Proteasome-dependent degradation of intracellular carbamylated proteins

Evidence for the presence of carbamylated proteins inside human skin cells

Our previous studies have shown that skin HCit content increases with age in different species and that this accumulation is partly due to carbamylation of matrix proteins such as collagen and elastin [15]. In order to determine whether CDPs may also be formed intracellularly, immunohistolabelling analysis of HCit in human skin sections was performed (Fig. 1). This labelling showed the presence of HCit inside cells, providing evidence of intracellular carbamylated proteins.

Figure 1. Evidence for the presence of carbamylated proteins inside human skin cells. An anti-HCit immunolabelling was realized in a skin section from a 77-year old man counter-stained with DAPI and hematoxylin-eosin. Epidermal area was screened by light microscopy thanks to hematoxylin-eosin counterstaining (a). Nuclei were highlighted by DAPI staining (b, blue) permitting confirmation of the presence of HCit inside skin cells by immunofluorescence analysis (c, green). An example of a cell exhibiting a positive intracellular labelling for HCit is indicated by arrows. Observations were realized at 10x magnification.

Incubations with urea or cyanate lead to an increase of intracellular protein carbamylation level

In order to evaluate the level of protein carbamylation inside cells, fibroblasts were incubated for 4 weeks with 20 mmol/L urea or 0.5 mmol/L sodium cyanate. Firstly, the results showed a basal carbamylation level of intracellular proteins, since HCit was detectable even without incubation with a carbamylating agent (Fig. 2a). Basal concentrations (0.07±0.01 µmol/mol Lys) were 2-fold and 18-fold increased when fibroblasts were incubated with urea (0.13±0.01 µmol/mol Lys, p<0.01) and with cyanate (1.30±0.02 µmol/mol Lys, p<0.01), respectively.

Figure 2. Intracellular accumulation of carbamylated proteins after long-term incubations with urea or cyanate. Confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions, open bars) or with 20 mmol/L urea (grey bars) or 0.5 mmol/L cyanate (black bars). HCit content was determined by LC-MS/MS in total cell extracts (a) and in cytoplasmic and membrane fractions (b). The data are presented as means ± SEM (n=6) compared using the Mann-Whitney U test (ns: non significant, **: p<0.01).

As results obtained from total cell extracts could be explained only by the carbamylation of membrane proteins exposed to the extracellular medium, another experiment was performed in which membrane and cytosolic fractions were separated. Similar carbamylation levels were found in both membrane and cytoplasmic protein fractions (Fig. 2b) and the same trends as in total extracts were observed in both fractions when the different conditions were compared. In membrane proteins, no significant difference was found between control and urea-incubated cells (0.17±0.02 µmol/mol Lys vs 0.19±0.01 µmol/mol Lys) whereas the carbamylation level was significantly increased when cyanate was used as carbamylating agent (1.38±0.03 µmol/mol Lys, p<0.01). In cytoplasmic proteins, HCit content was 0.10±0.01 µmol/mol Lys in basal conditions and reached 0.16±0.02 µmol/mol Lys after incubation with urea and 1.58±0.01 µmol/mol Lys when incubated with cyanate. These results showed that urea as well as cyanate are able to enter cells and induce a significant level of protein carbamylation.

Actin cytoskeleton and nuclear proteins constitute preferential targets for carbamylation

In order to determine which intracellular proteins are most prone to carbamylation, fibroblasts were incubated for a shorter period (5 days) with 5 mmol/L cyanate and carbamylation measured using an anti-HCit immunofluorescence labelling assay (Fig. 3). An intense labelling of the nucleus was observed whereas a more diffused signal was detected in the cytoplasm. These results indicated that cyanate is able to cross the nuclear membrane and thereafter carbamylate nuclear proteins (Fig. 3a). For cytoplasmic proteins, as cytoskeleton proteins exhibit among the longest half-lives among the intracellular proteins [17], a phalloidin-based staining of actin was performed in order to search for colocalization points with HCit labelling (Fig. 3b). Many colocalization points were found (Fig. 3c) demonstrating that actin is one of the preferential targets for carbamylation among the intracellular proteins. To complete these results, β-actin was immunoprecipitated from total cell extracts and HCit was quantified in precipitates in order to determine its carbamylation level (Fig. 3d). Actin was found to be 8.5-fold more carbamylated when fibroblasts were incubated with cyanate in comparison with control conditions (0.23±0.06 vs 1.95±0.53 µmol/mol Lys, p<0.05). However, other HCit labelling areas were not colocalized with actin, especially in the perinuclear zone, suggesting that other intracellular proteins are also carbamylated.

