mM, pH 7.4). This solution was transferred to a potentiometric titrator (902 Titrando; Metrohm) and incubated at 25°C with stirring. A freshly made solution of TEO tetrafluoroborate (Et 3 OBF 4 ; 280 mg, 1.47 mmol) in phosphate buffer (1 ml) was added stepwise with a constant pH being maintained (pH 7.4) through the slow addition of 1 M NaOH solution via the automatic burette. The reaction mixture was stirred, with the pH being maintained, for 1 hour after the final Et 3 OBF4 addition to ensure that all TEO was hydrolyzed. The reaction mixture was then dialyzed against dH 2 O (1 liter) overnight.

Mass spectrometry
The water was removed from a postdialysis-trapped sample supernatant using a centrifugal vacuum concentrator. Protein was resuspended in urea solution (8 M, 500 l), and disulfide bonds in the sample were reduced using dithiothreitol (DTT; 25 mM final concentration) at 37°C for 1 hour. The resulting free thiol groups were alkylated using iodoacetamide (40 mM) in the dark for 1 hour at room temperature. The sample was diluted to 1 M urea with ammonium bicarbonate buffer and digested with trypsin gold [mass spectrometry grade, Promega; 1:25 (w/w) ratio to protein] overnight at 37°C. The solution of digested proteins was desalted and resolved using a C18 column (ZipTip, Merck Millipore), dried down, and resuspended in 4% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid. The eluted peptides were analyzed by LC-MS-MS on a QStar Pulsar QTOF mass spectrometer (Sciex) coupled to nano-LC instrument. Peptides were eluted from an LC gradient from 3 to 80% (v/v) acetonitrile and injected online to the mass spectrometer [information dependent acquisition (IDA) mode, mass range of 300 to 1600 Da, MS accumulation time of 1 s, ion source voltage of 2300 V, three MS-MS spectra per cycle, MS-MS mass range of 100 to 1600 Da, and MS-MS accumulation time of 3 s]. The post-ESI-MS-MS raw data files were converted into .mgf files using the freeware MSConvert provided by ProteoWizard (50) and analyzed using PEAKS Studio 10.5 software (51) including the variable modifications ethylation (28.0313 Da at D or E), carboxyethylation [72.0211 for 12 CO 2 or 73.0211 for 13 CO 2 at K or protein N-terminal groups], oxidation (M), acetylation (N terminus), and the fixed modification carbimidomethyl (C). These data were then refined using a false discovery rate of 1% and a PTM AScore of 50.

NMR spectroscopy
NMR experiments were performed on a Bruker Avance-III 800 MHz spectrometer equipped with a QCI cryoprobe. All carbamylation experiments were performed at 37°C in a buffer containing 20 mM sodium phosphate, 0.02% (w/v) NaN 3 , and 10% (v/v) D 2 O at pH 7.4. The buffer was prepared with 100 mM NaH 13 CO 3 unless otherwise indicated. 13 C spectra were acquired with a 45° pulse, spectral width of 12820.5 Hz, an acquisition time of 847 ms for 21,738 points, and a recycle delay of 2.5 s. Spectra with 13 C-and 15 N-labeled Ub were acquired with 15 N decoupling during an 80-ms acquisition period. All processing was performed with Mestrelab MNova within an NMRBox (52) environment. Ub Lys pK aH values were determined by following either N  or C  /C  chemical shifts measured via triple-resonance H  2C  N  experiments (H2CN) (53) or 1 H-13 C HSQC experiment, respectively, within the pH range from 8.6 to 12.3 at 23°C. Initially, 13 C/ 15 N Ub was exchanged into 5 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES)/5 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer at pH 10.5 and separated into two 500-l 1 mM Ub aliquots that were run as two divergent samples for lower or higher pH measurements. After each experiment, these samples were consecutively exchanged into the same buffer with either increasing or decreasing pH by the addition of 0.1 M HCl and 0.1 M KOH in approximately 0.3 (H2CN) or 0.4 (HSQC) pH unit steps. D 2 O was added to each sample to a final concentration of 5% (v/v). The pK aH values were obtained by fitting the observed signal shifts to a Henderson-Hasselbalch model. The errors in reported pK aH values (Table 1) for each residue were assessed by fitting 2000 Monte Carlo-generated synthetic datasets, in which Gaussian noise was added to the chemical shifts according to the resolution in the respective H2CN or 1 H-13 C HSQC spectra.

