Association of PADI2 and PADI4 polymorphisms in COVID-19 host severity and non-survival.

Background: Enzymes of the peptidylarginine deiminase family (PADs) play a relevant role in the pathogenesis of COVID-19. However, the association of single nucleotide polymorphisms (SNPs) in their genes with COVID-19 severity and death is unknown. Methodology: We included 1045 patients who were diagnosed with COVID-19 between October 2020 and December 2021. All subjects were genotyped for PADI2 (rs1005753 and rs2235926) and PADI4 (rs11203366, rs11203367, and rs874881) SNPs by TaqMan assays and their associations with disease severity, death, and inflammatory biomarkers were evaluated. Results: 291 patients presented had severe COVID-19 according to PaO2/FiO2, and 393 had a non-survival outcome. Carriers of the rs1005753 G/G genotype in the PADI2 gene presented susceptibility for severe COVID-19, while the heterozygous carriers in rs11203366, rs11203367, and rs874881 of the PADI4 gene showed risk of death. The GTACC haplotype in PADI2-PADI4 was associated with susceptibility to severe COVID-19, while the GCACC haplotype was a protective factor. The GCGTG haplotype was associated with severe COVID-19 but as a protective haplotype for death. Finally, the GTACC haplotype was associated with platelet-to-lymphocyte ratio (PLR), the GCACC haplotype with neutrophil-to-hemoglobin and lymphocyte and the GCGTG haplotype as a protective factor for the elevation of procalcitonin, D-dimer, CRP, LCRP, NHL, SII, NLR, and PLR. Conclusions: Our results suggest that the haplotypic combination of GTACC and some individual genotypes of PADI2 and PADI4 contribute to the subjects' susceptibility for severity and death by COVID-19.

PADI4 negatively regulates RIG-I-mediated antiviral response through deacetylation of IFN-ß promoter via HDAC1.

The production of type I interferon (IFN) is precisely modulated by host to protect against viral infection efficiently without obvious immune disorders. Elucidating the tight control towards type I IFN production would be helpful to get insight into natural immunity and inflammatory diseases. As yet, however, the mechanisms that regulate IFN-ß production, especially the epigenetic regulatory mechanisms, remain poorly explored. This study elucidated the potential function of Peptidylarginine deiminases (PADIs)-mediated citrullination in innate immunity. We identified PADI4, a PADIs family member that can act as an epigenetic coactivator, could repress IFN-ß production upon RNA virus infection. Detailed experiments showed that PADI4 deficiency increased IFN-ß production and promoted antiviral immune activities against RNA viruses. Mechanistically, the increased PADI4 following viral infection translocated to nucleus and recruited HDAC1 upon binding to Ifnb1 promoter, which then led to the deacetylation of histone H3 and histone H4 for repressing Ifnb1 transcription. Taken together, we identify a novel non-classical role for PADI4 in the regulation of IFN-ß production, suggesting its potential as treatment target in inflammatory or autoimmune diseases.

hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50.

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody.

BACKGROUND: Peptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer. METHODS: The subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes. RESULTS: PADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated. CONCLUSIONS: PADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.

Molecular Factors in PAD2 (PADI2) and PAD4 (PADI4) Are Associated with Interstitial Lung Disease Susceptibility in Rheumatoid Arthritis Patients.

Around 50% of rheumatoid arthritis (RA) patients show some extra-articular manifestation, with the lung a usually affected organ; in addition, the presence of anti-citrullinated protein antibodies (ACPA) is a common feature, which is caused by protein citrullination modifications, catalyzed by the peptidyl arginine deiminases (PAD) enzymes. We aimed to identify single nucleotide variants (SNV) in PADI2 and PADI4 genes (PAD2 and PAD4 proteins, respectively) associated with susceptibility to interstitial lung disease (ILD) in RA patients and the PAD2 and PAD4 levels. Material and methods: 867 subjects were included: 118 RA-ILD patients, 133 RA patients, and 616 clinically healthy subjects (CHS). Allelic discrimination was performed in eight SNVs using qPCR, four in PADI2 and four in PADI4. The ELISA technique determined PAD2 and PAD4 levels in serum and bronchoalveolar lavage (BAL) samples, and the population structure was evaluated using 14 informative ancestry markers. Results: The rs1005753-GG (OR = 4.9) in PADI2 and rs11203366-AA (OR = 3.08), rs11203367-GG (OR = 2.4) in PADI4 are associated with genetic susceptibility to RA-ILD as well as the ACTC haplotype (OR = 2.64). In addition, the PAD4 protein is increased in RA-ILD individuals harboring the minor allele homozygous genotype in PADI4 SNVs. Moreover, rs1748033 in PADI4, rs2057094, and rs2076615 in PADI2 are associated with RA susceptibility. In conclusion, in RA patients, single nucleotide variants in PADI4 and PADI2 are associated with ILD susceptibility. The rs1748033 in PADI4 and two different SNVs in PADI2 are associated with RA development but not ILD. PAD4 serum levels are increased in RA-ILD patients.

