Regulation of Protein Citrullination through p53/PADI4 Network in DNA Damage Response

Mutations in the p53 gene are the most common genetic alteration observed in human cancers (1). Because ∼90% of them were detected within its DNA-binding domain (2, 3), the crucial function of p53 is considered as a sequence-specific transcription factor. Recently, extensive analyses were conducted to clarify the role of posttranslational modifications (PTM) in diverse cellular signaling pathways. Proteome analyses using two-dimensional gel electrophoresis showed that activated p53 altered the amounts of various protein spots without affecting their mRNA levels (4), suggesting that p53 could change the biological properties of proteins through modulation of PTMs. However, the role of p53 in the regulation of PTMs has not yet been elucidated.

Citrullination (also referred to as deimination) is one form of PTMs which converts an arginine residue in protein into a citrulline residue (5). This reaction is mediated by a Ca²⁺-dependent enzyme called peptidylarginine deiminase. Citrullination of proteins results in a loss of positive charge and causes a significant biochemical change. For example, PADI4 was shown to convert monomethyl-arginine residues of histone H3 and H4 to citrullines and negatively regulate gene expression (6, 7).

On the other hand, citrullinated peptides/proteins are recognized as non–self-proteins and subsequently activate immune systems. Citrullinated myelin basic protein in the brain was considered as a putative autoantigen associated with multiple sclerosis (8). Autoantibodies against citrullinated peptides derived from filaggrin or fibrin have been established as specific markers for rheumatoid arthritis (9–11). We have also revealed a functional haplotype of the PADI4 gene associated with rheumatoid arthritis (12). Hence, protein citrullination is now considered to play a pivotal role in various human diseases (13), but the role(s) of citrullination in carcinogenesis, if any, have not yet been reported.

Among five members belonging to the peptidylarginine deiminase family (14–16), we found PADI3 and PADI4 to be transactivated by ectopic expression of p53 in U373MG cells through our cDNA microarray analysis (17, 18). Therefore, we investigated the possible roles of p53 in the regulation of protein citrullination in this study.

cDNA microarray analysis was performed as described previously (19). Briefly, U373MG cells were infected with viral solutions and incubated at 37°C until the time of harvest. Polyadenylated RNA were isolated from U373MG cells using standard protocols. Each RNA sample was labeled and hybridized to a microarray consisting of 36,864 cDNA fragments. The results of the microarray analysis were deposited at the Gene Expression Omnibus database (accession no. GSE14953).⁴

Plasmids expressing PADI3 and PADI4 were kindly provided by Dr. Akari Suzuki (RIKEN, Yokohama, Japan). Catalytically inactive mutant PADI4 (D350A and D473A) was generated using the QuickChange site-directed mutagenesis kit (Stratagene). Primers are shown in Supplementary Table S1.

Each cell line was purchased from American Type Culture Collection, Lonza Biologics, Inc., or Japanese Collection of Research Bioresources. HCT116 (p53+/+ and p53−/−) cell lines were gifts from B. Vogelstein (Johns Hopkins University, Baltimore, MD). Cells were transfected with plasmids using FuGENE6 (Roche). Replication-deficient recombinant viruses Ad-p53 or Ad-LacZ, expressing p53 or LacZ, respectively, were generated and purified, as described previously (20, 21). U373MG cells were infected with viral solutions at various amounts of multiplicity of infection and incubated at 37°C until the time of harvest. For treatment with genotoxic stress, cells were continuously incubated with Adriamycin (ADR) for 2 h as indicated in the respective figure legends. Small interfering RNA (siRNA) oligonucleotides, commercially synthesized by Sigma Genosis, were transfected with LipofectAMINE 2000 reagent (Invitrogen) for 4 h. Sequences of oligonucleotides are shown in Supplementary Table S1. For treatment with calcium ionophore, cells were incubated with 5 μmol/L of A23187 (Calbiochem) for 1 h at 37°C.