Figure 3. Localization of intracellular carbamylated proteins. Fibroblasts were seeded in chambered coverglass system and incubated for 5 days with DMEM containing 0.5% (v/v) FBS and 5 mmol/L cyanate. At the end of incubation, cells were fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.25% (v/v) Triton X-100 before immunolabelling of carbamylated proteins using an anti-HCit polyclonal antibody (a). Cells were also labelled using ActinRed 555 ReadyProbes® in order to identify actin fibers (b). Colocalization points between HCit and actin labelling were identified using ImageJ software (c). In a second set of experiments, fibroblasts were incubated in the same conditions without (control) or with 5 mmol/L cyanate before preparing total cell extracts which were then used for β-actin immunoprecipitation. The immunoprecipitates were submitted to acid hydrolysis before HCit quantification by LC-MS/MS (d). The data are presented as means ± SEM (n=4) compared using the Mann-Whitney U test (**: p<0.01).

Carbamylation of intracellular proteins alters fibroblast motility but does not induce their senescence

After demonstrating that carbamylated proteins accumulate intracellularly, this study aimed at evaluating whether these carbamylated proteins could modify various cell functions. Firstly, after long-term incubation (4 weeks) in the presence of urea or cyanate, fibroblasts did not show any morphological change (data not shown). Moreover, viability rates close to 97% were observed in all conditions using the Trypan blue exclusion test (data not shown). A proliferation test was then carried out using cells previously incubated in the presence or absence of carbamylating agents and no significant difference was observed between these treatments (Fig. 4a). As actin was identified as a particular target of carbamylation, the impact of intracellular carbamylation on cell motility was evaluated by performing migration assays (Fig. 4b). The results showed that incubation with carbamylating agents led to a significant decrease of fibroblast migration speed (0.23±0.01 µm/min, -12%, p<0.05 for urea and 0.21±0.01 µm/min, -19%, p<0.01 for cyanate) in comparison with control conditions (0.26±0.01 µm/min). As one of the major functions of fibroblasts is the synthesis of matrix proteins, we determined the levels of expression of COL1A1 and COL1A2 genes (i.e. genes encoding α₁ and α₂ chains of type 1 collagen) after long-term exposure to carbamylating agents. As shown in Fig. 4c, the expression of these two genes was not modified after 4 weeks exposure to urea or cyanate. Finally, as it had been previously demonstrated that carbamylated proteins can induce cell senescence [18], we assessed whether the presence of higher levels of intracellular carbamylated proteins could promote fibroblast senescence. The flow cytometry-based assay using a fluorogenic substrate of the senescence-associated β-galactosidase showed that the cells incubated with the carbamylating agents did not show accelerated senescence when compared with control conditions (Fig. 4d).

Figure 4. Effect of intracellular protein carbamylation on cell function and senescence. Confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions) or with 20 mmol/L urea or 0.5 mmol/L cyanate. (a) Proliferation: cells were then seeded in 96-well plates at a density of 1,500 cells per well and incubated for 1, 2, 4 and 7 days with DMEM with 10% (v/v) FBS and carbamylating agents. Cell number was evaluated using a WST-1 assay by measuring absorbance at 450 nm. The data presented are means ± SEM (n=6) compared using the Mann-Whitney U test. No significant difference was found between the three conditions (control: dotted line,•; urea: grey line ■; cyanate: black line, ■). (b) Cell migration: cells were seeded in 24-well plates at a density of 15,000 cells per well and incubated for 24h at 37°C with DMEM containing 0.5% (v/v) FBS. Pictures of cells were taken every 30 min over the incubation period and each cell (n=58) was followed separately in order to calculate the migration speed. The data are presented as means ± SEM compared using the Mann-Whitney U test (*:p<0.05, **:p<0.01). (c) Expression of type I collagen mRNAs: at the end of the 4 weeks-incubation, RNA was isolated from confluent cells and then submitted to RT-qPCR analysis for evaluating the expression of COL1A1 and COL1A2 genes. Data represent the relative mRNA expression normalized to EEF1A1 gene and are expressed as means ± SEM (n=4). The Mann-Whitney U test was used to compare the three conditions: control (open bars), urea (grey bars) and cyanate (black bars). ns: not significant. (d) Senescence: cell senescence was determined by measuring the SA-β-galactosidase activity using a C₁₂FDG fluorogenic substrate and by detection of senescent cells by flow cytometry. Each plot represents the results of 20,000 events acquired per condition. Incubation of cells with rotenone was used as a positive control of cell senescence whereas a negative control without addition of the fluorogenic substrate was performed.