Polymorphic Analysis of Genes PADI4 (rs2240340, rs1748033) and HLA-DRB1 (rs2395175) in Arthritis Patients in Pakistani Population.

Genes are an important factor for the initiation of any disease. Many genes are associated with rheumatoid arthritis (RA) other than environmental factors. The main objective of the study was to evaluate the association of genes PADI4 (peptidylarginine deiminases 14) (rs2240340, rs1748033) and Human leukocyte antigen class II histocompatibility, D-related beta chain (HLA-DRB1) (rs2395175) polymorphisms in RA patients from Punjab, Pakistan. Blood samples of RA patients were collected from different hospitals of Sargodha. DNA was extracted, followed by PCR. Polymorphic analysis was performed in 300 rheumatoid arthritis patients and 300 healthy controls on PADI4 (rs2240340, rs1748033) and HLA-DRB1 (rs2395175). In PADI4 gene, both homozygous mutant genotype (TT) and heterozygous (CT) of SNP rs2240340 showed significant association by increasing the risk of RA up to two fold (OR 2.55; 95% CI 1.57-4.15; p = 0.0002). In case of rs1748033 polymorphism, homozygous mutant genotype (TT) showed significant association with RA by increasing the risk of disease up to three fold (OR 3.46; 95% CI 1.97-6.07; p = 0.0001), while heterozygous genotype (CT) of the same SNP showed significant association with RA by playing a protective role (OR 0.57; 95% CI 0.36-0.91; p = 0.0197). In HLA-DRB1 gene, homozygous mutant genotype (GG) of SNP rs2395175 showed no significant association with RA, while heterozygous genotype (AG) of the same SNP showed significant association with RA by playing a protective role (OR 0.44; 95% CI 0.27-0.71; p = 0.0009). Highly significance association of genes PADI4 (rs2240340, rs1748033) and HLA-DRB1 (rs2395175) polymorphisms with RA was observed in Pakistani population.

PLA2G2E-mediated lipid metabolism triggers brain-autonomous neural repair after ischemic stroke.

The brain is generally resistant to regeneration after damage. The cerebral endogenous mechanisms triggering brain self-recovery have remained unclarified to date. We here discovered that the secreted phospholipase PLA2G2E from peri-infarct neurons generated dihomo-γ-linolenic acid (DGLA) as necessary for triggering brain-autonomous neural repair after ischemic brain injury. Pla2g2e deficiency diminished the expression of peptidyl arginine deiminase 4 (Padi4), a global transcriptional regulator in peri-infarct neurons. Single-cell RNA sequencing (scRNA-seq) and epigenetic analysis demonstrated that neuronal PADI4 had the potential for the transcriptional activation of genes associated with recovery processes after ischemic stroke through histone citrullination. Among various DGLA metabolites, we identified 15-hydroxy-eicosatrienoic acid (15-HETrE) as the cerebral metabolite that induced PADI4 in peri-infarct-surviving neurons. Administration of 15-HETrE enhanced functional recovery after ischemic stroke. Thus, our research clarifies the promising potential of brain-autonomous neural repair triggered by the specialized lipids that initiate self-recovery processes after brain injury.

New insights into cancer: MDM2 binds to the citrullinating enzyme PADI4.

PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. MDM2 is an E3 ubiquitin ligase which is crucial for down-regulation of degradation of the tumor suppressor gene p53. Given the relationship between both PADI4 and MDM2 with p53-signaling pathways, we hypothesized they may interact directly, and this interaction could be relevant in the context of cancer. Here, we showed their association in the nucleus and cytosol in several cancer cell lines. Furthermore, binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that MDM2 could bind to the active site of PADI4, as confirmed by in silico experiments. In vitro and in silico studies showed that the isolated N-terminal region of MDM2, N-MDM2, interacted with PADI4, and residues Thr26, Val28, Phe91 and Lys98 were more affected by the presence of the enzyme. Moreover, the dissociation constant between N-MDM2 and PADI4 was comparable to the IC50 of GSK484 from in cellulo experiments. The interaction between MDM2 and PADI4 might imply MDM2 citrullination, with potential therapeutic relevance for improving cancer treatment, due to the generation of new antigens.