Quantitative real-time PCR was conducted with the SYBR Green I Master on a LightCycler 480 (Roche). Primer sequences are indicated in Supplementary Table S1.

Chromatin immunoprecipitation (ChIP) assay was performed using ChIP Assay kit (Upstate Biotechnology) as described previously (18). PCR amplifications of PADI4 intron 1, containing the consensus p53-binding sites, were performed on immunoprecipitated chromatin using the primers indicated in Supplementary Table S1.

DNA fragments, including potential p53-binding sites of PADI4, BAX, FAS, or p21^WAF1 were amplified and subcloned into the pGL3-promoter vector (Promega). Primers for amplification are shown in Supplementary Table S1. To make a series of mutant vectors, a point mutation “T” was inserted into the site of the 4th and the 14th nucleotide “C” and the 7th and the 17th nucleotide “G” of the consensus p53-BS using the QuickChange site-directed mutagenesis kit (Stratagene). Reporter assays were performed using the Dual Luciferase assay system (Promega) as described previously (18).

To develop antibodies against Cit197 NPM1, a NPM1 peptide (KKSICitDTPAKN) was chemically synthesized and used to immunize rabbits. The positive antisera were further purified by immune-affinity purification using a NPM1 Cit197 peptide. Polyclonal anti–modified citrulline antibody (17–347) was purchased from Upstate and the modification of citrulline residues was carried out according to the instructions of the supplier. Anti–β-actin monoclonal antibody (A5441) and anti-Flag monoclonal (F3165) antibody were purchased from Sigma. Anti-p53 monoclonal antibodies (OP140 and OP43) and anti-p21^WAF1 monoclonal antibody (OP64) were purchased from Calbiochem. Anti-B23 (NPM1) polyclonal (sc-6013) and monoclonal (sc-32256) antibodies, anti-HA monoclonal (sc-7392) and polyclonal (sc-805) antibodies, anti-Myc monoclonal antibody (sc-40), anti-HSP90 monoclonal antibody (sc-13119), anti-PARP1 monoclonal antibody (sc-8007), and anti–glutathione S-transferase (GST) monoclonal antibody (sc-138) were purchased from Santa Cruz Biotechnology. Anti-PADI4 polyclonal antibody (ab50332) and anti-nucleolin polyclonal antibody (ab22758) were purchased from Abcam.

GST-PADI4 and GST-NPM1 were generated by cloning of their coding sequences into pGEX6P-1 (Amersham). Proteins were expressed in Escherichia coli and purified on glutathione-Sepharose beads (Amersham) by standard methods. Deimination reactions were carried out using purified protein in 0.1 mol/L of Tris-HCl (pH 7.4), 5 mmol/L of DTT, and up to 1 mmol/L of CaCl₂ at 37°C or using bead-bound substrate in 50 mmol/L of HEPES (pH 7.4), 2 mmol/L of DTT, 150 mmol/L of NaCl, 0.5% NP40, and 1 mmol/L of CaCl₂ at 30°C. HEK293T cells were transfected with constructs expressing HA-tagged wild-type or mutant NPM1 (R13K, R101K, R142K, R197K, and R221K). At 48 h after transfection, cells were lysed in 50 mmol/L of HEPES (pH 7.4), 2 mmol/L of DTT, 150 mmol/L of NaCl, 0.5% NP40, and immunoprecipitated using an anti–HA-agarose (Sigma). The immunoprecipitated bead-bound protein was washed with lysis buffer and reactions were carried out as described above.

Cell extracts from HEK293T cells transfected with a plasmid encoding HA-PADI4 or U373MG cells infected with Ad-p53 were prepared by adding lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 0.5% NP40]. Lysates were precleared by incubation with protein G-Sepharose 4B (Zymed) and mouse IgG at 4°C for 1 h. Then, precleared cell extracts were incubated with anti-B23 (NPM1) antibody (sc-32256) and protein G-Sepharose 4B at 4°C for 2 h. The beads were washed four times with 1 mL of ice-cold lysis buffer and immunoprecipitated proteins were released from the beads by boiling in sample buffer for 2 min.

