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Peptidyl arginine deiminases (PAD) are proteins that modify arginine residues to citrulline. In humans there are five isoforms PAD1-4 and PAD6. PAD4 is involved in several human diseases such as Alzheimer, breast cancer, rheumatoid arthritis and therefore has been recognized as a potential pharmacological target. In this work, 3175 molecules retrieved from ZINC12 Food and Drug Administration (FDA) catalog database were assayed through a structure-based virtual screening to identify potential PAD4 binders. Three were confirmed through enzyme inhibition studies. Flow cytometry analysis were used to check for drug cytotoxic effects on blood cells from three healthy donors. Molecular docking in a wild-type PAD4 structure showed the top 20 FDA molecules with the best binding energies. Those molecules were distributed in clusters of mixed binding energies but with similar chemical structures. These patterns along with the FDA information were used to aid in the rational selection of methotrexate, testosterone and leucovorin compounds for in vitro evaluations. Time course fluorescent enzyme kinetics confirmed that methotrexate (0.01–1 mmol) and testosterone enanthate (49.9–12.5 mmol) inhibit PAD4 at similar levels to the known inhibitor BB-Cl-amidine (8.8 mmol), except leucovorin. The chosen concentrations lack cytotoxic effects on blood cells. These findings encourage repositioning campaigns in the quest of PAD4 inhibitors. [ABSTRACT FROM AUTHOR]

This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted NET release, while NADPH oxidase and mitochondrial reactive oxygen species only played a role in PMA-induced NETs. Neutrophil elastase inhibition modestly affected PAF-induced NET liberation but not in PMA or A23187-induced NET, while myeloperoxidase did not contribute to NET release. We expect to provide a foundation for future investigations into the role of NETs in equine health and disease and the search for potential therapeutic targets. • PMA, A23187, and PAF induce extracellular trap release in equine neutrophils. • Reactive oxygen species are only necessary for extracellular traps induced by PMA. • Neutrophil elastase is only relevant for PAF-induced extracellular traps. • Myeloperoxidase is not required for neutrophil extracellular trap release. • Peptidyl-arginine deiminase inhibition halted neutrophil extracellular trap release. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests a beneficial role of rhizobacteria in ameliorating plant disease resistance in an environment-friendly way. In this study, we characterize a rhizobacterium, Bacillus cereus NJ01, that enhances bacterial pathogen resistance in rice and Arabidopsis. Transcriptome analyses show that root inoculation of NJ01 induces the expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes in Arabidopsis leaves. Genetic evidence showed that EDS1 , PAD4 , and WRKY18 are required for B. cereus NJ01-induced bacterial resistance. An EDS1-PAD4 complex interacts with WRKY18 and enhances its DNA binding activity. WRKY18 directly binds to the W box in the promoter region of the SA biosynthesis gene ICS1 and ABA biosynthesis genes NCED3 and NCED5 and contributes to the NJ01-induced bacterial resistance. Taken together, our findings indicate a role of the EDS1/PAD4-WRKY18 complex in rhizobacteria-induced disease resistance. [Display omitted] • Rhizobacterium Bacillus cereus NJ01 enhances plant resistance against bacterial pathogens • EDS1 enhances WRKY18 DNA-binding activity for the NJ01-enhanced disease resistance • ICS1 and NCED3/5 are downstream of EDS1-WRKY18 for NJ01-enhanced disease resistance The plant microbiome helps to shape the immune system to counter infection of pathogens. Wang et al. show that the root-associated Bacillus cereus NJ01 significantly enhances plant resistance. The EDS1-WKRY18 module is required for NJ01-enhanced disease resistance through activation of SA- and ABA-mediated immunity. [ABSTRACT FROM AUTHOR]

