Citrullination of NF-kB p65 enhances its nuclear localization and TLR-induced expression of IL-1β and TNFα
Associated Data
Abstract
Citrullination converts peptidyl-arginine to peptidyl-citrulline and is mediated by the enzymes peptidyl arginine deiminases (PADs), including PAD1–4 and PAD6. The physiological role of citrullination in immune cells is poorly understood. Here we report that suppression of PAD activity attenuates TLR-induced expression of IL-1β and TNFα by neutrophils in vivo and in vitro but not their global transcription activity. Mechanistically, PAD4 directly citrullinates NF-kB p65 and enhances the interaction of p65 with importin α3, which brings p65 into the nucleus. The citrullination-enhanced interaction of p65 with importin α3, and its nuclear translocation and transcriptional activity can be attributed to citrullination of four arginine residues located in the Rel homology domain of p65. Furthermore, a rheumatoid arthritis-prone variant of PAD4, carrying three missense mutations, is more efficient in interacting with p65 and enhancing NF-kB activity. Together, these data demonstrate a critical role of citrullination in NF-kB-dependent expression of IL-1β and TNFα.
Introduction
Citrullination, also called deimination, is a form of posttranslational modification of proteins in which peptidyl-arginine residues are converted to citrulline. This process is mediated by peptidyl arginine deiminases (PADs), including PAD1–4 and PAD6, in a calcium dependent manner (1). These enzymes have different tissue distributions and overlapping substrates (2, 3). By converting positively charged arginines to neutral citrullines, citrullination can alter protein folding and function. Indeed, citrullination critically regulates early embryogenesis (4), pluripotency of stem cells (5), and epithelial-to-mesenchymal transformation of mammary cells (6).
Much less is known regarding the role of citrullination and the targets of PADs in hematopoietic cells, which mainly express PAD2 and PAD4. Both PAD enzymes are found in the nucleus and are capable of citrullinating nuclear proteins, such as histones. PAD4-mediated citrullination of histones is essential for the formation of neutrophil extracellular traps (NETs) (7–9). NETs are enriched with bactericidal proteins, such as LL37 and defensin, and can trap and kill invading bacteria. Indeed, PAD4-deficient mice are more susceptible to bacterial infection in an animal model of necrotizing septic fasciitis (9). In addition, citrullination of extracellular proteins can modulate the function of the proteins and impact the behavior of cells. For example, PAD-citrullinated CXCL8 was less potent than native CXCL8 in attracting neutrophils after injecting into the peritoneum (10).
Citrullination also plays a pathogenic role in several human diseases. One of those diseases is rheumatoid arthritis (RA), which is characterized by the presence of anti-citrullinated protein antibodies and is mediated by inflammatory cytokines, such as TNFα, IL-1, and IL-6 (11, 12). Several RA environmental and genetic risk factors are known to increase the activity or level of PADs. For example, protein tyrosine phosphatase PTPN22 interacts with and suppresses the activity of PAD4 independently of its phosphatase activity (13). A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human PTPN22 cDNA carries the highest risk of RA among all non-HLA genetic variations (14–17). This C1858T SNP, converting an arginine to a tryptophan, renders PTPN22 unable to interact with and suppress PAD4 activity, leading to cellular hypercitrullination (13). In addition, a haplotype of padi4 (tentatively called the type 1 haplotype), encoding a PAD4 protein possessing three missense mutations in its N-terminus (called SNP-PAD4), is associated with a higher risk of RA (18–21).
How abnormal citrullination increases RA risk is still not fully understood. Existing data suggest that aberrant citrullination expands the pool of citrullinated antigens and promotes the formation of NETs, which are not only a rich source of citrullinated antigens but also propagate joint inflammation (22). However, it is unclear if citrullination also intrinsically regulates other functions of immune cells in addition to promoting NETosis. If it does, what are the mechanisms of action?
Here we report that inhibition of PADs in neutrophils results in a profound defect in TLR-induced expression of TNFα and IL-1β, two potent inflammatory cytokines involved in the pathogenesis of RA and several other autoimmune/inflammatory diseases. This effect is due to impaired nuclear localization of NF-kB p65, which is a direct substrate of PAD4. Citrullination of p65 at 4 arginine residues located at the Rel homology domain (RHD) augments the interaction of p65 with importin α3 and nuclear localization. We further demonstrate that SNP-PAD4 is more efficient in interacting with p65 and enhancing NF-kB activity than its wild type counterpart. Our data depict a novel mechanism by which citrullination promotes inflammation.
Results
Inhibition of LPS-induced transcription of inflammatory cytokines by suppressing PAD activity
To determine whether citrullination played a role in regulating LPS-induced expression of inflammatory cytokines, we pre-treated primary human neutrophils with BB-Cl-amidine (BB-Cl), a pan-PAD inhibitor (23), prior to LPS stimulation, which is known to induce citrullination in neutrophils (24). Expectedly, the level of citrullinated histone H3 (cit-H3) was enhanced by LPS but reduced by BB-Cl (Figure 1A). We also found that BB-Cl inhibited LPS-induced transcription of IL-1β and TNFα in human neutrophils from three different donors (Figure 1B). Dose dependent inhibition by BB-Cl was also seen in mouse neutrophils (Figure 1C). Near complete inhibition was observed at 5 µM concentration, a dose that did not have cytotoxic effect on neutrophils even after 8 hours of incubation and had no effect on the transcript level of a few house-keeping genes, such as HPRT (Figure S1A and S1B), which are not induced by LPS. Mouse neutrophils were used in subsequent experiments. BB-Cl also inhibited the induction of IL-1β and TNFα by other MYD88-dependent TLRs, such as Pam3CSK and flagellin (Figure S1C and S1D). However, BB-Cl did not globally inhibit transcription because it had no effect on GMCSF-induced expression of RhoH (Figure 1D) and IFNγ-induced expression of IRF7 (Figure 1E). Furthermore, BB-Cl did not alter LTB4-mediated transmigration (Figure 1F), PMA-induced production of ROS (Figure 1G), or bactericidal activity of neutrophils (Figure 1H).
A–E. Primary human (A and B) or mouse (C–H) neutrophils were stimulated with LPS (A–C), GMCSF (D), or IFNγ (E) in the absence or presence of indicated concentration of BB-Cl for 2 hours. Whole cell extract was analyzed by western blotting for the levels of cit-histone H3 (cit-H3) and total histone H3 (H3) (A). The transcript levels of indicated genes were quantified with real time PCR in duplicate (B–E). The results from at least three independent experiments are shown in B–E. Data in B are from three different donors. F. Mouse neutrophils were pretreated with BB-Cl in the upper chambers of transwells, then allowed to migrate toward LTB4 in the bottom chambers for 1 and 2 hours. The numbers of neutrophils in the bottom chambers from one of the two triplicate experiments are shown. G. Mouse neutrophils were stimulated with PMA in the presence or absence of BB-Cl pre-treatment. The generation of ROS is quantified. The data from two independent experiments is shown. H. Mouse neutrophils were subjected to in vitro bactericidal assay with or without BB-Cl pre-treatment. The survival of bacteria from one of the two triplicate experiments is shown. I. Mice (5 mice per group) were subjected to LPS-induced lung injury with or without BB-Cl pre-treatment. The transcript level of IL-1β and TNFα in lung homogenate was quantified with real time PCR (the left and the middle panels). The numbers of neutrophils in bronchial lavage are shown in the right panel. The data shown are from one of two experiments.