Fibroblasts are able to degrade intracellular carbamylated proteins

In order to investigate the intracellular metabolism of carbamylated proteins, carbamylation was first induced by incubating fibroblasts with 0.5 mmol/L cyanate for 7 days, which resulted in a carbamylation level of 0.55±0.03 µmol/mol Lys (Fig. 5). When the incubation with cyanate was continued for two additional weeks, the carbamylation level increased slightly to 0.61±0.02 µmol/mol Lys at 14 days, and reached 0.83±0.03 µmol/mol Lys at 21 days incubation.

Figure 5. Intracellular degradation of carbamylated proteins. Confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS and 0.5 mmol/L cyanate in order to induce intracellular protein carbamylation. Cells were then incubated in the same conditions (with 0.5 mM cyanate, solid line) or without cyanate (dotted line) for two additional weeks. HCit content was determined at each time point. The data are presented as means ± SEM (n=6) and the two conditions (with or without cyanate) were compared using the Mann-Whitney U test (**: p<0.01).

By contrast, when cyanate was removed from the culture medium, a significant (p<0.01) decrease of the carbamylation level was observed. This decrease was equal to -40% and -75% 7 and 14 days after cyanate withdrawal, respectively. HCit values obtained at 21 days were close to those of basal carbamylation conditions (0.14±0.01 µmol/mol Lys). These results demonstrate that fibroblasts were able to degrade intracellular carbamylated proteins.

26S proteasome is involved in the degradation of intracellular carbamylated proteins

As 26S proteasome is a key actor in the degradation and the renewal of intracellular proteins [19], we aimed to determine whether this proteasome was able to degrade carbamylated proteins.

Firstly, as it has been reported that carbamylation may lead to a partial or complete loss of activity of different enzymes [20,21], we hypothesized that long-term incubations with urea and cyanate could directly impair proteolytic activities of the proteasome by carbamylation of its catalytic subunits. To test this hypothesis, fibroblasts were incubated for 4 weeks with 20 mmol/L urea or 0.5 mmol/L cyanate, and the proteolytic activities of proteasome (i.e. caspase-like, chymotrypsin-like and trypsin-like activities) were then evaluated in cell extracts (Fig. 6a). Chymotrypsin-like activity was increased in cells incubated with urea in comparison with control conditions (1139±48 a.u. vs 891±19, p <0.05) but no variation was found after incubation with cyanate. No differences were observed in the other activities (i.e. caspase-like and trypsin-like) whatever the conditions, which suggests that a long-term incubation of the cells with these two carbamylating agents does not exert a direct inhibitory effect on 26S proteasome activity.

Figure 6. Impact of carbamylation on proteasome proteolytic activities and on the ubiquitination process. (a) Evaluation of proteasome proteolytic activity after incubation of cells with urea or cyanate: confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions, open bars) or with 20 mmol/L urea (grey bars) or 0.5 mmol/L cyanate (black bars). Chymotrypsin-like, caspase-like and trypsin-like activities have been measured in cell extracts using the corresponding Proteasome-Glo™ assays. The data are presented as means ± SEM (n=6) and compared using the Mann-Whitney U test (ns: non significant, **: p<0.01). (b) Ubiquitination level of intracellular proteins after incubation of cells with urea or cyanate: confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions) or with 20 mmol/L urea or 0.5 mmol/L cyanate, and cell extracts were prepared and submitted to western-blot analysis using an anti-ubiquitin antibody. (c) Anti-HCit and anti-ubiquitin immunolabellings were performed using fibroblasts previously seeded (10,000 cells/well) in chambered coverglass system and incubated for 2 days with DMEM containing 0.5% (v/v) FBS and 5 mmol/L cyanate. At the end of incubation, cells were fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.25% (v/v) Triton X-100 before immunolabelling of proteins using both anti-HCit and anti-ubiquitin antibodies. Colocalization points between HCit and ubiquitin labelling were identified using ImageJ software.