Recently, specific interaction of anthrax protective antigen domain 4 (PAD4) and lethal factor domain 1 (LFD1) have been considered for the design of novel diagnostic and therapeutic systems in medicine. In this study, theoretical and experimental approaches were used to monitor the interactions of PAD4 and LFD1. CLusPro server and Dimplot software were used to predict the interaction of these domains. Results, revealed interactive sites between PAD4 and LFD1 on loop regions of both C and N terminal of PAD4. In experimental methods, PAD4 and LFD1 were expressed in Escherichia coli and purified for usage in Magnetic Bead (MB) and Multi-Walled Carbon Nanotubes (MWCNTs) based bio-sensing platforms. In the magnetic-based system, the magnetic sedimentation of QD-PAD4 by MBs-LFD1 and the observation of the fluorescence spectrum related to QD-PAD4 in the precipitated materials confirmed the interaction of PAD4 with LFD1 protein. In the MWCNTs-based method, the QD-PAD4 fluorescence was quenched by absorption on MWCNTs. Upon the addition of LFD1, fluorescence emission was recovered, indicating interaction of LFD1 with QD-PAD4, which results the separation of QD-PAD4 from MWCNTs surfaces and fluorescence restoration. Finally, new approaches showed the interaction of PAD4 and LFD1, which can be used as an attractive model in medicine. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages (TAMs) shape tumor immunity and therapeutic efficacy. However, it is poorly understood whether and how post-translational modifications (PTMs) intrinsically affect the phenotype and function of TAMs. Here, we reveal that peptidylarginine deiminase 4 (PAD4) exhibits the highest expression among common PTM enzymes in TAMs and negatively correlates with the clinical response to immune checkpoint blockade. Genetic and pharmacological inhibition of PAD4 in macrophages prevents tumor progression in tumor-bearing mouse models, accompanied by an increase in macrophage major histocompatibility complex (MHC) class II expression and T cell effector function. Mechanistically, PAD4 citrullinates STAT1 at arginine 121, thereby promoting the interaction between STAT1 and protein inhibitor of activated STAT1 (PIAS1), and the loss of PAD4 abolishes this interaction, ablating the inhibitory role of PIAS1 in the expression of MHC class II machinery in macrophages and enhancing T cell activation. Thus, the PAD4-STAT1-PIAS1 axis is an immune restriction mechanism in macrophages and may serve as a cancer immunotherapy target. [Display omitted] • PAD4 is among the most active PTM enzymes in TAMs • Loss of PAD4 enhances MHC class II and anti-tumor immunity • PAD4 citrullinates STAT1, facilitating the STAT1-PIAS1 interaction • STAT1 citrullination negatively correlates with IFNγ signaling and response to ICB Pitter et al. demonstrate that the PAD4-mediated citrullination of STAT1 in macrophages enforces the STAT1-PIAS1 interaction restraining STAT1 transcriptional activity and MHC class II machinery expression and, consequently, limits T cell-mediated anti-tumor immunity. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) airway disease is characterized by the long-term presence of neutrophil granulocytes. Formation of neutrophil extracellular traps (NETs) and/or autoantibodies directed against extracellular components of NETs are possible contributors to neutrophil-mediated lung damage in CF. The goal of this study was to measure their levels in CF adults compared to healthy controls and subjects with rheumatologic diseases known to develop NET-related autoantibodies and pathologies, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Sera were analyzed from the following number of subjects: 37 CF, 23 healthy controls (HC), 20 RA, and 21 SLE. CF had elevated serum myeloperoxidase (MPO) concentrations (347.5±56.1 ng/ml, mean+/-S.E.M., p =.0132) compared to HC (144.5±14.6 ng/ml) but not of neutrophil elastase (NE) complexed with alpha-1-antitrypsin, cell-free DNA or NE-DNA complexes. The peptidylarginine deiminase 4 (PAD4) enzyme is required for NET formation and associated DNA release in neutrophils. Serum levels of anti-PAD4 antibodies (Ab) were elevated in CF (p =.0147) compared to HC and showed an inverse correlation with a measure of lung function, FEV1% predicted (r = −0.5020, p =.015), as did MPO levels (r = −0.4801, p =.0026). Anti-PAD4 Ab levels in CF sera associated with lung infection by P. aeruginosa, but not that by S. aureus, age, sex, CF-related diabetes or the presence of musculoskeletal pain. Serum levels of anti-citrullinated protein Abs (ACPAs) and anti-nucleosome Abs were not elevated in CF compared to HC (p =.7498, p =.0678). In summary, adult CF subjects develop an autoimmune response against NET components that correlates with worsening lung disease. • CF serum contains elevated levels of anti-PAD4 antibodies • Anti-PAD4 antibody levels in CF serum correlate with lung disease • Anti-PAD4 antibody levels in CF are associated with Pseudomonas aeruginosa lung infection • Levels of NETs, cell-free DNA, anti-nucleosome antibodies or ACPAs are not elevated in CF • Pattern of autoantibodies in CF is different from that of RA or SLE, two diseases with autoantibody-linked pathogeneses [ABSTRACT FROM AUTHOR]