SNP in PTPN22, PADI4, and STAT4 but Not TRAF1 and CD40 Increase the Risk of Rheumatoid Arthritis in Polish Population.

Single nucleotide polymorphisms in non-HLA genes are involved in the development of rheumatoid arthritis (RA). SNPS in genes: PADI4 (rs2240340), STAT4 (rs7574865), CD40 (rs4810485), PTPN22 (rs2476601), and TRAF1 (rs3761847) have been described as risk factors for the development of autoimmune diseases, including RA. This study aimed to assess the prevalence of polymorphisms of these genes in the Polish population of patients with rheumatoid arthritis as compared to healthy controls. 324 subjects were included in the study: 153 healthy subjects and 181 patients from the Department of Rheumatology, Medical University of Lodz who fulfilled the criteria of rheumatoid arthritis diagnosis. Genotypes were determined by Taqman SNP Genotyping Assay. rs2476601 (G/A, OR = 2.16, CI = 1.27-3.66; A/A, OR = 10.35, CI = 1.27-84.21), rs2240340 (C/T, OR = 4.35, CI = 2.55-7.42; T/T, OR = 2.80, CI = 1.43-4.10) and rs7574865 (G/T, OR = 1.97, CI = 1.21-3.21; T/T, OR = 3.33, CI = 1.01-11.02) were associated with RA in the Polish population. Rs4810485 was also associated with RA, however after Bonferroni's correction was statistically insignificant. We also found an association between minor alleles of rs2476601, rs2240340, and rs7574865 and RA (OR = 2.32, CI = 1.47-3.66; OR = 2.335, CI = 1.64-3.31; OR = 1.88, CI = 1.27-2.79, respectively). Multilocus analysis revealed an association between CGGGT and rare (below 0.02 frequency) haplotypes (OR = 12.28, CI = 2.65-56.91; OR = 3.23, CI = 1.63-6.39). In the Polish population, polymorphisms of the PADI4, PTPN22, and STAT4 genes have been detected, which are also known risk factors for RA in various other populations.

Intrinsically Disordered Chromatin Protein NUPR1 Binds to the Enzyme PADI4.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 µM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.

A common missense variant rs874881 of PADI4 gene and rheumatoid arthritis: Genetic association study and in-silico analysis.

The peptidylarginine-deiminase 4 (PADI4) is involved in the post-translational catalytic conversion of arginine into citrulline. The autoantibodies including anti-citrullinated protein antibodies (ACPAs) produced in response to hypercitrullinated proteins are a hallmark of rheumatoid arthritis (RA) autoimmunity. Therefore, the role of a missense variant rs874881 (Gly112Ala) of PADI4 in RA susceptibility was analyzed, along with in-silico analysis of structural and functional impacts of this substitution. We did a case-control association study and in-silico analysis. For the case-control study, confirmed RA cases and healthy controls were recruited. Genotyping for rs874881 (n = 750) was performed through polymerase chain reaction-restriction fragment length polymorphism. Multivariate logistic regression analysis was employed to determine association. The in-silico analysis was carried out through HOPE, VarMap, MutationAssessor, MutPred2, SIFT, PolyPhen, CADD, REVEL and MetaLR. In the case-control study, the rs874881 exhibited a strong association with increased RA susceptibility (G vs C odds ratio = 3.85, 95 % confidence interval = 2.81-5.27). Interaction analysis revealed significant interaction of genotype with smoking and gender (p < 0.05). Significant results (p < 0.05) were also obtained in stratified analysis by presence/absence of comorbidities and radiographic damage. According to in-silico pathogenicity prediction analysis, this Gly112Ala substitution does not exert a major effect on protein structure and function including its enzymatic activity. We report a significant association of PADI4 rs874881 with overall RA susceptibility. To our knowledge, this is the first study to do the interaction and stratified analyses on the PADI4 rs874881 in RA. Similar detailed studies should also be performed in other populations.

The Role of Peptidyl Arginine Deiminase IV(PADI4) in Cancers.