To examine the effect of BB-Cl on LPS-induced expression of IL-1β and TNFα in vivo, we injected WT mice with BB-Cl or DMSO intraperitoneally 4 hours prior to an intraperitoneal LPS injection. Intraperitoneal injection of LPS is known to lead to infiltration of neutrophils into the lung (25). The infiltrating neutrophils also produce a large amount of IL-1β and TNFα, resulting in acute lung injury. We found that pre-treatment with BB-Cl significantly reduced the level of LPS-induced transcription of IL-1β and TNFα in the lung but did not affect the number of infiltrating neutrophils in bronchial lavage (Figure 1I). Thus, BB-Cl inhibits LPS-induced transcription of IL-1β and TNFα in vitro and in vivo without broad effects on neutrophils.
Because BB-Cl is a pan-PAD inhibitor, we investigated if a single PAD was required for LPS-induced IL-1β or TNFα expression or if the PADs are functionally redundant. Neutrophils express the highest level of PAD4 among hematopoietic cells and also express PAD2 and PAD3 (Figure S2A) (3), but we saw no defect in the expression of IL-1β or TNFα in LPS-stimulated PAD4-deficient (KO) or PAD2KO bone marrow neutrophils (Figure S2B) suggesting functional redundancy.
Enhancement of NF-kB activity by the enzymatic activity of PADs
LPS-induced expression of TNFα and IL-1β depends on NF-kB. Thus, the data shown in Figure 1 strongly suggest that BB-Cl attenuates NF-kB activity. We therefore used HL60 cells in transient transfection assays to examine the effect of citrullination on NF-kB activity. HL60 are human promyelocytic leukemia cells, which can be differentiated to granulocyte-like cells (tentatively called differentiated HL60, dHL60) with DMSO. As expected, LPS treatment of dHL60 cells induced the expression of TNFα and IL-1β and their induction was blocked by BB-Cl (Figure 2A). By contrast, BB-Cl had no effect on IFNγ-induced expression of IRF7 in dHL60 (Figure 2B). A recent study showed that bacterial DNA also induces IL-1β expression in neutrophils through the Sox5-NF-kB pathway (26). In agreement with this observation, transfection of dHL60 with plasmid DNA induced the expression of IL-1β and TNFα in a BB-Cl-sensitive manner (Figure 2C).
A & B. dHL60 cells were stimulated with LPS (A) or IFNγ (B) for 2 hours in the presence or absence of BB-Cl pre-treatment. The expression of the indicated genes was quantified with real time PCR. C. dHL60 cells were pretreated with BB-Cl, and then transfected with empty plasmid vectors. The transcript levels of IL-β and TNFα were quantified with real time PCR 4 hours later. D. dHL60 cells were pretreated with BB-Cl then transfected with a NF-kB, CRE, or NFAT luciferase reporter. The normalized luciferase activities were measured 4 hours later. E & F. dHL60 cells were transfected with indicated amount (6 µg in E) of an expression vector of His-tagged WT PAD4 (E & F), mutant PAD4 (F) or the corresponding empty vector (E) along with indicated reporters (NF-kB reporter in F). The normalized luciferase activities were measured 4 hours later. The levels of exogenous PAD4 and endogenous tubulin in the transfected cells were also analyzed by western blotting (F). G & H. Primary human neutrophils were pretreated with 5 µM BB-Cl for 20 minutes, then stimulated with LPS for indicated periods of time. Whole cell (G) and nuclear (H) extract was prepared separately and examined by western blotting using indicated antibodies. The density of IkB in whole cell extract (G) and p65 in nuclear extract (H) was normalized against that of tubulin and histone H3 (H3), respectively. The data shown in A–H are from at least three independent experiments. I. dHL60 cells were left unstimulated or stimulated with LPS in the presence or absence of BB-Cl pre-treatment. The cells were stained with anti-p65 (in red) and Draq5 (in blue), and visualized with a confocal microscope. The mean intensity of nuclear p65 in more than 50 cells from two independent experiments is shown in the bar graph.
To further examine the effect of BB-Cl on NF-kB activity, we pre-treated dHL60 with BB-Cl and then transfected the pre-treated cells with a reporter driven by NF-kB binding sites. We found that BB-Cl suppressed the transcriptional activity of NF-kB in a dose-dependent manner (Figure 2D). By contrast, the activity of a reporter driven by CRE sites, which had basal activity comparable to the NF-kB reporter, or by NFAT sites, which was slightly less active than the NF-kB reporter, was not inhibited by BB-Cl. To ensure that our findings were not an off-target effect of BB-Cl and to further examine if PAD activity influences NF-kB activity, we co-transfected dHL60 cells with the NF-kB reporter and an expression vector containing PAD4, the dominant PAD in neutrophils and dHL-60 cells. We found that forced expression of PAD4 increased NF-kB activity in a dose-dependent manner but had no effect on the activity of the CRE or NFAT reporter (Figure 2E and 2F). Furthermore, conversion of Cys645 of PAD4 to a serine, which has been shown to attenuate its enzymatic activity (6), attenuated the effect of PAD4 on NF-kB activity (Figure 2F). Taken together, our data indicate that the enzymatic activity of PAD4 enhances the activity of NF-kB.
Inhibition of nuclear localization of NF-kB p65 by BB-Cl-amidine
Citrullination of histones can epigenetically influence gene expression (27, 28). However, plasmid DNA, such as the NF-kB reporter, is not integrated into chromatin in the transient transfection assay. Thus, the impact of BB-Cl on NF-kB activity is unlikely to be due to alterations in the status of histone citrullination. We therefore sought out other explanations. LPS activates the canonical NF-kB pathway, in which MYD88, TRAF6, and the IKK complex are sequentially activated, eventually leading to the phosphorylation and degradation of IkB. Degradation of IkB frees p65/p50 dimers, which are then brought into the nucleus by importins. However, intracellular bacterial DNA bypasses MYD88 to activate NF-kB. Therefore, BB-Cl very likely acts on a step downstream of MYD88. Indeed, pre-treatment with BB-Cl had no impact on LPS-induced degradation of IkB in primary human neutrophils (Figure 2G). We then examined the level of nuclear p65, which was barely detectable in the nucleus of unstimulated dHL60 cells. Nuclear p65 became clearly visible 30 minutes after LPS stimulation and its level peaked at 1 hour (Figure 2H). However, pre-treatment with BB-Cl reduced the level of nuclear p65 by at least 50% in all time points examined. By contrast, BB-Cl had no impact on the total level of p65 or regeneration of IkB after LPS stimulation (Figure 2G). The effect of BB-Cl on LPS-induced nuclear localization of p65 was also observed in dHL60 cells with immunocytochemistry (Figure 2I).
Physical interaction between p65 and PAD4
The data shown in Figure 2 strongly suggest that PAD activity regulates the nuclear localization of p65. We therefore tested if p65 physically interacted with PAD4 by co-immunoprecipitation using anti-p65. Anti-p65 but not control IgG was able to immunoprecipitate a 74 kD protein that is recognized by anti-PAD4 from unstimulated primary human neutrophils (Figure 3A). Interestingly, the co-immunoprecipitation of p65 and PAD4 was diminished upon LPS stimulation but was partially preserved by BB-Cl. Reciprocally, GST-fused PAD4, but not GST, was able to pull down exogenous p65 expressed in 293T cells (Figure 3B), confirming the physical interaction between p65 and PAD4.