Secondly, given that the targeting of altered proteins towards the 26S proteasome is dependent on the protein ubiquitination process, we examined whether the increase of protein carbamylation level could interfere with ubiquitination since both reactions occur on lysine residues. For this purpose, cell extracts were prepared following the 4-week incubation of cells with urea or cyanate, and an anti-ubiquitin western-blot analysis performed (Figure 6b). A similar ubiquitination level of proteins was found under all conditions (control, urea and cyanate), which suggests that the carbamylation of intracellular proteins does not interfere with the ubiquitination process. To confirm this result, anti-HCit and anti-ubiquitin immunofluorescence labellings were performed. As seen in Figure 6c, many colocalization points were observed between both labellings suggesting that carbamylation did not greatly interfere with the ubiquitination process, and enough free lysine residues remained for ubiquitin binding.

Finally, to determine the participation of the 26S proteasome in the degradation of intracellular carbamylated proteins, fibroblasts were incubated for three weeks with 0.5 mmol/L cyanate and proteasome inhibitors, namely 10 nmol/L Bortezomib or 500 nmol/L MG-132. Cell viability assessed using the Trypan blue exclusion test was greater than 85% in all conditions (data not shown). In this experiment, results were expressed as µmol HCit per 10⁶ cells because proteasome inhibition also impairs the degradation of non-carbamylated proteins, leading to an increase in lysine content.

HCit concentrations were significantly (p<0.01) higher in the presence of the inhibitors at each point of the kinetics for Bortezomib and after only 14 days for MG-132 (Fig. 7). For example, after 14 days of incubation, the carbamylation level was 1.6-fold higher when cells were incubated with Bortezomib than in control conditions (2.16±0.05 vs 1.39±0.03 µmol HCit/10⁶ cells, p<0.01) and 2.3-fold higher when cells were incubated with MG-132 (1.43±0.05 vs 0.61±0.10 µmol HCit/10⁶ cells, p<0.01). These results demonstrate that the 26S proteasome plays a major role in the degradation of intracellular carbamylated proteins.

Figure 7. Role of proteasome in the degradation of carbamylated proteins. Confluent fibroblasts were incubated for 7 and 14 days at 37°C with DMEM containing 0.5% (v/v) FBS, 0.5 mmol/L sodium cyanate with or without proteasome inhibitors (10 nmol/L Bortezomib (Bz) or 500 nM MG-132). HCit content in total cell extracts was determined by LC-MS/MS. The data are presented as means ± SEM (n=6) and compared using the Mann-Whitney U test (ns: non significant, **: p<0.01).

Reagents

Dulbecco’s Modified Eagle’s Medium (DMEM), Penicillin and Streptomycin (P/S, PenStrep™), Bovine Serum Albumin (BSA), Phosphate Buffered Saline (PBS), Trypsin-EDTA (15400054), Alexa Fluor™488 donkey anti-rabbit IgG, Alexafluor-568 goat anti-rabbit IgG and ActinRed™ 555 ReadyProbes™ reagent (R37112) were purchased from Thermo Fisher Scientific (Villebon-sur-Yvette, France). Triton X-100 (T9284), EDTA (E-5134), urea (EU0014-B), SIGMAFAST™ proteinase inhibitor coktail (s8820) and formaldehyde solution (F8775) were from Sigma-Aldrich (St Louis, MO, USA), and sodium cyanate (NaCNO, A12413) from VWR (Radnor, PA, USA), and Bortezomib and MG-132 from Selleckchem (Souffel Weyersheim, France). Rabbit polyclonal antibody anti-HCit was produced by Covalab (Villeurbanne, France) and purified in the laboratory [15] whereas anti-ubiquitin monoclonal antibody (sc-8017) was from Santa-Cruz (Dallas, TX, USA). Control rabbit polyclonal IgG (ab27478), anti-β-actin monoclonal antibody (ab8226) and protein A/G sepharose beads were purchased from Abcam (Cambridge, UK). Fetal Bovine Serum (FBS) was from Dutscher (Issy-les-Moulineaux, France), Trypan blue from Sciencetec (Villebon-sur-Yvette, France) and DAPI-Fluoromount-G™ from SouthernBiotech (Birmingham, AL, USA). The Proteasome-Glo™ Chymotrypsin-Like, Trypsine-Like and Caspase-Like Cell-Based Assays were from Promega (Madison, WI, USA). Bradford protein assay was from Bio-Rad (Marnes-la-Coquette, France).