BACKGROUND: Peptidyl arginine deiminase IV (PADI4, also called PAD4), a Ca2+-dependent posttranslational modification enzyme, catalyzes the conversion of arginine residues to non-coded citrulline residues. Dysregulation of PADI4 is involved in a variety of diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), Alzheimer's disease (AD) and many kinds of malignant tumors. OBJECTIVE: The roles of PADI4 in different tumors and the underlying molecular mechanisms are presented in this article. RESULTS: PADI4-mediated citrullination is associated with either transcriptional activation or repression in different contexts. Abnormal expression of PADI4 exists in a variety of malignant tumors and affects tumor progression and metastasis. Epithelial-to-mesenchymal transition (EMT), apoptosis, and neutrophil extracellular traps (NETs) may be the underlying molecular mechanisms. CONCLUSION: PADI4 plays crucial role in the occurrence, development, and metastasis of tumors, and PADI4 may be an effective biomarker for cancer prognosis and a potential target for cancer treatment.

The armadillo-repeat domain of Plakophilin 1 binds to human enzyme PADI4.

Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a key structural component of cell-cell adhesion scaffolds, although it can also be found in other cell locations, including the cytoplasm and the nucleus. PADI4 (peptidyl-arginine deiminase 4) is one of the human isoforms of a family of enzymes engaged in the conversion of arginine to citrulline, and is present in monocytes, macrophages, granulocytes, and in several types of cancer cells. It is the only family member observed both within the nucleus and the cytoplasm under ordinary conditions. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with PADI4, by using several biophysical methods, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), isothermal titration calorimetry (ITC), and molecular simulations; furthermore, binding was also tested by Western-blot (WB) analyses. Our results show that there was binding between the two proteins, with a dissociation constant in the low micromolar range (∼ 1 µM). Molecular modelling provided additional information on the possible structure of the binding complex, and especially on the binding hot-spot predicted for PADI4. This is the first time that the interaction between these two proteins has been described and studied. Our findings could be of importance to understand the development of tumors, where PKP1 and PADI4 are involved. Moreover, our findings pave the way to describe the formation of neutrophil extracellular traps (NETs), whose construction is modulated by PADI4, and which mediate the proteolysis of cell-cell junctions where PKP1 intervenes.

Role of miRNA-4298/PADI4 axis in the apoptosis of leukemia cells induced by Nrf2 inhibitor 4f / 中华微生物学和免疫学杂志

Objective:To investigate the role of miRNA-4298/PADI4/p53 signal axis in mediating 4f-induced apoptosis of leukemia cells.Methods:The cell growth density was observed under inverted microscope and the proliferation of leukemia cells was detected by CCK-8 counting assay. The expression of PADI4 and P53 at mRNA level was detected by qRT-PCR. Cell cycle and apoptosis were measured with flow cytometry. The expression of PADI4, P53, Bcl-2, Bax, caspase-3 and caspase-9 at protein level was detected by Western blot. Differential miRNA and mRNA expression profiles was detected by next generation sequencing. Databases such as TargetScan were used to predict the potential upstream and downstream genes of PADI4. A luciferase reporter assay was used to detect the 3′UTR of PADI4 targeted by miRNA-4298. Cell transfection assay was used to detect the effect siRNA, PADI4 vector, miRNA mimics and miRNA inhibitor in interference and rescue.Results:Nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor 4f could inhibit the proliferation of THP-1, K562 and U937 cells, and induce the apoptosis of these leukemia cells. It downregulated the expression of PADI4 mainly through the binding activity of miRNA-4298 to miRNA sponges, which resulted in the proliferation inhibition and apoptosis of leukemia cells. The inhibited proliferation and apoptosis of leukemia cells by 4f were associated with the increase of P53 expression after the decrease of PADI4 expression. The PADI4-dependent upregulation of P53 led to the ratio inversion of downstream Bcl-2/Bax, which activated caspase-3 or caspase-9 to induce the apoptosis of leukemia cells.Conclusions:The apoptosis of leukemia cells induced by Nrf2 inhibitor 4f was mainly associated with the miRNA-4298/PADI4/p53 axis, suggesting that it might be a novel signaling pathway for targeted therapy.

A Screening of Epigenetic Therapeutic Targets for Non-Small Cell Lung Cancer Reveals PADI4 and KDM6B as Promising Candidates.