A. Primary human neutrophils were stimulated with LPS with or without BB-Cl-amidine (BB-Cl) pre-treatment. Whole cell extract was subjected to immunoprecipitation with anti-p65 or control IgG. The immunoprecipitant was probed with anti-PAD4. A fraction of the un-manipulated whole cell extract was probed with anti-PAD4 and anti-p65 (total). The normalized density of precipitated PAD4 is shown in the bottom bar graph. B. GST or GST-PAD4 was used to pull down His-tagged p65 expressed in 293T cells. Whole cell extract from the transfected 293T cells (the top panel) and pulldown extract (the bottom panel) was probed with anti-His. C–E. GST-PAD4 (C) or GST-p65 (D & E) was used to pull down indicated His-p65 (C), His-PAD4 (D & E), or His-SNP-PAD4 (E) expressed in 293T cells. Whole cell extract from the transfected 293T cells (input) and pulldown extract was probed with anti-His. Schematic diagrams of the truncation mutants are show in the top panels of C and D. The *s in the western blots of C and D mark exogenous His-p65 (C) or His-PAD4 (D). The arrowheads in E mark His-PAD4 and His-SNP-PAD4. The normalized density of pulldown His-p65 (C), His-PAD4 (D & E), and His- SNP-PAD4 (E) is shown in the bar graphs. F. dHL60 cells were transfected with indicated amounts of vectors expressing WT PAD4 or SNP-PAD4 along with the NF-kB reporter. The normalized luciferase activity was measured 4 hours later. All data shown in Figure 3 are from at least three independent experiments.
To map the structural domains that are required for the interaction between PAD4 and p65, we overexpressed full-length or various truncation mutants of p65 in 293T cells (Figure 3C), and used GST-PAD4 to trap the overexpressed p65 from 293T cell extract. We found that fragments 1–190 (p65-N), 190–384 (p65-M), and 1–384 (p65-N+M) interacted with PAD4 stronger than full-length p65 did. By contrast, fragment 286–551 (p65-C) was not pulled down by PAD4 at all. Interestingly, deletion of 1–190 (p65-M+C) or 190–285 (p65-N+C) had little effect on the interaction between p65 and PAD4. These data suggest that p65 interacts with PAD4 through two independent domains: 1–190 and 190–285. Either one is sufficient to mediate the interaction between p65 and PAD4. Interestingly, 1–384 encompasses the RHD that is also critical for DNA binding and dimerization of p65.
Reversely, we used GST-tagged p65 to pull down full-length PAD4 and a series of its truncation mutants expressed in 293T cells. We found that amino acid residues 1–119 (PAD4-N) interacted with p65 much more strongly than full-length PAD4 did (Figure 3D). Deletion of 1–119 (PAD4-M+C) modestly reduced the interaction between PAD4 and p65. Interestingly, fragment 119–523 (PAD4-M) was also pulled down by GST-p65 as efficiently as full-length PAD4. These results suggest that either 1–119 or 119–523 is sufficient to mediate the interaction between PAD4 and p65. However, 1–119 very likely is the dominant interacting domain.
Augmentation of PAD4/p65 interaction and NF-kB activity by a RA-prone variant of padi4
The type 1 haplotype of padi4, encoding a variant of PAD4 that contains three mis-sense mutations, S55G/A82V/A112G, is associated with a higher risk of RA. These three mutations are located at the N-terminus of PAD4, far away from its enzymatic domain and do not have a significant effect on the intrinsic catalytic activity of PAD4 (29, 30). The observation that p65 interacts mainly with the N-terminus of PAD4 promoted us to propose that the S55G/A82V/A112G may affect the interaction between PAD4 and p65. We therefore expressed WT or the S55G/A82V/A112G (SNP) PAD4 in 293T cells and then used GST-p65 to pull down the exogenous SNP-PAD4 protein. We found that exogenous SNP-PAD4 was more efficiently pulled down by GST-p65 than WT PAD4 (Figure 3E). In addition, SNP-PAD4 enhanced NF-kB activity more robustly than WT PAD4 in the luciferase reporter assay (Figure 3F). At a low dose (1.5 ug), SNP-PAD4 is almost twice as effective as WT PAD4 in boosting the activity of NF-kB. Our data suggest that the three mis-sense mutations increase the expression of IL-1β and TNFα by strengthening the interaction between PAD4 and p65.
Modeling the physical interaction between PAD4 and p65
Our data indicate that PAD4 interacts mainly with the RHD of p65, which is divided into the DNA binding domain and the dimerization domain. The crystal structures of PAD4 (PDB ID: 4DKT) and the RHD of p65 (PDB ID: 2I9T) have been separately solved. We therefore used BioLuminate to model the PAD4/p65 complex. The biological unit of PAD4 is thought to be a dimer, which was used as the receptor. The top 1000 of 70,000 poses were retained, and clustered by similarity of structural orientation. The top 30 clusters were evaluated. Eight of the 30 clusters showed interaction with the N-terminus of PAD4. The first cluster of the 8 (the 15th most populated of the 30) showed the DNA binding domain of p65 interacting with the N-terminus of one PAD4, whereas the dimerization domain of p65 interacting with the mid-region of another PAD4 (Figure 4A). The docked model is consistent with the data showing that there are two potential interaction points between PAD4 and p65 (Figure 3C and 3D). Models within this cluster also show favored hydrophobic contacts between A82 of PAD4 and P168 of p65 with distances between 3.2 to 4.8Å (Figure 4B). The docked model predicts that the A82V mutation encoded by the type 1 haplotype of padi4 will lead to more favorable hydrophobic contacts between residue 168 and 82 suggesting a stronger association between PAD4 and p65 while the A112G in close contact with A82V would alter the flexibility in the region. This prediction is consistent with the data shown in Figure 3E.
A. A docking model of the PAD4 dimer (individual monomers show in green and orange) with p65 (DNA binding and dimerization domains, purple). The model shows an interface between the N-terminus of PAD4 and the DNA binding domain of p65 while the dimerization domain of p65 interacts with the adjacent PAD4. B. The interfacial regions between PAD4 (green) and p65 (purple) indicate an interaction between P168 in the DNA binding domain of p65 with A82 in the N-terminus of PAD4. A82 and A112 are in close contact. The V82 encoded by the type 1 haplotype of padi4 is modeled in pink.
Citrullination of p65 in vivo and in vitro
One potential mechanism by which PAD4 regulates the activity of NF-kB is by citrullinating p65. We therefore set to determine if p65 was citrullinated in vivo. We stimulated dHL60 cells with LPS in the absence or presence of BB-Cl pre-treatment. Whole cell extract from the cells was labeled with biotin-phenylglyoxal (PG), a chemical probe that specifically modifies peptidyl citrulline under acidic conditions (31, 32). The biotin-PG-labeled proteins were pulled down with streptavidin-agarose beads and then examined by western blotting. When we probed the pulldown extract with anti-H3, we found that the level of H3 was increased by more than 2-fold after LPS stimulation (Figure 5A). This increase was suppressed by pre-treatment with BB-Cl. This pattern was almost identical to what is shown in Figure 1A, validating this approach to detect in vivo citrullinated proteins. We then probed the pulldown extract with anti-p65. A protein band of approximately 65 kD was detected in extracts from LPS-stimulated cells but not in unstimulated cells (Figure 5B). The level of this protein band was reduced by BB-Cl pre-treatment. This result demonstrates that p65 is citrullinated in LPS-stimulated dHL-60 cells and its citrullination is inhibited by BB-Cl.