Protein arginine deiminase IV (PAD4) is a potential target for diseases including rheumatoid arthritis and cancers. Currently, GSK199 is a potent, selective yet reversible PAD4 inhibitor. Its derivative, GSK106, on the other hand, was reported as an inactive compound when tested against PAD4 assay. Although they had similar skeleton, their impact towards PAD4 structural and flexibility is unknown. In order to fill the research gap, the impact of GSK199 and GSK106 binding towards PAD4 stability and flexibility is investigated via a combination of computational methods. Molecular docking indicates that GSK199 and GSK106 are capable to bind at PAD4 pocket by using its back door with −10.6 kcal/mol and −9.6 kcal/mol, respectively. The simulations of both complexes were stable throughout 100 ns. The structure of PAD4 exhibited a tighter packing in the presence of GSK106 compared to GSK199. The RMSF analysis demonstrates significant changes between the PAD4-GSK199 and PAD4-GSK106 simulations in the regions containing residues 136, 160, 220, 438, and 606. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis shows a marked difference in binding free energies, with −11.339 kcal/mol for the PAD4-GSK199 complex and 1.063 kcal/mol for the PAD4-GSK106 complex. The hydrogen bond analysis revealed that the GSK199 and GSK106 binding to PAD4 are assisted by six hydrogen bonds and three hydrogen bonds, respectively. The visualisation of the MD simulations revealed that GSK199 remained in the PAD4 pocket, whereas GSK106 shifted away from the catalytic site. Meanwhile, molecular dockings of benzoyl arginine amide (BAEE) substrate have shown that BAEE is able to bind to PAD4 catalytic site when GSK106 was present but not when GSK199 occupied the site. Overall, combination of computational approaches successfully described the behaviour of binding pocket of PAD4 structure in the presence of the active and inactive compounds. [Display omitted] • The refined PAD4-GSK199 is geometrically similar to the crystal structure of 4X8G. • Three additional residues (Ala581, Asn585, and Cys645) assisting GSK199 binding towards PAD4 through hydrogen bonds. • No contact and binding free energy from the catalytic residues in PAD4-GSK106 simulation, causing the compound to shift away. • BAEE substrate has higher probability of binding to PAD4 when GSK106 is present than GSK199 according to molecular docking. • The highlighted PAD4 pocket residues offer an opportunity for non-covalent modulation, especially when PAD4 is inactive. [ABSTRACT FROM AUTHOR]

Résumé La NETose suicidaire diffère des autres morts cellulaires par l’expulsion d’ADN nucléaire mélangé à des protéines préalablement citrullinées par l’arginine déiminase 4 des neutrophiles. Ces « filets » (NETs) sont sertis de protéines des granules cytoplasmiques à activité bactéricide. Lors de la NETose vitale, le neutrophile anucléé survit à cette expulsion et garde une capacité à migrer et à être infecté par des bactéries vivantes. Lors des NEToses mitochondriales, seul les enzymes et de l’ADN mitochondrial oxydés sont éjectés. On retrouve au sein des NETs, les cibles de la plupart des auto-anticorps des polyarthrites rhumatoïdes (PR), lupus et vascularites. Les chevauchements cliniques et biologiques entre bronchectasies, PR, lupus et vascularites, désignent la NETose comme un cofacteur inducteur probable de ces ruptures de tolérance. La NETose accroît la probabilité d’une association entre ces auto-antigènes et des antigènes infectieux au sein des biofilms, et d’une réaction auto-immune en cas de défaut de clairance de l’ensemble. La NETose aggrave le risque de lésions endothéliales et de thrombose. Cependant, la pathogénie des PR, lupus et vascularites ne se résume pas à ces auto-anticorps contre les NETs, et d’autres stimuli que les NETs pourraient aussi contribuer à leur induction, notamment d’autres circonstances d’hypercitrullination. La contribution d’infections intracellulaires à la rupture de tolérance aux NETs, pourrait être plus étudiée, comme les infections « dormantes » qui favorisent aussi la citrullination pour entretenir leur dormance. La seule inhibition de la NETose pourrait en effet ne pas suffire à stopper les pathologies dysimmunitaires si des infections latentes persistent, dont au sein des neutrophiles. [ABSTRACT FROM AUTHOR]