Cell culture

Human dermal fibroblasts were purchased from PromoCell (batch number: 0083002.2, Heidelberg, Germany). Cells were cultured in DMEM containing glucose 1g/L with 0.5% (v/v) FBS and 1% (v/v) P/S in 75 cm² or 150 cm² culture flasks (Corning, Life Sciences, Durham, NC, USA) in a humid atmosphere (5% CO2, 95% air). Different carbamylating agents were added in the culture medium: urea at 20 mmol/L or sodium cyanate (NaCNO) at 0.5 mmol/L or 5 mmol/L. The lack of toxicity was checked by using different assays (trypan blue and WST-1 assays) and by assessing the absence of morphological changes of cells (data not shown). No cellular toxicity was observed up to 80 mM urea and 10 mM cyanate for the corresponding incubation times. For the experiments aiming to determining the role of the proteasome in the degradation of carbamylated proteins, cells were incubated with 10 nmol/L Bortezomib or 500 nmol/L MG-132.

Cell proliferation

Fibroblasts previously incubated for 4 weeks in DMEM containing 0.5% (v/v) FBS and carbamylating agents (0.5 mmol/L cyanate or 20 mmol/L urea) were seeded in 96-well plates at a density of 1,500 cells per well. They were then incubated for 1, 2, 4 and 7 days with DMEM with 10% (v/v) FBS and carbamylating agents. At the end of incubation, the number of cells was evaluated using a WST-1 assay (Roche, Switzerland) by measuring absorbance at 450 nm.

Cell migration

Fibroblasts previously incubated for 4 weeks in DMEM containing 0.5% (v/v) FBS and carbamylating agents (0.5 mmol/L cyanate or 20 mmol/L urea) were seeded in 24-well plates at a density of 15,000 cells per well. Cell migration was analyzed by time-lapse videomicroscopy using an inverted microscope Axioobserver ZEISS (Carl Zeiss SAS, Germany) equipped with a small transparent environmental chamber Climabox (Zeiss), at 37˚C in an humidified atmosphere containing 5% (v/v) CO₂. The microscope was driven by the Metamorph software (Roper Scientific, Evry, France) and images were recorded with a charge-coupled device camera CoolsnapHG2 bining 2 (Roper Scientific). Pictures of cells were taken every 20 min over a 24h period and cell migration speed was quantified (μm/min) for each individual non-mitotic cell (n=16) using an interactive tracking method (Fiji-Manual Tracking software).

RNA isolation and RT-qPCR analysis

Total RNAs were isolated from fibroblasts previously incubated for 4 weeks in DMEM containing 0.5% (v/v) FBS and carbamylating agents (0.5 mmol/L cyanate or 20 mmol/L urea) using the RNeasy Plus Mini Kit 50 (QIAGEN) according manufacturer's instructions. Reverse transcription (RT) was then performed from 1 µg RNA using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (ThermoFisher). Quantitative polymerase chain reaction (qPCR) was thereafter conducted in 20 μL reaction mixture using Maxima SYBR Green/ROX qPCR (ThermoFisher) and the following primers for COL1A1 (Forward: 5’AAG ACG AAG ACA TCC CAC CA 3’, reverse: 5’ GCA GTT CTT GGT CTC GTC AC 3’), COL1A2 (Forward: 5’ TTT CCC TGG AAC TCC TGG AC 3’, reverse: 5’ AGG TTC ACC CTT CAG ACC AG 3’) and EEF1A1 (Forward: 5’ TAT CCA CCT TTG GGT CGC TT 3’, reverse: 5’ ACC GTT CTT CCA CCA CTG AT 3’). The relative expression of the different genes was calculated by the ΔΔCt method. The Ct of any gene of interest was normalized to the Ct of the reference gene (EEF1A1).