A & B. Whole cell extract from dHL60 cells stimulated with LPS in the presence or absence of BB-Cl-amidine (BB-Cl) pre-treatment was labeled with biotin-PG probe, pulled down with streptavidin beads, and probed with anti-histone H3 (H3) (A) or anti-p65 (B) in western blotting (the top panels). A fraction of the biotin-PG-labeled extract prior to pull-down was also analyzed to serve as input controls. The normalized density of pulldown H3 or p65 from two experiments is shown in the dot graph. Data from the same experiment are connected with lines. C–E. Recombinant GST, GST-p65, and various R-to-K mutants were incubated with (C–E) or without (C) PAD4, fractionated by SDS-PAGE gels, stained with Coomassie blue (the left panels) or probed with F95 (the right panels of C and E, and the middle panel of D) in western blotting. Schematic diagrams of predicted recombinant GST-p65 proteins are shown in the right panel of D. Arginine residues are marked with *. The diagrams and the location of the arginine residues are not to scale. F. The density of the citrullinated 50–80 kD bands detected with F95 was normalized against that of the corresponding bands detected with Coomassie blue. The normalized density of citrullinated WT GST-p65 was arbitrarily set as 1. Cumulated results from all experiments are shown. Each dot represents one experiment.
We subsequently performed an in vitro citrullination reaction to confirm that p65 is a direct substrate of PAD4. We first generated GST-p65 and enriched the protein on glutathione beads (Figure 5C, the left panel). Purified GST-p65 was incubated with PAD4 and then probed with F95, a mouse monoclonal IgM raised against a deca-citrullinated peptide (33). Several dense F95-reactive bands were detected in PAD4-treated GST-p65 but not in untreated GST-p65 (Figure 5C, the right panel). By contrast, there was very little or no F95 reactivity of PAD4-treated GST, confirming that GST-p65 is a direct substrate of PAD4.
Identification of citrullination sites within p65
The purified GST-p65 protein contains several degradation products. As GST was fused at the N-terminal end of p65, these degradation products are the result of truncations in the C-terminus of p65. The degradation products actually correlated with several major groups of citrullinated GST-p65: 30 kD, 35 kD, and 50–80 kD (schematic diagrams on the right of Figure 5D). The observation that the degraded GST-p65 products are also citrullinated suggests the presence of citrullination sites within the N-terminus of p65. There are five arginines (R30, R33, R35, R41, and R50) within the first 50 amino acid residues of p65. We therefore converted each of the arginines to a lysine (Figure 5D, the left panel), which maintains the positive charge but cannot be citrullinated, and subjected the mutants to in vitro citrullination. We then examined the effect of the R-to-K mutation on the level of 50–80 kD citrullinated p65, the dominant species. R30K, R33K, and R41K mutations had little impact on the density of the 50–80 kD forms of citrullinated p65. By contrast, R50K mutation and, to a lesser degree, the R35K mutation modestly attenuated the level of citrullination (the middle panel of Figure 5D). A mutant containing both R35K and R50K mutations (R35/50K) was still citrullinated to approximately 50% of the WT level (Figure 5E and 5F), suggesting the presence of additional citrullination sites.
To identify additional citrullination sites beyond the N-terminus of p65, we prepared native and in vitro citrullinated GST-p65 and performed in-gel digestion and high-resolution mass-spectrometry (LC-MS/MS) analysis on the ~80 kD bands (Figure S3). The citrullinated form of two peptides, INGYTGPGTVRISLVTK (containing R73) and IQTNNNPFQVPIEEQRGDYDLNAVR (containing R149), were identified with higher spectral counts in cit-GST-p65 compared to native GST-p65 (Table S1 and Figure 6A). Manual interrogation of the high-resolution MS1 spectra confirmed the presence of the ~1 Da heavier citrullinated species for each of these peptides (Figure 6A). The lack of the corresponding monoisotopic peak for the uncitrullinated peptide within the isotope envelope confirmed the assignments of these citrullinated species (Figure 6A). Generation of high-resolution MS2 fragmentation spectra localized the site of citrullination to R73 and R149 based on the presence of matching y6 and y7 pairs for the R73 peptide and b15 and b16 pairs for the R149 peptide. In each case, the high-resolution fragment masses unambiguously confirmed the presence of a citrulline residue based on the ~1 Da mass shift.
A. Extracted MS1 chromatograms and high-resolution isotope envelops (inset) of the R73 and R149 peptides of citrullinated GST-p65 are shown in the left panels. Annotated MS2 fragmentation spectra of the citrullinated R73 and R149 peptides are shown in the right panels. B–D. Recombinant GST-p65 and indicated R-to-K mutants were citrullinated with PAD4, fractionated in SDS-PAGE gels, stained with Coomassie blue (the left panels of B & C) or probed with F95 in western blotting (the right panels of B & C). The density of the citrullinated 50–80 kD bands detected with F95 was normalized against that of the corresponding bands detected with Coomassie blue. The normalized density of WT GST-p65 was arbitrarily set as 1. Cumulated results from two experiments are shown in D.
However, conversion of R73 and/or R149 to lysines also only modestly reduced the citrullination of p65 (Figure 6B). We therefore created a R35K/R50K/R73K/R149K quadruple mutant (4R-K) and found that this mutant was much more resistant to PAD4-mediated citrullination; this mutant was citrullinated to only 20% of the WT level (Figure 6C and 6D). One possible explanation for the reduced citrullination was that the 4R-K mutation interfered the interaction between p65 and PAD4. According to the model shown in Figure 4, these 4 arginine residues do not participate in the p65/PAD4 interaction. In addition, we found that the 4R-K mutant still physically interacted with PAD4 as tightly as WT p65 in GST pulldown assay (Figure S4), suggesting that its resistance to citrullination is not due to a weakened interaction with PAD4. Instead, our data demonstrate that R35, R50, R73, and R149 are the major, but not the only, in vitro citrullination sites of p65
Citrullination of p65 promotes its interaction with importin α3
Our data so far suggest that citrullination of p65 facilitates its nuclear localization. To determine if cit-p65 was present in the nucleus, we separately prepared cytoplasmic and nuclear extract from primary human neutrophils. Cit-p65 was pulled down with biotin-PG and detected with anti-p65 according to the method described in Figure 5B. Indeed, we detected cit-p65 in both cytoplasmic and nuclear extract only in LPS-stimulated cells, but not resting or BB-Cl-treated cells (Figure 7A). As the rate of disappearance of nuclear p65 after LPS stimulation was very comparable between BB-Cl-treated and untreated cells (Figure 2H), we postulated that citrullination mainly promoted the nuclear entry of p65 but did not affect its nuclear export by IkB or degradation by the proteasome. The nuclear entry of p65 is mediated by importin α3 (34). We therefore postulated that citrullination of p65 facilitated its interaction with importin α3. In agreement with published data, anti-importin α3 was able to co-immunoprecipitate p65 from LPS-stimulated primary human neutrophils and dHL60 cells (Figure 7B). This co-immunoprecipitation was attenuated by BB-Cl. Conversely, in vitro citrullinated GST-p65 was more efficient than native GST-p65 in pulling down importin α3 from dHL60 cells (Figure 7C). No importin α3 was pulled down by native or PAD4-treated GST. Citrullination neutralizes the positive charge of arginine and might affect the binding of p65 to DNA. We found that citrullinated GST-p65, but not PAD4-treated GST, bound to the NF-kB site of the IL-1β promoter as well as, if no better than, native p65 (Figure S5). By contrast, citrullinated GST-p65 did not bind to a control sequence that does not contain a consensus NF-kB site.