Recombinant protein production
WT human Ub derived from pET15 (54) was cloned into the Nde I and Bam HI restriction sites of pET28a with a stop codon introduced to preclude expression of the His 6 affinity tag. Mutant human Ub-expressing constructs were derived from the WT plasmid by site-directed mutagenesis. Recombinant WT and mutant Ub were expressed as untagged proteins from pET28a in E. coli BL21(DE3) at 20°C for 16 hours with 0.4 mM isopropyl--d-thiogalactoside (IPTG). Pelleted bacteria (10 ml) were suspended in sonication buffer (phosphate-buffered saline, 50 ml) including SIGMAFAST Protease Inhibitor Cocktail Tablets, lysed by sonication (180 s on ice), and centrifuged (50,000g, 40 min, 4°C). The supernatant was incubated on ice with the addition of 70% (w/v) perchloric acid with vigorous stirring until solution pH dropped to 4.5. The solution was left to stir for 1 hour and then centrifuged to remove precipitate (5000g, 40 min, 4°C). The remaining supernatant was dialyzed against 50 mM ammonium acetate buffer overnight (1 liter, 4°C). Sample was centrifuged (50,000g, 40 min, 4°C), and supernatant was dialyzed against purification buffer for 8 hours (10 mM tris, pH 7.6). Protein from this sample was then purified using size exclusion chromatography (Superdex 75). mE1 was expressed from pET28a in E. coli BL21(DE3) at 16°C for 20 hours with 0.5 mM IPTG (55). Pelleted bacteria (10 ml) were suspended in sonication buffer [50 ml; 50 mM tris-HCl (pH 8), 150 mM NaCl, 0.1% (v/v) Triton X-100, 1 mM EDTA, 1 mM DTT, phenylmethylsulfonyl fluoride (PMSF; 0.1 mg/ml)], lysed by sonication (180 s on ice), and centrifuged (50,000g, 40 min, 4°C). Protein was affinitypurified from the supernatant using a 5-ml HisPrep HP Ni-NTA column (GE Healthcare) on an AKTA Pure chromatography system at 2 ml min −1 (GE Healthcare). Eluted protein was concentrated and buffer-exchanged with 10 mM tris-HCl (pH 8), 1 mM EDTA, and 1 mM DTT before additional purification by ion exchange chromatography and size exclusion chromatography at 0.5 ml min −1 .

Western blotting
Polyacrylamide gels [1.0 mm; 10% (v/v) bis-acrylamide resolving and 5% (v/v) bis-acrylamide stacking] were poured using the Mini-PROTEAN Tetra Electrophoresis System. Samples were mixed 1:1 (v:v) with loading buffer [50 mM tris-HCl (pH 6.8), 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol, and 100 mM DTT], incubated at 95°C for 5 min, and run at 20 V cm −1 in running buffer [25 mM tris-HCl (pH 6.8), 200 mM glycine, 0.1% (w/v) SDS]. Proteins were transferred at 2 V cm −1 at 4°C overnight in transfer buffer [25 mM tris-HCl (pH 8.5), 190 mM glycine, and 15% (v/v) methanol]. Membranes were washed for 5 min in TBS-T [25 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (v/v) Tween 20] and incubated in blocking buffer [5% (w/v) nonfat milk in TBS-T] for 2 hours at room temperature. Membranes were washed three times in TBS-T for 10 min each and then probed with primary antibody diluted in blocking buffer. Membranes were washed again with TBS-T and then probed with secondary antibody diluted in blocking buffer. Membranes were again washed with TBS-T before developing with ECL Western Blotting Detection Reagent at room temperature. Blots were imaged and quantified using ImageJ (58). The signal for the -HA (human influenza hemagglutinin) antibody used for the quantitative Western blot of Fig. 4C was confirmed to lie on a straight line of a plot of Western blot signal versus loaded protein lysate for which the slope was significantly nonzero (fig. S6). The conditions of the Western blot were therefore suitable for quantitation.
GFP reporter assay NF-B/293/GFP-Luc cells (System Biosciences) were cultured until 80% confluency in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated newborn calf serum, 100 M nonessential amino acids, 50 U of penicillin, and 50 g of streptomycin. Cells were passaged into 24-well plates and allowed to adhere for 6 hours before transfection. Transfections were performed using Lipofectamine 3000 (Invitrogen) in Opti-MEM media (Gibco) with 1 g of DNA per well. Human Ub WT and K48R were expressed from pRK5 (60). A plasmid expressing human Ub K63R was generated from the WT template by site-directed mutagenesis. Cells were incubated at 37°C and 5% (v/v) CO 2 for 20 hours, and then the medium was exchanged for DMEM without phenol red containing 25 mM Hepes buffer (pH 7.4) supplemented with 10% (v/v) heatinactivated newborn calf serum, 100 M nonessential amino acids, and 25 mM or 35 mM sodium bicarbonate. Cells were incubated at 37°C at 5 or 10% (v/v) CO 2 , respectively, with TNF- (30 ng ml −1 ) for 20 hours before lysis with M-PER extraction reagent and fluorescence counting in a microplate reader.

Immunoprecipitation
Antibody immunoprecipitation columns were produced using the Pierce Coimmunoprecipitation Kit (Thermo Fisher Scientific). Briefly, aminolink coupling resin was washed with coupling buffer and incubated with 70 g of -Ub antibody (Abcam ab134953) and sodium cyanoborohydride for 2 hours at room temperature with mixing. The column was washed and incubated with quenching buffer and sodium cyanoborohydride for 30 min with mixing. The column was washed and stored at 4°C before immediate use. Cells were incubated at 37°C with 5 or 10% (v/v) CO 2 , respectively, with TNF- (30 ng ml −1 ) for 20 hours before lysis with M-PER extraction reagent (Thermo Fisher Scientific, 78501). Input Western blots were carried out as described using 5 g of total cell lysate and probed with both -Ub and -IB antibodies. Input material for immunoprecipitation was normalized to total protein assessed by Bradford assay. Cell lysate from the same experiment was incubated with the -Ub resin overnight at 4°C with rolling. Columns were centrifuged at 500g for 30 s to remove buffer, samples were eluted with 50 l of elution buffer (pH 2.8), and the pH was neutralized with the addition of 1 M tris (pH 9.5). Eluted samples were then probed using an -IB antibody and the described Western blot protocol. p65 assay NF-B nuclear p65 was assayed by enzyme-linked immunosorbent assay (ELISA) (abcam133112) according to the manufacturer's instructions.

SUPPLMENTARY MATERIALS
Supplementary material for this article is available at https://science.org/doi/10.1126/ sciadv.abi5507 View/request a protocol for this paper from Bio-protocol.