To prepare whole cell extracts, cells were collected and lysed in chilled radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% NP40] for 30 min on ice and centrifuged at 16,000 × g for 15 min. Samples were subjected to SDS-PAGE and immunoblotting using a standard procedure. Blots of deiminated proteins were treated with medium for chemically modifying citrulline residues at 37°C for 3 h and then modified citrulline residues were detected with a modified citrulline-specific antibody (anti-MC). Immunocytochemistry was performed as described previously (18).

Each sample was loaded onto SDS-PAGE followed by CBB-R250 staining. The excised protein bands were reduced in 10 mmol/L of tris (2-carboxyethyl)phosphine with 50 mmol/L of ammonium bicarbonate for 30 min at 37°C and alkylated in 50 mmol/L of iodoacetamide with 50 mmol/L of ammonium bicarbonate for 45 min in the dark at 25°C. Porcine trypsin (Promega) was added for a final enzyme to protein ratio of 1:20. The digest was conducted at 37°C for 16 h and then desalted on an Oasis HLB cartridge (Waters). The resulting peptide mixture was separated on a 75 μm × 150 mm L-column (Chemicals Evaluation and Research Institute, Japan) using a 30-min linear gradient from 5.4% to 29.2% acetonitrile in 0.1% formic acid with a total flow of 200 nL/min. The eluting peptides were ionized by electrospray ionization and analyzed by a QSTAR Elite QqTOF system (Applied Biosystems/MDS Sciex). Peptide tandem mass spectrometry (MS/MS) analyses were acquired in an information-dependent mode using the Analyst QS software 2.0 acquisition features (rolling collision energy and dynamic exclusion; Smart Exit). Peak list files generated by the Protein Pilot version 1.0 software (Applied Biosystems) using default parameters were exported to a local MASCOT version 2.2.03 (Matrix Science) search engine for protein database searching, in which an additional modification file “citrullination” [H(−1) N(−1) O] was set for a variable modification. All MS/MS analyses indicating citrullinated peptide fragments were reconfirmed manually.

Nucleoli were purified using a procedure described previously (22). In brief, cells were resuspended in 15 volumes of a hypotonic buffer [10 mmol/L Tris-HCl (pH 7.4), 10 mmol/L NaCl, and 1 mmol/L MgCl₂] and incubated on ice for 30 min. Then Nonidet P40 (Roche) was added (final concentration of 0.3%) and homogenization was performed with seven strokes through a 21-gauge needle. Nuclei were collected by centrifugation at 1,200 × g for 5 min and resuspended in 10 volumes of 0.25 mol/L sucrose containing 10 mmol/L of MgCl₂. The supernatant containing the cytoplasmic fraction was harvested for further analyses. Nuclei were then purified by centrifugation at 1,200 × g for 10 min through a 0.88 mol/L sucrose cushion containing 0.05 mmol/L of MgCl₂. Purified nuclei were resuspended in 10 volumes of 0.34 mol/L sucrose containing 0.05 mmol/L of MgCl₂ and sonicated on ice. Nucleoli were then purified from the resulting homogenate by centrifugation at 2,000 × g for 20 min through a 0.88 mol/L sucrose cushion containing 0.05 mmol/L of MgCl₂. The supernatant containing the nucleoplasmic fraction devoid of nucleoli was harvested for further analyses. Purified nucleoli were resuspended in 0.34 mol/L of sucrose containing 0.05 mmol/L of MgCl₂ for further analyses.

Colony formation assays were carried out in six-well culture plates. Cells transfected with pCAGGS/PADI4 or mock plasmid were cultured in the presence of geneticin (Invitrogen) for 2 wk. Colonies were stained with crystal violet (Sigma) and scored using ImageJ software. U373MG cells or U-2 OS cells were transfected with siRNA oligonucleotide designed to suppress the expression of PADI4 or EGFP (control) 6 h before treatment with adenoviral vector or ADR. Apoptotic cells or viable cells were quantified by fluorescence-activated cell sorting analysis or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis as described previously (18).