A. Cytoplasmic and nuclear extract was separately prepared from primary human neutrophils stimulated with LPS in the presence or absence of BB-Cl-amidine (BB-Cl). The detection of citrullinated p65 was performed according to the methods described in Figure 5B. Fractions of the biotin-PG-labeled extract prior to pull-down were also analyzed in western blotting using indicated antibodies to serve as input controls. The density of pulldown p65 was normalized against that of input tubulin (for cytoplasmic extract) or histone H3 (for nuclear extract). The normalized density from two experiments is shown in the dot graph. Data points from the same experiments are connected with lines. B. Cell extract from primary human neutrophils (PMN) or dHL60 cells stimulated with LPS in the absence (−) or presence (+) of BB-Cl was subjected to immunoprecipitation with anti-importin α3 (αImp) or control IgG. The immunoprecipitant and a fraction of the un-manipulated extract (total) was probed with anti-p65 in western blotting. The density of anti-importin α3 co-immunoprecipitated p65 was normalized against that of total p65. The normalized density from four independent experiments (two with dHL60 and two with human PMN) is shown in the dot graph. C & D. Recombinant GST (C), GST-p65 (C & D), and indicated R-to-K mutants (D) were incubated (+) or not incubated (−) in vitro with PAD4. A fraction of the recombinant proteins was fractionated on SDS-PAGE gels and stained with Coomassie blue (the bottom panel of C & D). The remaining recombinant proteins were used to pull down importin α3 from whole cell extract prepared from LPS-stimulated dHL60 cells. The pulldown extract and a fraction of un-manipulated dHL60 extract (input) was probed with anti-importin α3 in western blotting (the top and the middle panels of C and D). The density of pulldown importin α3 was normalized against that of input importin α3. The normalized density from at least three independent experiments is shown in the dot graph in C and the bar graph in D. E. dHL60 cells were transfected with empty vectors (−) or vectors expressing WT or 4R-K His-p65 along with the NF-kB reporter. The luciferase activity was measured 4 hours later. The normalized luciferase activity (NF-kB activity) from three independent experiments is shown in the bar graph. A fraction of the cell extract was probed with indicated antibodies in western blotting. The arrow marks the location of exogenous His-p65.
We then examined if citrullination of R35, R50, R73, and/or R149 are responsible for the enhanced interaction between cit-p65 and importin α3. We found that native WT GST-p65, R35K, R50K, R73K/R149K, and 4R-K mutants all interacted weakly and comparably with importin α3 (Figure 7D). Citrullination of WT, R35K, R50K, and R73K/R149K comparably increased their interaction with importin α3 in the GST pull-down assay. However, no such an increase was observed in citrullinated 4R-K mutant. Furthermore, 4R-K mutant was less efficient than WT p65 in transactivating the NF-kB reporter in dHL-60 cells (Figure 7E).
Discussion
Our data have expanded the role of protein citrullination in neutrophils beyond facilitating the formation of NETs. Several studies have also suggested a role for citrullination in promoting inflammation. Rabadi et al. recently reported that PAD4KO mice were more resistant to ischemic renal injury due to attenuated expression of several NF-kB-dependent inflammatory cytokines, including MIP-2 and TNFα (35). They further showed that over-expression of PAD4 in mouse proximal renal tubule cells promoted nuclear localization of NF-kB p65. Although the mechanism of this finding was not investigated, we believe that PAD4-mediated citrullination of p65 also increases its interaction with importins in proximal renal tubule cells.
Sharma et al. demonstrated that PAD4 siRNA or Cl-amidine, a pan-PAD inhibitor, attenuated the transcription of TNFα in MCF7 breast cancer cells (36). They proposed that citrullination of R8 of histone H3 weakens the binding of transcriptional repressor HP1a to tri-methylated K9 of histone H3, thereby epigenetically augmenting the transcription of TNFα. This mechanism very likely also operates in neutrophils and is consistent with several reports demonstrating that histones are targets of PAD2 and PAD4. However, this epigenetic mechanism should not affect nuclear localization of p65. In addition, the transient transfection assay shown in Figure 2E and 2F is not subjected to epigenetic regulation. Furthermore, Ghari et al. showed that citrullination of the transcription factor E2F facilitated its binding with BRD4 (bromodomain-containing protein 4) and recruitment to the promoter of several inflammatory cytokine genes, including IL-1β and TNFα, thereby augmenting the expression of these cytokines (37). Thus, citrullination can regulate the expression of IL-1β and TNFα by more than one mechanism: epigenetically modifying their genetic loci, facilitating the recruitment of E2F to their promoters, and promoting the nuclear translocation of p65. This scenario could also explain why BB-Cl nearly shuts down the transcription of IL-1β and TNFα but reduces the level of nuclear p65 only by 50% (Figure 2H). While BB-Cl did not affect the activity of NFAT or CRE, it is very possible that citrullination can also directly modulate the function of other transcription factors in response to different stimuli. Comparing the "citrullinome" between resting and stimulated neutrophils would clarify this issue.
In contradiction to the aforementioned data, Lee et al. reported that PAD2 physically interacted with IKKγ and that overexpression of PAD2 modestly inhibited LPS-induced NF-kB activity in RAW 264.7 cells (38). There are several possible explanations for the discrepancy. Overexpression of PAD2 may lead to citrullination of peptidyl arginines that are not targets of PAD2 under physiological conditions. Our approach of pharmacologically suppressing PAD activity is less likely to cause such artifacts. Unlike other hematopoietic cells, such as monocytes and lymphocytes, neutrophils express a very high level of PAD4. Thus, the functional impact of PADs in RAW264.7 macrophage-like cells may differ from that in neutrophils and HL-60 cells. Finally, BB-Cl-amidine has no effect on the degradation of IkB (Figure 2G), the downstream event of IKK activation. Thus, the functional significance of IKKγ citrullination is still unclear.
Pan-PAD inhibitors have shown therapeutic effects in animal models of human autoimmune diseases, including RA, inflammatory bowel disease, and lupus (23, 39, 40). The current dogma indicates that the therapeutic effect of the pan-PAD inhibitors comes from their ability to attenuate the formation of NETs. As IL-1β and TNFα, two potent inflammatory cytokines, are pathogenic in many of those diseases, our data provide an additional potential explanation for the therapeutic effect of PAD inhibition, i.e. by suppressing the expression of these two cytokines. As citrullination also critically regulates embryogenesis and the pluripotency of embryonic stem cells, global inhibition of PADs can have unwanted effects. Targeting the interaction between p65 and PAD will enable us to specifically inhibit the citrullination of p65 without the unwanted effects of pan-PAD inhibitors. Analysis of the crystal structure of the p65/PADs complexes will facilitate the development of such therapeutic reagents.