Cell senescence

Cell senescence was determined by measuring the senescence-associated (SA)-β-galactosidase activity. This method is based on the use of a fluorogenic substrate (C₁₂FDG: 5-dodecamoylaminofluorescein di-β-galactopyranoside) of SA-β-galactosidase and the detection of senescent cells by flow cytometry. For this purpose, fibroblasts were incubated for 4 weeks with carbamylated agents as described above. A positive control of senescence state was performed by incubating control fibroblasts for 72h with 0.1 µM rotenone.

To induce lysosomal alkalinisation, cells were incubated for one hour with 100 nM bafilomycin in fresh culture medium at 37°C, before the addition of the C₁₂FDG substrate (final concentration: 33 µmol/L) for one hour. Culture medium was removed and cell monolayers were washed twice for 30 s with a PBS solution at room temperature. After incubation with trypsin-EDTA, cells were resuspended in 200 µL of ice-cold PBS solution and analyzed by flow cytometry (LSR Fortessa instrument, BD Biosciences, Franklin Lakes, NY, USA). Results of 20,000 events acquired per condition were represented in a plot where the x-axis indicates C₁₂-fluorescein fluorescence intensity in a log scale. The β-galactosidase activity was estimated using the median fluorescence intensity.

Total protein extraction

Total protein extraction was performed from fibroblasts cultured for 4 weeks in DMEM containing 0.5% (v/v) FBS and 1% (v/v) P/S, with or without 20 mmol/L urea or 0.5 mmol/L cyanate. At the end of the incubation in a 150 cm² flask, medium was removed and cells were washed with 10 mL of PBS. Cells were collected after 10 min incubation at 37°C with 10 mL of 0.05% (m/v) trypsin and 0.53 mmol/L EDTA in PBS. Cell viability was measured using Trypan blue exclusion test. After centrifugation (300g, 5 min, 20°C) the cell pellet was lysed by adding 600µL distilled water and stored at -80°C until LC-MS/MS quantification of HCit.

Membrane and cytoplasmic protein extraction

After fibroblasts had been cultured in the same conditions as described above, membrane and cytoplasmic proteins have been extracted using the mem-PER™ Plus Kit (ref. number 89845, ThermoScientific, Rockfrod, IL, USA). Briefly, after the 4-weeks incubation at 37°C, cells were washed with 10 mL PBS and collected after 10 min incubation at 37°C in 10 mL of PBS containing 1 mmol/L EDTA. Cells were then washed with PBS and centrifuged (300g, 5 min, 20°C). The supernatant was discarded and the cell pellet was mixed with 650 µL of permeabilization buffer, vortexed and incubated for 10 min at 4°C under constant agitation. After centrifugation (16,000 g, 15 min, 4°C), the supernatant, which corresponds to the cytosolic fraction, was kept for further analyses and the pellet was homogenized with 650 µL of solubilization buffer and incubated for 30 min at 4°C under constant agitation. After centrifugation (16,000 g, 15 min, 4°C), the supernatant (corresponding to the membrane fraction) was transferred into a new microtube. In order to remove solvents contained in permeabilization and solubilization buffers, the two fractions were dialyzed for 24 h against distilled water using MIDI GeBaFlex-tubes (cutoff 8 kDa, Gene Bio-Application, Kfar-Hanagid, Israel).