To validate the results of our previous cDNA microarray analyses, we analyzed the expression of PADIs in U373MG cells infected with adenovirus vector expressing either p53 (Ad-p53) or LacZ (Ad-LacZ; refs. 17, 18). As a result, we found that both PADI3 and PADI4 were transactivated by p53 (Fig. 1A), indicating the possible roles of p53 in the regulation of PTMs.

To examine the involvement of p53 in protein citrullination, we infected U373MG cells with Ad-p53 or Ad-LacZ. Subsequent Western blot analysis using an antibody against a chemically modified form of citrulline (23) revealed multiple additional citrullinated proteins in the cells infected with Ad-p53 (Fig. 1B). To activate endogenous p53, we treated U-2 OS osteosarcoma cells with ADR and observed several citrullinated proteins in cells treated with ADR in a dose-dependent manner (Fig. 1C). Similar results were obtained in ADR-treated HCT116 cells (Supplementary Fig. S1). In addition, treatment of siRNA designed to suppress p53 expression (sip53) remarkably inhibited the citrullination of these proteins (Fig. 1C), implicating that DNA damage could induce protein citrullination in a p53-dependent manner.

Next, we treated U373MG cells with siRNA designed to suppress PADI3 or PADI4 expression (siPADI3 or siPADI4) prior to Ad-p53 treatment (Supplementary Fig. S2). Treatment of the cells with siPADI4 almost completely inhibited the citrullination of the 37-kDa protein in Ad-p53–infected U373MG cells, whereas that with siEGFP or siPADI3 revealed no effect on citrullination (Fig. 2A). Furthermore, introduction of PADI4 in HEK293T cells induced citrullination of various proteins including the 37-kDa protein, whereas that of PADI3 or catalytically inactive mutant-PADI4 (D350A and D473A; ref. 24) induced weak or no protein citrullination (Fig. 2B). We then treated U-2 OS cells with ADR and found the induction of PADI4 mRNA (Fig. 2C), in concordance with the results of protein citrullination. To investigate p53-dependent activation of PADI4 expression, we treated HCT116 p53+/+ and HCT116 p53−/− cells with ADR and found the induction of PADI4 only in HCT116 p53+/+ cells in an ADR dose-dependent manner (Fig. 2C). The results of Western blot analysis also revealed the increased expression of PADI4 protein in Ad-p53–infected U373MG or ADR-treated U-2 OS cells, respectively (Fig. 2D). Taken together, we suspect that PADI4 is a key mediator of p53-induced protein citrullination.

To further investigate whether PADI4 is a direct transcriptional target of p53, we surveyed genomic sequences of the PADI4 gene on chromosome 1p36.13 and identified two potential p53-binding sequences (p53BS-A and p53BS-B) within the first intron (Fig. 3A). ChIP assays using U373MG cells that were infected with either Ad-p53 or Ad-LacZ indicated the binding of p53 protein to the genomic fragment including p53BS-A (Fig. 3B). We subsequently analyzed the promoter/enhancer activity of the DNA fragment including p53BS-A and p53BS-B (p53BS-AB) using a reporter assay and found that wild-type p53 significantly enhanced luciferase activity (Supplementary Fig. S3). The DNA fragment containing the base substitutions within BS-A completely diminished the enhancement of luciferase activity (Fig. 3C), indicating that p53 directly regulates PADI4 expression through p53-responsible element, p53BS-A in intron 1.