The type 1 haplotype of padi4 encoding the SNPs-PAD4 is associated with a higher risk of RA (18, 19, 41). The S55G/A82V/A112G SNP does not affect the intrinsic enzymatic activity of PAD4, but instead stabilizes the PAD4 transcript (18, 29, 30). However, we found that exogenous WT and SNP-PAD4 proteins reached comparable levels when expressed in dHL-60 cells, a finding that somewhat argues against the published data. Instead, our data uncover a novel mechanism for the S55G/A82V/A112G mutant to modulate the function of PAD4. By strengthening the interaction between PAD4 and p65, this haplotype results in an enhancement of p65 activity. Notably, SNP-PAD4 was also shown to preferentially interact with HDAC1, adding another layer to how this mutant might regulate gene expression(30). It will be of great interest to examine the nuclear level of p65 and NF-kB activity in blood cells of healthy donors carrying type 1 haplotype of padi4.
We have identified at least 4 major citrullination sites within p65. There appears to be functional redundancy among these four sites because mutation of each site had little effect on the p65/importin α3 interaction. Interestingly, while all four arginine residues are conserved between mouse and human, only R35 and R73 are conserved across all species. The significance of this observation is still unclear. It is also unclear if all 4 arginine residues are citrullinated in response to LPS and, if so, if the citrullination of these four arginine residues takes place simultaneously or sequentially. Development of citrullination site-specific antibodies will help address these questions.
There is still residual citrullination of 4R-K p65 after incubation with PAD4, suggesting the presence of additional citrullination sites. Unfortunately, our mass spectrometry analysis did not cover the C-terminal third of p65 due to difficulty in obtaining a sufficient amount of full-length recombinant p65. Despite this uncertainty, the role of the yet-to-be-determined sites is likely minimal because citrullination of 4R-K p65 no longer enhances its interaction with importin α3. It remains unclear how citrullination of R35, R50, R73, and R149 can enhance the p65/importin α3 interaction. Importins are thought to interact with the nuclear localization signal of p65 (34), which is located at position 301–304. Our data strongly suggest the presence of additional contact points between p65 and importin α3 involving R35, R50, R73, and R149. Solving the crystal structure of p65/importin α3 dimer will provide additional insight into how citrullination of the four arginines might facilitate this interaction.
Although PAD4 is able to enhance NFkB signaling, neither PAD4 nor PAD2 is uniquely required to upregulate the expression of IL-1β and TNFα. This observation indicates functional redundancy between these two PADs. Other PADs, such as PAD1, PAD3, and PAD6, albeit expressed at a very low level, may also compensate for the loss of PAD2 or PAD4. Unfortunately, it is nearly impossible to generate mice deficient in both PAD2 and PAD4 by crossing PAD2KO and PAD4KO mice due to the close proximity of these two genes in the mammalian genome. The CRISPR-Cas gene-editing system may allow the generation of neutrophils deficient in more than one PAD gene in the future to address this issue of redundancy.
Material and Methods
Human subjects
Peripheral blood of healthy donors was obtained through the Partners HealthCare Biobank, an enterprise biobank of consented patients samples at Partners HealthCare (Massachusetts General Hospital and Brigham and Women’s Hospital), and leukoreduction collars were obtained from Brigham and Women's Hospital Specimen Bank according to IRB-approved protocols.
Mouse
The generation of PAD2KO and PAD4KO mice was described previously (9, 42). The mice were backcrossed to DAB/1J mice for 12 generation before use. Littermates were used for all experiments
Preparation and stimulation of neutrophils
Human neutrophils were enriched from peripheral blood or leukoreduction collars of healthy donors according to a previously published protocol (43). Mouse neutrophils from bone marrow were isolated on a Percoll gradient. The enriched neutrophil fraction was recovered at the interface of 65 and 55% Percoll. Neutrophils thus purified were washed and re-suspended in HBSS (without Ca++/Mg++, Gibco, Thermo Fisher, Waltham, MA) in the presence of 0.5% heat-inactivated human serum (Sigma-Aldrich, St. Louis, MO). The purity of the enriched neutrophils was >80%. Neutrophils (2 × 106/ml) were pre-treated with BB-Cl-amidine (5–10 uM) or DMSO for 40 minutes at 37°C before stimulation with LPS (1 ug/ml, Sigma-Aldrich), GMCSF (50 ng/ml, Biolegend, San Diego, CA), or IFNγ (50 ng/ml, Biolegend) for 2 hours.
Real time PCR
RNA isolation, reverse transcription, and real time PCR were performed as described (44). Transcript level thus detected was normalized against that of actin (Figure 1B–1E, S1, and S2) or HPRT (Figure 1I and Figure 2) from the same sample. The sequences of the primers used are: mouse IL-1β, forward 5'-TGG GAA ACA ACA GTG GTC AGG-3' and reverse 5'-CCA TCA GAG GCA AGG AGG AA-3'; mouse TNF-α, forward 5'-GAG TGA CAA GCC TGT AGC C-3' and reverse 5'-CTC CTG GTA TGA GAT AGC AAA-3'; mouse HPRT, forward 5'-AGT ACA GCC CCA AAA TGG TTA AG-3' and reverse 5'-CTT AGG CTT TGT ATT TGG CTT TTC-3'. human IL-1β, forward 5'-ATA AGC CCA CTC TAC ACC T-3' and reverse 5'-ATT GGC CCT GAA AGG AGA GA-3'; human TNF-α, forward 5'-CAG CCT CTT CTC CTT CCT GA-3' and reverse 5'-GGA AGA CCC CTC CCA GAT AGA-3'; human HPRT, forward 5'- CAA GGA TGT GGA TGA GAA AGC AGA CA-3' and reverse 5'- ATG ATG GCG TCG GTC TGG ATG TAG TC-3'.
Neutrophil Chemotaxis assay
Chemotaxis was performed in 5-µm 24-well microchamber (Corning, Corning, NY). Mouse bone marrow neutrophils (2 × 105 cells), pretreated with DMSO or BB-Cl-amidine (10 µM) for 40 min at 37 °C with 5% CO2, were seeded into the upper chamber and allowed to migrate toward LTB4 (100 nM, Santa Cruz) or medium alone in the lower chamber at 37°C. After indicated time, the upper chamber was removed and the cells migrating into the lower chamber were counted under a light microscope. The results are expressed as mean number of neutrophils per well from triplicate measurements.
Measurement of ROS
Neutrophils (1 × 106/ml) were incubated with 10 µM H2DCFDA (C-400, Life Technologies) in HBSS for 15 minutes at 37°C. Cells were then washed and seeded at 4 × 105 cells per well in a 96-well microplate containing HBSS with 1 mM Ca++. Fluorescence was read at 530 nm in triplicate using Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek, VT).
In vitro neutrophil bactericidal assay
Pseudomonas aeruginosa strain PAO1 was grown overnight at 37°C, washed and re-suspended to a concentration of 5 ×106 /µL. Mouse bone marrow neutrophils (2 × 106) were pretreated with DMSO or BB-Cl-amidine and incubated with PAO1 (5 ×106) in flat-bottom 24-well plates in 1mL RPMI 1640 medium for 2 hours at 37°C with 5% CO2. The wells were then treated with 0.01% Triton X-100 for 5 min to lyse neutrophils and 10 µl lysate was applied to nutrient agar plates which were then incubated overnight at 37°C. The survival of PAO1 was calculated by the colony number of PAO1 with neutrophils incubation versus the colony number of PAO1 without neutrophils incubation. The results are expressed as the mean of survival of PAO1 from triplicate measurements.