HCit and lysine quantification

Samples were hydrolyzed by 6 mmol/L (final concentration) hydrochloric acid for 18h at 110°C. Hydrolysates were evaporated to dryness under a nitrogen stream. After addition of 1 mL of distilled water, a new evaporation was performed in order to remove residual acidity. Dried samples were resuspended in 100 µL ammonium formate 125 mmol/L containing 1 µmol/L of d₇-citrulline and 65 µmol/L of d₈-lysine used as internals standards (IS), and filtered using Uptidisc PTFE Filters (0.45 µm, Phenomenex, Le Pecq, France). For HCit quantification, samples were 5-fold diluted in 125 mmol/L ammonium formate containing the IS. For lysine quantification, samples were 10-fold diluted in 125 mmol/L ammonium formate containing the IS and then 5-fold in 5 mmol/L ammonium formate (pH 2.9) containing 65 µmol/L d₈-lysine. HCit and lysine were assayed in diluted hydrolysates by LC-MS/MS (Shimadzu, API 4000; ABSciex) as previously described [15,41]. Results were expressed as ratios of HCit to lysine.

HCit, ubiquitin and actin labelling

Anti-HCit immunofluorescence labelling was performed using fibroblasts seeded (10,000 cells per well) in a chambered cover glass system (Lab-Tek, ThermoFisher Scientific, Rochester, NY,USA) and incubated for 5 days in DMEM containing 0.5% (v/v) FBS, 1% (v/v) P/S and 5 mmol/L cyanate. At the end of incubation, the medium was removed and the cells were washed with PBS and fixed with 4% (m/v) paraformaldehyde for 10 min at room temperature. Fixed cells were washed three times for 5 min with PBS and permeabilized with 0.25% (v/v) Triton X-100 in PBS for 10 min at room temperature. Cells were washed three times with PBS and nonspecific sites were saturated by incubation with 3% (m/v) BSA in PBS for 30 min. Cells were then incubated for 12h at 4°C with anti-HCit polyclonal antibody or the corresponding control IgG, both diluted 120-fold in PBS containing 1% (m/v) BSA. After three washes with PBS, the secondary antibody (AlexaFluor™ 488 donkey anti-rabbit 100-fold diluted in BSA 1% (m/v) in PBS) was added for one hour at room temperature. After another set of three washes with PBS, cells were incubated 30 min at room temperature in darkness with ActinRed 555 ReadyProbes™ reagent diluted 50-fold in PBS containing 1% (m/v) BSA. Finally, cells were washed 3 times in PBS, and were incubated in DAPI-Fluoromount-G™ at 4°C in darkness. Cells were observed with a LSM 710-NLO confocal microscope coupled to laser Chameleon femtosecond Titanium-Sapphire (Platform of Cellular and Tissular Imagery, University of Reims Champagne-Ardenne). Image treatment was performed with the public open source software ImageJ (Bethesda, MD, USA). Co-localization points correspond to a 90% rate of pixel overlay between HCit and actin labelling in a single cell z-cross-section.

For the simultaneous anti-HCit and anti-ubiquitin immunofluorescence labelling, fibroblasts were seeded (10,000 cells per well) in Lab-Tek coverglass system, and incubated for 5 days in DMEM containing 0.5% (v/v) FBS, 1% (v/v) P/S and 5 mmol/L cyanate. At the end of incubation, washes, fixation of cells and incubation with antibodies were performed as described in the above paragraph. The anti-ubiquitin monoclonal antibody used for this experiment was directly coupled with Alexafluor 488 and diluted 120-fold (v/v) in PBS containing 1% (m/v) BSA. The secondary antibody raised against anti-HCit antibody (Alexafluor-568 goat anti-rabbit IgG) was 100-fold (v/v) diluted in BSA 1% (m/v) in PBS. Cells were then observed as described above with a confocal microscope and image treatment performed with ImageJ software. Co-localization points correspond to a 90% rate of pixel overlay between HCit and ubiquitin labelling in a single cell z-cross-section.

Immunohistolabelling of HCit in human skin

HCit immunolabelling was realized in several skin samples obtained from humans after protocol of body donation to science following death according to French regulations. The skin sample from a 77-year old man used for this article is an example showing a typical labelling. The samples were cleaned of adipose tissue and embedded in a cryomatrix (Shandon, Thermo Scientific) then frozen in liquid nitrogen before storage at -80°C. From cryomatrix block, 6-µm thick sections were performed with a microtome (MICROM Cryo-Star HM 560). After rehydration and washes with PBS, sections were incubated with anti-HCit antibody (rabbit polyclonal antibody, dilution 1:40, Covalab) overnight at 4°C. After serial washes with PBS, sections were incubated with anti-rabbit IgG antibody labelled with Alexa Fluor 488 (dilution 1:100, Invitrogen) for 30 min at room temperature. The sections were counterstained with DAPI (Dapi-Fluoromount G, Clinisciences) and hematoxylin-eosin staining.