Nucleophosmin (NPM1), a ubiquitously-expressed nucleolar protein, was previously reported to be possibly citrullinated with treatment of calcium ionophore in HL-60 cells (14). The molecular weight of NPM1 was almost identical to that of the 37-kDa citrullinated protein (Supplementary Fig. S4). Therefore, we suspect that NPM1 is one of the candidate substrates for PADI4. To examine whether PADI4 could directly interact with and citrullinate NPM1, cell extracts from PADI4-transfected HEK293T cells or Ad-p53–infected U373MG cells were immunoprecipitated with anti-NPM1 antibody or control mouse IgG. Subsequent Western blot analyses indicated the binding of endogenous NPM1 with exogenous or endogenous PADI4 (Fig. 4A). To investigate in vivo citrullination of NPM1, we transfected HEK293T cells with plasmid expressing wild-type or mutant PADI4. Then, endogenous NPM1 protein was purified with anti-NPM1 antibody and subjected to Western blotting with anti–modified citrulline antibody. As a result, we found citrullination of NPM1 in wild-type PADI4-transfected cells although not in mock-transfected or mutant PADI4–transfected cells (Fig. 4B). Moreover, knockdown of NPM1 with siRNA against NPM1 (siNPM1) reduced the amount of the 37 kDa citrullinated protein in ADR-treated U-2 OS cells (Fig. 4C). Then we purified recombinant NPM1 and PADI4 proteins, both fused to GST. We incubated GST-NPM1 with GST-PADI4 for 1 hour at 37°C with different concentrations of Ca²⁺ and found that PADI4 citrullinated NPM1 under intracellular Ca²⁺ concentrations of 10⁻⁷ mol/L in vitro (Fig. 4D). These data clearly indicated that PADI4 citrullinates NPM1 under physiologic conditions.

To identify the citrullinated site(s) of NPM1, we immunopurified endogenous NPM1 proteins from PADI4-transfected HEK293T cells. Following liquid chromatography MS/MS analysis, we identified the citrullination of NPM1 at the 197th arginine residue (data not shown). Then we constructed plasmid expressing mutant NPM1 (R197K NPM1) and introduced HEK293T cells. Wild-type and mutant-NPM1 protein were purified from cell extracts and subjected to in vitro citrullination assay. Concordant with the results of liquid chromatography MS/MS analysis, substitution of the 197th arginine residue caused a significant reduction of protein citrullination by GST-PADI4 (Fig. 5A).

To examine the in vivo citrullination of NPM1 at the 197th arginine residue, we raised an antibody that could recognize citrullinated NPM1 using a peptide corresponding to amino acids 193 to 202. Antibody titers and specificity were shown by ELISA using citrullinated peptide (Cit197: KKSI_Cit_DTPAK) and noncitrullinated peptide (Arg197: KKSI_R_DTPAK; Supplementary Fig. S5). Western blotting using these antibodies clearly indicated the citrullination of NPM1 in PADI4-transfected HEK293T cells (Fig. 5B) as well as ADR-treated U-2 OS cells in a p53/PADI4-dependent manner (Fig. 5C). These findings strongly implied that cellular DNA damage could induce the citrullination of NPM1 at the 197th arginine residue.

Protein citrullination reduces the positive charge of proteins and may subsequently influence their structure and function. NPM1 was shown to continuously shuttle between the nucleus and the cytoplasm and translocate from the nucleoli to the nucleoplasm as a response to DNA damage (25). Therefore, we investigated the effect of protein citrullination on subcellular localization of NPM1. Immunocytochemical analysis revealed that cotransfection of NPM1 with PADI4 altered the subcellular localization of NPM1 from the nucleoli to the nucleoplasm in HEK293T cells (Fig. 6A). Moreover, coexpression of p53 with NPM1 in H1299 cells changed subcellular localization of NPM1 from the nucleoli to the nucleoplasm (Supplementary Fig. S6A). In contrast, PADI4 did not induce the translocation of nucleolin (nucleolar protein) as well as mutant NPM1 (R197K; Supplementary Fig. S6B and S6C). To validate these findings, we transfected either wild-type or mutant PADI4 into HEK293T cells and separated the cells into three fractions—cytoplasm, nucleoli, and nucleoplasm—using sucrose gradient centrifugation (22). The results of Western blot analysis clearly indicated the translocation of NPM1 from the nucleoli to the nucleoplasm in cells transfected with wild-type PADI4, whereas NPM1 was dominantly located at nucleoli in the cells transfected with mock or mutant-PADI4 plasmid (Fig. 6B). The expressions of PARP1 (nuclear protein) and HSP90 (cytoplasmic protein) were used as quality and quantity controls. These findings clearly indicated that the p53-PADI4 pathway would regulate subcellular localization of NPM1.