LPS-induced acute lung injury
Mice were first injected intraperitoneally with BB-Cl-amidine (5 mg/kg) or DMSO, and then received an i.p injection of LPS (5 mg/g of body weight) 4 hours later to induce lung injury. Two hours after the LPS injection, mice were sacrificed and the lungs were removed. RNA was prepared from lungs using Trizol (Ambion, Foster City, CA) and subjected to real time PCR analysis. In some experiments, bronchoalveolar lavage (BAL) was performed and the total cell number and neutrophil number in BAL fluid were counted. All mice survived to the end of the experiments.
Cell lines, plasmid, mutagenesis, transfection, and luciferase assay
HL-60 human promyelocytic leukemia cells were maintained in RPMI medium containing 10% heat-inactivated fetal bovine serum (Hyclone, GE Healthcare Life Science, Chicago, IN) and 1% (v/v) penicillin–streptomycin (Gibco). Differentiation of HL-60 to dHL-60 was induced with DMSO (1.3%, v/v, Thermo Fisher) for 6–7 days. 3xNF-κB-Luc, 3xNF-AT-Luc, 3xCRE-Luc, and pTK-Renilla luciferase vector were described previously (45). The cDNA of mouse p65 was cloned into pCNDA3.1/HisA vector (Invitrogen, Carlsbad, CA). R-to-K mutants of p65 were generated with QuikChange XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to manufacturer’s instructions. The primers used are 5'-CAG CCC AAG CAG AAG GGC ATG CGC TTC-3' (for R30K), 5'-CAG CGG GGC ATG AAG TTC CGC TAC AAG-3' (for R33K), 5'-GGC ATG CGC TTC AAG TAC AAG TGC GAG-3' (for R35K), 5'-AAG TGC GAG GGG AAG TCC GCG GGC AGC-3' (for R41K), 5'-ATC CCA GGC GAG AAG AGC ACA GAT ACC-3' (for R50K), 5'-CCA GGG ACA GTG AAG ATC TCC CTG GTC-3' (for R73K), and 5'-ATA GAA GAG CAG AAG GGG GAC TAC GAC-3' (for R149K). R35/50K was generated from R50K; R73/149K was generated from R73K; R35/50/73K was generated from R35/50K; and 4R-K was generated from R35/50/73K. Human PAD4 and SNPs-PAD4 cDNA are a gift from Dr. Antony Rosen at Johns Hopkins Medical Institute (46). The cDNA was then cloned into pcDNA3.1/HisA plasmid.
Transfection of dHL-60 cells was performed with Amaxa cell line nucleofector kit V (Amaxa Biosystems, Gaithersburg, MD). Briefly, dHL-60 cells (2 × 106/ml) were pretreated with DMSO or BB-Cl-amidine (10 µM) in RPMI 1640 medium for 40 min at 37°C with 5% CO2, and then transfected with an indicated reporter. In all transfection/luciferase experiments, the pTK-Renilla luciferase reporter (1 ug) was included to serve as internal control for transfection efficiency. Luciferase activity was detected with the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI) according to the manufacturer’s protocol. Experiments were performed in triplicates. Values obtained from firefly luciferase signals were normalized to renilla luciferase activity.
Western blotting
Whole cell extract was obtained by lysing cells with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% TritonX-100, 0.5% DOC, 0.1% SDS, and 1 mM EDTA) containing 0.5 mM PMSF and complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Upper Bavaria, Germany). Cytoplasmic and nuclear extracts were prepared by washing cells with cold PBS and resuspending them in hypotonic lysis buffer (10 mM HEPES, pH7.9, 1 mM MgCl2, 10 mM KCl, 0.1% TritonX-100, 20% Glycerol, 0.5 mM PMSF, and protease inhibitors) on ice for 10 min. The supernatant, corresponding to cytoplasmic fraction, was collected by centrifugation at 12, 000 g for 10 min. The nuclear pellet was washed with hypotonic lysis buffer and then resuspended in hypertonic lysis buffer (10 mM HEPES, pH7.9, 400 mM NaCl, 1 m EDTA, 0.1% TritonX-100, 20% Glycerol, 1 mM PMSF, and protease inhibitors) and then incubated on ice for 20 min. Nuclear extract was collected by centrifugation. Protein extract was analyzed by immunoblot. The following antibodies were used: anti-p65 (8242S) and anti-histone H3 (4620S) from Cell Signaling Technology (Danvers, MA); anti-PAD4 (ab128086), anti-KPNA4 (importinα3, ab176585), and anti-citrullinated histone H3 (ab5103) from Abcam (Cambridge, MA), and anti-Tubulin (T5168) from Sigma-Aldrich (St. Louis, MO). Densitometry readings of western blots were obtained and analyzed with UN-SCAN-IT 6.0 software (Silk Scientific, Inc., Orem, UT) and normalized against those of indicated loading controls.
Immuocytochemistry and confocal analysis
Differentiated HL-60 cells were allowed to settle onto poly-L-lysine-coated glass coverslips for 15 mins at 37°C. Cells were pretreated with BB-Cl-amidine (10 uM) for 40 min, stimulated with LPS for 2 hours, and fixed with 2% paraformaldehyde in PBS. Coverslips were blocked with 10% FBS, 1% BSA, 0.05% Triton X-100 and 2mM EDTA in PBS over night. Staining was done with rabbit anti-NF-kB p65 (Cell Signaling, Cat # 8242S) at 1:100 dilution for 2 hours. Antibody binding was detected with Donkey anti-rabbit IgG-AF594 (Jackson Immunoresearch). Draq5 (Cell Signaling) was used to stain DNA. Confocal Microscopy was performed on a Nikon TE2000-U inverted microscope.
Immunoprecipitation
Cells were lysed in lysis buffer containing 20 mM Tris (pH 8.0), 138 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM NaF, 2 mM NaVO4, 1 mM pyrophosphoric acid and CompleteTM protease inhibitors (Roche Applied Science), and centrifuge at 12,000 rmp at 4°C for 15 min. The supernatant was collected and incubated with indicated antibodies (2 ug/sample) overnight at 4°C, then incubated with Protein A/G plus-agarose (Santa Cruz) for 4 hours at 4°C. The bound proteins were eluted by boiling for 10 min at 1× loading buffer and subjected to western blotting.
GST pulldown assay
The cDNA of p65 or PAD4 was cloned into pGEX-4T3 or pGEX-6P1 vector (GE Healthcare, Marlborough, MA), respectively, and expressed in bacteria (BL21, EMD Millipore, Billerica, MA) as GST-fusion proteins. Soluble GST-fused proteins were pre-bound to glutathione–agarose beads (Sigma). The full length and truncation mutants of p65 or PAD4 were subcloned into pcDNA3.1/HisA vector (Invitrogen, Carlsbad, CA), and expressed in 293T cells as His-tagged proteins through transfection. Whole cell lysate was prepared 48 hours after transfection in lysis buffer containing 20 mM Tris (pH 8.0), 138 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM NaF, 2 mM NaVO4, 1 mM pyrophosphoric acid and CompleteTM protease inhibitors (Roche Applied Science). The lysate was incubated with GST-p65 or GST-PAD4 bound beads overnight at 4°C. The captured proteins were then eluted and subjected to Western blotting analysis.