Suicidal NETosis differs from other mechanisms of cell death by the release of a lattice, composed of DNA associated with proteins citrullinated by protein-arginine deiminase 4, from neutrophils. These 'NETs' are composed of granule-derived proteins with microbicidal activity. A similar type of release occurs during vital NETosis, in which anuclear neutrophils maintain their chemotactic ability and imprison live bacteria, even after NET extrusion. Mitochondrial NETosis is limited to the expulsion of oxidised mitochondrial DNA and cytoplasmic enzymes. NETs include the targets of most autoantibodies found in rheumatoid arthritis, lupus, and vasculitis. The clinical and biological overlaps sometimes observed between bronchectiasis and RA, RA and SLE, or SLE and vasculitis, implicate NETosis as a major triggering event common to these disorders. NETosis increases the possibility of association between autoantigens and infectious antigens in mucosal biofilms, impairing the clearance of pathogens and possibly triggering autoimmune reactions. NETosis aggravates these three conditions and increases endothelial damage and the risk of thrombosis. However, the pathogenesis of RA, SLE, and vasculitis is not confined to autoantibodies against NET components, and other mechanisms have been suggested to explain the breakdown of tolerance to NET autoantigens, such as hypercitrullination. The question of whether continuous presentation of autoantigens mixed with antigens from dormant intracellular pathogens (released following suicidal, vital, or mitochondrial NETosis) is required to induce and sustain autoimmunity must be addressed. Inhibiting NETois may not be sufficient to improve autoimmune disorders whereas such latent infections remain uncontrolled. [ABSTRACT FROM AUTHOR]

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) are intracellular immune receptors for pathogen recognition and signaling. They include sensor NLRs (sNLRs) that detect pathogens, and helper NLRs, which transduce downstream immune signals. During immune responses, both membrane-localized pattern recognition receptors (PRRs) and sNLRs rely on helper NLRs for signal transduction. The Arabidopsis helper NLRs, ADR1s and NRG1s, along with their interacting lipase-like protein dimers, are differentially required by sNLRs. Recent structural and biochemical analyses suggest that they assemble into oligomeric resistosomes with lipase-like protein dimers upon perception of small molecules produced from enzymatic activities of upstream TIR-type sNLRs. As a result, ADR1s and NRG1s form membrane calcium channels to induce immune responses and cell death. In contrast, Solanaceous NRC clade helper NLRs transduce signals from many sNLRs and some PRRs. Here, we summarize the recent advances in plant helper NLR research, with a focus on their structural and biochemical mechanisms in immune signaling. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer is associated with ulcerative colitis (UC). The infiltration of neutrophils is the main cause of DNA damage produced by inflammation in the intestinal epithelium. Under the action of peptidyl arginine deaminase 4 (PAD4), neutrophils dissociate chromatin and form neutrophil extracellular traps (NETs), which can aggravate tissue inflammation and encourage tumor development. Although Huang Qin Decoction (HQD) was found to be useful in treating UC and was used to gradually prevent and treat digestive tract cancers, the underlying reasons were unclear.Methods: To demonstrate HQD could inhibits the initiation of colitis associated carcinogenesis by controlling NETs related inflammation, we first performed an AOM/DSS-generated colitis-associated carcinogenesis model to assess the efficacy of HQD in reducing neutrophil infiltration and anti-tumor activity. Then, using network pharmacology research, we investigated the potential mechanisms underlying those medicinal effects, as demonstrated by the detection of NETs aggregation and PAD4 expression changes in the colon.Results: HQD substantially reduced the number of colon cancers and the expression of Ki67, restored the level of intestinal tight junction protein occludin and ZO-1, and relieved the intestinal inflammation caused by TNF-α, IL-1β. At the same time, it inhibited neutrophil infiltration in the colon and improved the immunosurveillance of CD8+T cells. The potential mechanisms of HQD intervention against UC and UC with neoplasia (UCN) were studied using network pharmacology, and 156 conjunct genes as well as numerous inflammation-related pathways were identified. Protein-protein interaction (PPI) analysis indicated that HQD inhibition of intestinal tumors might be related to the deactivation of PAD4, which was verified by the down-regulation of NETs, MPO-DNA complex levels, and PAD4 expression after HQD treatment.Conclusion: Huang Qin Decoction inhibits the initiation of colitis associated carcinogenesis by controlling PAD4-dependent neutrophil extracellular traps. [ABSTRACT FROM AUTHOR]