Finally, we assessed the possible role of PADI4 in the p53-dependent growth-suppressive pathway. We conducted colony formation assays using several cancer cell lines and found that ectopic expression of PADI4 significantly inhibited the growth of cancer cells (A549, HCT116, and U-2 OS cells; Fig. 6C). We then treated U373MG cells with either siPADI4 or siEGFP prior to infection with Ad-p53 and found that the proportion of apoptotic cells was decreased in siPADI4-treated cells (Fig. 6D). Knockdown of PADI4 also inhibited the ADR-induced growth suppression in U-2 OS cells (Fig. 6D). Taken together, our findings clearly showed that PADI4 would play critical roles in a p53 downstream pathway.

Although a number of p53 targets related to cell cycle arrest and apoptosis have been characterized, the role of p53 in the regulation of PTMs has not yet been well clarified. Here, we revealed that DNA damage induces the citrullination of various proteins in a p53/PADI4-dependent manner. We also showed that PADI4 citrullinates NPM1 at arginine 197 residue and regulates subcellular localization of NPM1. Moreover, our findings revealed that knockdown of PADI4 attenuated the growth-suppressive effect of p53. Considering that PADI4 is a direct target of p53, PADI4 would be one of the important mediators in the p53-signaling pathway.

In several hematologic malignancies, the NPM1 locus on chromosome 5q35 is frequently translocated and forms oncogenic fusion proteins such as NPM-ALK, NPM-RARa, and NPM-MLF1 (26). NPM1 is also overexpressed in various solid tumors such as colorectal and bladder cancers, and its overexpression is associated with tumor progression (27, 28). On the other hand, mutations of the NPM1 gene were found in about one-third of adult de novo acute myelogenous leukemias causing aberrant cytoplasmic expression (29). Thus, the role of NPM1 in carcinogenesis still remains unclear and controversial. NPM1 is essential for the development of the mouse embryo (30) and is also implicated in multiple functions including ribosomal biogenesis (31), centrosome duplication (32), and regulation of tumor suppressor pathways such as p53 (33) and CDKN2A (34). At the nucleoli, NPM1 is involved in rRNA transcription, pre-rRNA processing, and ribosome subunit assembly which are essential for cell growth and maintenance of cellular homeostasis (35). Because PADI4 induced the nucleoli/nucleoplasm translocation of NPM1, the growth-suppressive effect mediated by the p53/PADI4 pathway would be attributed to the failure of ribosome biogenesis.

Furthermore, we showed the physiologic regulatory mechanism of protein citrullination. Most previous studies assessing the enzymatic activities of PADIs were conducted under nonphysiologic conditions using calcium ionophore or at higher calcium concentrations. Although activated PADI4 with calcium ionophore was shown to induce NPM1 citrullination in HL-60 cells similar to our findings (14), these results are not likely to reflect the physiologic function of PADI4. To our knowledge, this is the first report demonstrating the role of protein citrullination under physiologic conditions.

Taken together, our findings clearly revealed the novel role of p53 in the regulation of protein citrullination. Although further functional analyses are essential to fully characterize the p53/PADI4-mediated protein citrullination in carcinogenesis, our findings should shed light on the novel function of p53 that can directly or indirectly modulate the biological properties of various proteins through protein citrullination.

No potential conflicts of interest were disclosed.

Grant support: Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 18687012; K. Matsuda). C. Tanikawa is a JSPS Research Fellow.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank A. Suzuki for providing plasmid and helpful discussion, and K. Makino and A. Takahashi for technical assistance.