Computer modeling of the PAD4/p65 interaction
Protein-protein docking was accomplished by using BioLuminate (Schrodinger, Inc., New York, NY), which implements the program PIPER (47), a rigid body protein-protein docking algorithm. PAD4 (PDB ID: 4DKT) and the DNA and dimerization of p65 (PDB ID: 2I9C) were prepared use the Protein Preparation modular in Maestro (48). For PAD4 the biological unit was created using crystal symmetry mates and all crystallographic waters, buffer and salts were deleted from the structure. The DNA binding and dimerization domains of p65 were extracted from all other molecules present including DNA and p50. The number of orientations to explore was set to 70,000, which corresponds to sampling every 5° using Euler angles. The top 1000 poses were retained and clustered; the 30 most populous clusters were evaluated. Complexes were specifically evaluated for interactions between the N-terminus of PAD4 and p65. The first complex where this interaction was observed was energy minimized using Prime (49, 50) with the OPLS3 force-field (51).
Detection of in vivo citrullinated p65
Whole cell lysates were prepared by sonication in a Branson Sonifier 250 (Emerson Electric, Ferguson, MO) set at 50% output strength,15 cycles of pulse:On/Off =1 sec/10 sec in a buffer containing 50 mM HEPES. Citrullinated proteins were labeled with biotin-conjugated phenylglyoxal (biotin-PG, 0.1mM) in a buffer containing 50 mM HEPES and 20% trichloroacetic acid at 37°C for 30 min. After centrifuge, the pellet was resuspended in PBS containing 0.25% SDS, 0.14% BMe, 0.4 mM HEPES, 2 mM arginine, 2 mM NaCl. Biotin-PG labeled citrullinated proteins were captured with streptavidin–agarose beads (Thermo fisher, Cambridge, MA) over night at 4°C. The captured proteins were then eluted by boiling in 1× gel loading buffer for 10 mins and subjected to western blotting.
In vitro citrullination
Soluble GST-p65 fusion protein was pre-bound to glutathione–agarose beads and incubated with purified recombinant PAD4 (10mM) in a buffer containing 100 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 0.1 mM EDTA, and 2 mM DTT for 4 hrs at 37°C. After 3 times washing with PBS, the beads were boiled for 10 min and subjected to western blotting. The citrullinated p65 was detected with F95 antibody, a gift of Dr. Anthony Nicholas at University of Alabama at Birmingham.
Identification of citrullination sites by LC-MS/MS
In Gel Digestion
Extracted gel bands (1a–4a of Figure S2) were washed (slow vortexing) 2× for 15 minutes with 500 µL 100 mM ammonium bicarbonate. Following the washes, the supernatant was removed and 200 µL 10 mM TCEP was added and the samples were incubated at 37°C for 30 minutes. Samples were quickly spun down and TCEP was removed. 200 µL 55 mM iodoacetamide was added to the gel bands and incubated in the dark at room temperature for 30 minutes. The supernatant was removed and gel bands were washed (slow vortexing) 3× for 15 minutes in 500 µL 50:50 acetonitrile:100 mM ammonium bicarbonate each time. The supernatant was removed and 50 µL acetonitrile was added to dry the gel bands. Acetonitrile was removed via speed vac until gel bands were completely dry. 20 µg of sequencing-grade trypsin was resuspended in 40 µL of trypsin resuspension buffer. Trypsin was diluted to 10 ng/µL with 25 mM ammonium bicarbonate and 200 ng of trypsin (20 µL) was added to the gel bands. Gel bands were incubated in trypsin for 10 minutes to rehydrate the bands. 150 µL 25 mM ammonium bicarbonate (enough to cover the gel bands) was added and the samples were incubated at 37°C overnight. The following morning, the supernatant was transferred to Eppendorf LoBind microcentrifuge tubes and then 150 µL of 5% formic acid (enough to cover the gel bands) was added to the gel bands and incubated at room temperature for 15 minutes. The supernatant was transferred to the microcentrifuge tubes containing the supernatant from the overnight digestion. 150 µL acetonitrile was added to the gel bands and incubated at room temperature for 15 minutes. The supernatant was transferred to the microcentrifuge tubes containing the digested peptides. The acetonitrile elution step was repeated 3×. The samples were dried down to a volume of 20 µL via speed vac and aliquoted into two separate samples (10 µL each) and stored at −80°C until analysis by LC/LC-MS/MS.
Mass Spectrometry
LC-MS/MS analysis was performed on an LTQ-Orbitrap Discovery mass spectrometer (ThermoFisher) coupled to an Agilent 1200 series HPLC. Samples were pressure loaded onto a hand-pulled 100 µm fused-silica capillary column with a 5 µm tip packed with 10 cm Aqua C18 reverse phase resin (Phenomenex). Peptides were eluted using a 3-hour gradient 0–100% Buffer B in Buffer A (Buffer A: 95% water, 5% acetonitrile, 0.1% formic acid; Buffer B: 20% water, 80% acetonitrile, 0.1% formic acid). The flow rate through the column was set to ~0.25 µL/min and the spray voltage was set to 2.75 kV. One full MS scan (FTMS) was followed by 7 data dependent scans (ITMS) of the 7 most abundant ions. For high resolution runs, a full scan (FTMS) was followed by 4 data dependent scans (FTMS) limited to an inclusion mass list containing the masses of previously identified citrullinated peptides.
The tandem MS data were searched using the SEQUEST algorithm using a concatenated target/decoy variant of the human UniProt database. A static modification of +57.02146 on cysteine was specified to account for alkylation by iodoacetamide and a differential modification of 0.984 was specified on arginine. SEQUEST output files were filtered using DTASelect.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to manufacturer instructions in a 6% polyacrylaminde gel in 0.5× TBE. The sequences of the probe used are: NF-kB site from the human IL-1b promoter, 5'-TAACGTGGGAAAATCCAGTATT-3'; and control site, 5'-CCTCAGACTCCCGGATTCAAGCG-3', One microgram of native or citrullinated GST or GST-p65 described in Figure 7B was used per reaction of EMSA.
Statistical analysis
Statistical analysis was performed with Student's t test (Figure 2E, 2F, 3E, 3F, 7B, and 7C) or one-way ANOVA (Figure 1B, 1C, 1I, 2A, 2C, 2D, 2G, 2H, 2I, 3A, 3C, 3D, 7C, 7D, and 7E). The p values of Student's t test and one-way ANOVA analysis are shown. Multiple comparisons were also performed after one-way ANOVA, and the numbers of * were assigned automatically by GraphPad Prism.
Acknowledgments
This work is partly supported by an Innovative Research Grant from Rheumatology Research Foundation (to ICH), National Institutes of Health AR065500 (to MAS), GM079357 (to PT), GM109767 (to CAS), GM110394 (to PT and EW), and Joint Biology Consortium P30AR070253 (to ND). PRT is a paid consultant of Bristol Myers Squibb.
Footnotes
Author contributions:
ND conceived the idea of citrullination regulating the expression of inflammatory cytokines, generated the data shown in Figure 1A–1E, 1G, ,2I,2I, 5A, 5B, S1, and S2A, and co-wrote the manuscript. BS designed and carried out the experiments shown in Figure 1F, 1I, 2A–H, ,3,3, ,5,5, 6B–D, ,7,7, S3, S4, and S5, and co-wrote the manuscript. GL designed and carried out the experiment shown in Figure S5. MAS and MB designed and performed the experiments shown in Figure S2B and edited the manuscript; JLP and CAS generated and analyzed the model shown in Figure 4, and co-wrote the manuscript; TJB and EW carried out the experiment shown in Figure 6A and Table S1, and co-wrote the manuscript. AM and PRT produced BB-Cl-amidine, recombinant PAD4, and biotin-PG probe, and designed the experiments shown in Figure 5A–B. ICH participated in the design of all experiments and co-wrote the manuscript.