In neutrophils, caspase-11 cleaves gasdermin D (GSDMD), causing pyroptosis to clear cytosol-invasive bacteria. In contrast, caspase-1 also cleaves GSDMD but seems to not cause pyroptosis. Here, we show that this pyroptosis-resistant caspase-1 activation is specifically programmed by the site of translocation of the detected microbial virulence factors. We find that pyrin and NLRC4 agonists do not trigger pyroptosis in neutrophils when they access the cytosol from endosomal compartment. In contrast, when the same ligands penetrate through the plasma membrane, they cause pyroptosis. Consistently, pyrin detects extracellular Yersinia pseudotuberculosis ΔyopM in neutrophils, driving caspase-1-GSDMD pyroptosis. This pyroptotic response drives PAD4-dependent H3 citrullination and results in extrusion of neutrophil extracellular traps (NETs). Our data indicate that caspase-1, GSDMD, or PAD4 deficiency renders mice more susceptible to Y. pseudotuberculosis ΔyopM infection. Therefore, neutrophils induce pyroptosis in response to caspase-1-activating inflammasomes triggered by extracellular bacterial pathogens, but after they phagocytose pathogens, they are programmed to forego pyroptosis. [Display omitted] • Extracellular Yptb-ΔyopM drives caspase-1-GSDMD pyroptosis in neutrophils • Neutrophil inflammasomes discriminate plasma membrane from endosome membrane signals • Caspase-1-GSDMD pyroptosis induces PAD4-dependent H3 citrullination and NET extrusion • Pyroptosis-dependent NETs are critical to protect hosts against Yptb-ΔyopM infection Oh et al. demonstrate that neutrophil caspase-1 inflammasomes selectively drive pyroptosis in response to bacterial virulence factors that are translocated across the plasma membrane. Consequently, neutrophil caspase-1-GSDMD-driven pyroptosis promotes PAD4-mediated histone citrullination and NET extrusion to protect against Yptb-ΔyopM infection. [ABSTRACT FROM AUTHOR]

Peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme, catalyzes the conversion of arginine to citrulline and has been strongly associated with many malignant tumors. However, the molecular mechanisms of PAD4 in the development and progression of colorectal cancer (CRC) remain unclearly defined. In our study, PAD4 expression was increased in CRC tissues and cells, and was closely related to tumor size, lymph node metastasis. Moreover, the transcription factor KLF9 directly bound to PADI4 gene promoter, leading to overexpression of PAD4 in CRC cells, which augmented cell growth and migration. We revealed that PAD4 interacted with and citrullinated glycogen synthase kinase-3β (GSK3β) in CRC cells, and GSK3β Arg-344 was the dominating PAD4-citrullination site. Furthermore, IgL2 and catalytic domains of PAD4 directly bound to the kinase domain of GSK3β in CRC cells. Mechanistically, PAD4 promoted the transport of GSK3β from the cytoplasm to the nucleus, thereby increasing the ubiquitin-dependent proteasome degradation of nuclear cyclin-dependent kinase inhibitor 1 (CDKN1A). Our study is the first to reveal the details of a critical PAD4/GSK3β/CDKN1A signaling axis for CRC progression, and provides evidence that PAD4 is a potential diagnosis biomarker and therapeutic target in CRC. [ABSTRACT FROM AUTHOR]

Protein arginine deiminase 4 (PAD4) catalyzes the hydrolysis of a peptidylarginine residue to form a citrulline residue and ammonia during posttranslational modification. This process plays a pivotal role in rheumatoid arthritis (RA) and gene regulation. F-amidine belongs to a series of haloacetamidine compounds that are the most potent PAD4 inhibitors described to date. F-amidine acts as a mechanism-based inhibitor of PAD4, inactivating PAD4 by the covalent modification of the active site Cys645. In this manuscript, the fundamental mechanism of PAD4 inhibition by F-amidine is investigated using a QM/MM approach. Our simulations show that in the PAD4–F-amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated. This is consistent with the reverse protonation mechanism wherein the active site nucleophile, Cys645, in PAD4 exists as a thiolate in the active form of the enzyme. Inhibition of PAD4 by F-amidine is initiated by the nucleophilic addition of S γ to the C ζ of F-amidine, leading to the formation of a tetrahedral intermediate. His471 serves as a proton donor, helping F to leave the fluoroacetamidine moiety of F-amidine; meanwhile, S γ forms a three-membered ring with C ζ and C η of F-amidine. Subsequently, the three-membered sulfonium ring collapses and rearranges to the final thioether product. His471 acts as a proton donor in the transition state and facilitates the inhibition reaction of PAD4. [ABSTRACT FROM AUTHOR]