Despite advances in diagnostic and therapeutic approaches for lung cancer, new therapies targeting metastasis by the specific regulation of cancer genes are needed. In this study, we screened a small library of epigenetic inhibitors in non-small-cell lung cancer (NSCLC) cell lines and evaluated 38 epigenetic targets for their potential role in metastatic NSCLC. The potential candidates were ranked by a streamlined approach using in silico and in vitro experiments based on publicly available databases and evaluated by real-time qPCR target gene expression, cell viability and invasion assays, and transcriptomic analysis. The survival rate of patients with lung adenocarcinoma is inversely correlated with the gene expression of eight epigenetic targets, and a systematic review of the literature confirmed that four of them have already been identified as targets for the treatment of NSCLC. Using nontoxic doses of the remaining inhibitors, KDM6B and PADI4 were identified as potential targets affecting the invasion and migration of metastatic lung cancer cell lines. Transcriptomic analysis of KDM6B and PADI4 treated cells showed altered expression of important genes related to the metastatic process. In conclusion, we showed that KDM6B and PADI4 are promising targets for inhibiting the metastasis of lung adenocarcinoma cancer cells.

PADI4 Haplotypes Contribute to mRNA Expression, the Enzymatic Activity of Peptidyl Arginine Deaminase and Rheumatoid Arthritis Risk in Patients from Western Mexico.

Citrullination is catalyzed by the peptidyl arginine deiminase 4 (PAD4) enzyme, encoded by the PADI4 gene. Increased PAD4 activity promotes the onset and progression of rheumatoid arthritis (RA). This study aimed to evaluate the association of PADI4 haplotypes with RA risk, mRNA expression, and the PAD4 activity in patients with RA from Mexico. Methodology: 100 RA patients and 100 control subjects (CS) were included. Genotyping was performed by PCR-RFLP method, PADI4 mRNA expression was quantified by real-time PCR, the contribution of PADI4 alleles (PADI4_89 G>A, PADI4_90 T>C, and PADI4_92 G>C) to mRNA expression by the ASTQ method, and PAD4 activity by HPLC. Also, the anti-CCP and anti-PADI4 antibodies were quantified by ELISA. Results: The three PADI4 polymorphisms were associated with RA susceptibility (OR = 1.72, p = 0.005; OR = 1.62; p = 0.014; OR = 1.69; p = 0.009; respectively). The 89G, 90T, and 92G alleles have a higher relative contribution to PADI4 mRNA expression from RA patients than 89A, 90C, and 92C alleles in RA patients. Moreover, the GTG/GTG haplotype was associated with RA susceptibility (OR = 2.86; p = 0.024). The GTG haplotype was associated with higher PADI4 mRNA expression (p = 0.04) and higher PAD4 enzymatic activity (p = 0.007) in RA patients. Conclusions: The evaluated polymorphisms contribute to PADI4 mRNA expression and the enzymatic activity of PAD4 in leukocytes. Therefore, the GTG haplotype is a genetic risk factor for RA in western Mexico, and is associated with increased PADI4 mRNA expression and higher PAD4 activity in these patients.

Human Enzyme PADI4 Binds to the Nuclear Carrier Importin α3.

PADI4 is a peptidyl-arginine deiminase (PADI) involved in the conversion of arginine to citrulline. PADI4 is present in macrophages, monocytes, granulocytes, and several cancer cells. It is the only PADI family member observed within both the nucleus and the cytoplasm. PADI4 has a predicted nuclear localization sequence (NLS) comprising residues Pro56 to Ser83, to allow for nuclear translocation. Recent predictors also suggest that the region Arg495 to Ile526 is a possible NLS. To understand how PADI4 is involved in cancer, we studied the ability of intact PADI4 to bind importin α3 (Impα3), a nuclear transport factor that plays tumor-promoting roles in several cancers, and its truncated species (ΔImpα3) without the importin-binding domain (IBB), by using fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). Furthermore, the binding of two peptides, encompassing the first and the second NLS regions, was also studied using the same methods and molecular docking simulations. PADI4 interacted with both importin species, with affinity constants of ~1-5 µM. The isolated peptides also interacted with both importins. The molecular simulations predict that the anchoring of both peptides takes place in the major binding site of Impα3 for the NLS of cargo proteins. These findings suggest that both NLS regions were essentially responsible for the binding of PADI4 to the two importin species. Our data are discussed within the framework of a cell mechanism of nuclear transport that is crucial in cancer.

Anti-PADI4 antibody suppresses breast cancer by repressing the citrullinated fibronectin in the tumor microenvironment.