β-actin immunoprecipitation

Confluent fibroblasts were incubated for 5 days in DMEM containing 0.5% (v/v) FBS with or without 5 mmol/L cyanate in a 150 cm² flask. At the end of the incubation, the medium was removed, the cells were washed with 10 mL of PBS and collected after 10 min incubation at 37°C with 10 mL of 0.05% (m/v) trypsin and 0.53 mmol/L EDTA in PBS. After centrifugation (300g, 5 min, 20°C), the cell pellet was lysed by adding 500 µL lysis buffer containing 75 mM Tris, 2 mM EDTA, 12 mM MgCl₂, proteinase inhibitor cocktail, 10 mM NaF, 2 mM Na₃VO₄, 1% CHAPS, pH 7.5. After sonication, lysates were then centrifuged (20,000g, 45 min, 4°C) and protein content in the supernatant was determined using Bradford protein assay. In parallel, protein A/G sepharose beads were washed three times with lysis buffer and saturated with PBS containing 2% (m/v) BSA overnight at 4°C. One mg of proteins was mixed with 50 µg anti-β-actin antibody and 100 µL of saturated beads. After a 18h-incubation at 4°C, the beads were washed three times with lysis buffer and were then submitted to acid hydrolysis prior to HCit quantification by LC-MS/MS (acid hydrolysis and HCit quantification are described above).

Assessment of ubiquitination level of intracellular proteins by western-blot analysis

After a 4-week incubation of fibroblasts with carbamylating agents, cells were washed with PBS and lyzed using a specific buffer (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 1% Igepal CA-630; 0.5% (m/v) sodium deoxycholate; 0.1% sodium dodecyl sulphate) containing 1% (m/v) protease inhibitors cocktail (Hall Protease Inhibitor Cocktail, Thermo Scientific, Rockford, IL, USA). Protein content in cell lysates was determined using Bradford assay (Protein Assay Dye Reagent Concentrate, Bio-Rad, Hercules, CA, USA). Ten micrograms of proteins were then separated by 8% (m/v) SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with Tris-buffered saline (Tris HCl 20mM pH 7.5, NaCl 150mM) containing 0.1% Tween (v/v) (TBS-T) and 5% (w/v) fat-free dehydrated milk, at room temperature for 1 hour. After three washes with TBS-T, the membrane was incubated overnight at 4°C with an anti-ubiquitin antibody directly coupled to horseradish peroxidase (Santa Cruz, Dallas, USA, used at 1/120 dilution in TBS-T containing 1% (m/v) dry milk). Finally, detection was performed using an ECL Prime Western Blotting Detection kit (GE Healthcare) according to the manufacturer’s protocol.

Proteasome activity assays

Chymotrypsin-like, trypsin-like and caspase-like proteasome activities were measured separately using the Proteasome-Glo™ Cell-Based Assays. Each reagent contained a specific luminogenic substrate, which was recognized and cleaved by the proteasome, releasing aminoluciferin and allowing the luciferase reaction to produce a luminescent signal, proportional to the enzymatic activity.

Assays were performed in fibroblasts cultured for 4 weeks in DMEM containing 0.5% (v/v) FBS and 1% (v/v) P/S, with or without 20 mmol/L urea or 0.5 mmol/L cyanate. Ten thousand cells/well were incubated for 2 hours in a 96-well plate in a volume of 100 µL. Then, 100 µL of reagent Proteasome-Glo™ was added and luminescence measured using FLUOstar Omega (BMG Labtech, Champigny-sur-Marne, France) after 5 minutes of incubation at room temperature and expressed as arbitrary units (a.u.).

Statistical analysis

All statistical analyses were performed using GraphPad 6.0 Prism Software. Results were expressed as means ± standard errors of the mean (SEM). Data were compared using the Mann Whitney non-parametric U-test and were considered significantly different when p<0.05.