BACKGROUND: PADI4, an enzyme catalyzing arginine residues to citrulline residues, is highly expressed in malignant tumors. This study prepared a monoclonal anti-human PADI4 antibody and investigated the therapeutic effect of the antibody on breast tumors and the functional mechanism. METHODS: After treatment with PADI4 antibody, the changes in tumor-bearing mice were examined by PET-CT, pathological assays, biochemical tests, routine blood tests, cytokine assays and metabolic assays. We used PADI4 recombinant protein to catalyze fibronectin (Fn) and then used citrullinated fibronectin (Cit-Fn) to culture MDA-MB-231 cells. We also treated Cit-Fn cultured cells with PADI4 antibody. The cultured cells were examined using cell proliferation, apoptosis, colony formation, migration and glycolic ATP production. Citrullination in the tumor tissues and peripheral blood was measured using Western blotting and ELISA, respectively. RESULTS: Following PADI4 antibody treatment, tumor growth was significantly suppressed, and the number of apoptotic cells in tumor tissues was increased. The citrullination level in peripheral blood and tumor tissues was decreased, EMT-related gene expression in tumors was also decreased, and the spontaneous movement of tumor-bearing mice was increased following treatment. Following antibody treatment, the serum concentrations of IL-10, IL-12p70, IL-23, ALT and AST were significantly decreased. MDA-MB-231 cells treated with Cit-Fn showed increased cell proliferation, cell migration, colony formation and glycolytic ATP production and decreased apoptosis. The growth and migration of MDA-MB-231 cells were reduced following PADI4 antibody treatment, and PADI4 antibody inhibited the citrullination of fibronectin in vitro. CONCLUSIONS: The PADI4 antibody had a therapeutic effect on breast tumors by inhibiting the citrullination of fibronectin to change the tumor tissue microenvironment. PADI4 antibody is a potential means for tumor treatment.

Berberine regulates PADI4-related macrophage function to prevent lung cancer.

Coptis chinensis Franch (CCF) has been widely used by Chinese old herbalist doctor to treat internal and external diseases including malignant sore and cancer. Berberine (BBR) is a major bioactive compound in CCF and may exert anti-tumor and anti-inflammatory effects like CCF. However, the prevention effect of berberine against lung cancer and its relevance of anti-inflammation property to cancer-preventing effect are still obscure. Protein arginine deaminase 4 (PAD4) played an important role in macrophage related inflammatory response, the purpose of this study was to identify whether berberine can prevent lung cancer and explore its effect on PADI4-related macrophage function. In vitro, PADI4 overexpression affects cell-activated state in macrophages. PADI4 overexpressed macrophages promote epithelial-mesenchymal transition (EMT) of A549 lung cancer cells and inhibit cell apoptosis. Berberine at the experiment dose had no effect on cell viability of U937-derived macrophages, but could significantly inhibit PADI4 expression to reverse the macrophage-activated state and the lung cancer -promoting effect of PADI4-overexpressed macrophages. Unlike GSK484, berberine had a little effect on the PADI4 citrullination activity at the experimental doses, its IC50 for PADI4 inhibition is 45.07 µM (44.03-46.12 µM). In the mouse lung carcinogenetic model, PADI4 expression was directly related to the number of lung nodules. Berberine had the similar role to GSK484 in reducing the number of lung tumor nodules with the improved lung pathology in a dose-dependent manner and significantly inhibited PADI4 expression. Further, we found that PADI4 overexpression could inhibit IRF5 expression, up-regulate CD163 and CD206 and down-regulate CD86 in macrophages, which could be reversed by berberine. Our results suggest that berberine may regulate PADI4-related macrophage function to prevent lung cancer.

Intrinsic function of the peptidylarginine deiminase PADI4 is dispensable for normal haematopoiesis.

Peptidylarginine deiminases (PADIs) are strongly associated with the development of autoimmunity, neurodegeneration and cancer but their physiological roles are ill-defined. The nuclear deiminase PADI4 regulates pluripotency in the mammalian pre-implantation embryo but its function in tissue development is unknown. PADI4 is primarily expressed in the bone marrow, as part of a self-renewal-associated gene signature. It has been shown to regulate the proliferation of multipotent haematopoietic progenitors and proposed to impact on the differentiation of haematopoietic stem cells (HSCs), suggesting that it controls haematopoietic development or regeneration. Using conditional in vivo models of steady state and acute Padi4 ablation, we examined the role of PADI4 in the development and function of the haematopoietic system. We found that PADI4 loss does not significantly affect HSC self-renewal or differentiation potential upon injury or serial transplantation, nor does it lead to HSC exhaustion or premature ageing. Thus PADI4 is dispensable for cell-autonomous HSC maintenance, differentiation and haematopoietic regeneration. This work represents the first study of PADI4 in tissue development and indicates that pharmacological PADI4 inhibition may be tolerated without adverse effects.