[Display omitted] Peptidylarginine deiminase-4 (PAD4) is a calcium-dependent enzyme that catalyzes the conversion of arginine into citrulline of macromolecules in the body. It governs several processes including apoptosis, innate immunity (Netosis), and pluripotency. Dysregulated PAD4 plays a vital role in the occurrence and development of Rheumatoid arthritis (RA). Therefore, PAD4 is considered a promising target for diagnosing and treating RA. Over the last few years research has been carried out on PAD4 inhibitors. When administered it circulates to the entire body and inhibits PAD4 causing immunosuppression which may lead to infection. A growing number of studies have demonstrated infiltration and differentiation of monocytes and macrophages into the inflamed synovium, inducing overexpression of PAD4 levels in the inflamed joints. To overcome the above-mentioned critical issues, the targeted drug delivery systems inhibit PAD4 at the inflamed site. This review provides an update on the PAD4 inhibitors and emerging advanced drug delivery systems for the treatment of RA. Finally, we concluded that active targeting of PAD4 inhibitors to inflamed joints via hybrid nanocarriers provided an improved therapeutic efficacy, minimized extra synovial toxicity, and prevent the occurrence of inflammation in RA. [ABSTRACT FROM AUTHOR]

Abstract: The Arabidopsis cad1 (constitutively activated cell death 1) mutant shows a phenotype of hypersensitive response (HR)-like cell death and increased levels of endogenous salicylic acid (SA), indicating that the CAD1 protein negatively controls the SA-mediated pathway in plant immunity. To clarify the relationship between the CAD1 protein and SA-mediated defense pathways, a molecular genetic analysis was carried out using mutants related to SA biosynthesis. A genetic cross between the cad1 mutant and the pad4 and sid2 mutants only partially suppressed the cad1 phenotypes. These double mutants still showed acquired resistance to Pst DC3000 infection similar to the cad1 single mutant. These results indicate that the CAD1 negatively controls not only the SA-dependent defense pathway but also the SA-independent pathway. To clarify the CAD1-mediated SA-independent pathway, a revertant was isolated from an M2 population of the sid2 cad1 mutant. The revertant can1t mutant did not show cell death phenotype and the plant and leaf size recovered to those of the wild-type. The can1t mutant also showed pathogen susceptibility comparable to wild type, indicating that the can1t mutant looses the pathogen-resistance that the cad1 mutant acquires. There results suggested that CAN1 is a positive regulator of the novel plant immunity under the control of CAD1. [Copyright &y& Elsevier]

Abstract: Arabidopsis EDS1 and PAD4 are important regulators of plant defense signaling. We have constructed transgenic lines that constitutively overexpress tandem affinity purification (TAP)-tagged EDS1 and PAD4 in the respective eds1-1 and pad4-1 mutant backgrounds. Affinity purified EDS1 complexes contained not only SAG101 as recently reported but also PAD4. The PAD4 complexes, on the other hand, contained EDS1 but not SAG101. These results support that PAD4 and SAG101 interact with EDS1 but not with each other. To determine the effects of constitutive expression of EDS1 and PAD4 on plant disease resistance, we have compared these transgenic plants with the wild type and the respective eds1-1 and pad4-1 mutant plants for responses to the hemibiotrophic bacterial pathogen Pseudomonas syringae and necrotrophic fungal pathogen Botrytis cinerea. The eds1 and pad4 mutants were highly susceptible to virulent strains of P. syringae and constitutive overexpression of EDS1 and PAD4 enhanced resistance to the bacterial pathogen in a dosage-dependent manner. Enhanced resistance to the bacterial pathogen in transgenic EDS1- and PAD4-overexpressing lines was associated with a quicker and stronger induction of salicylate-regulated PR1 after infection of the bacterial pathogen. By contrast, the eds1-1 and pad4-1 mutants were highly tolerant to B. cinerea and their constitutive overexpression restored the susceptibility of the mutants to the fungal pathogen. The negative role of EDS1 and PAD4 in plant response to the necrotrophic fungal pathogen correlated with their negative effects on induction of jasmonate-regulated PDF1.2gene expression. [Copyright &y& Elsevier]

The conserved lipase-like protein EDS1 transduces signals from pathogen-activated intracellular nucleotide-binding leucine-rich repeat (NLR) receptors to transcriptional defences and host cell death. In this pivotal NLR signalling role, EDS1 works as a heterodimer with each of its partners, SAG101 and PAD4. Different properties of EDS1-SAG101 and EDS1-PAD4 complexes and functional relationships to sensor and helper NLRs have emerged. EDS1-SAG101 dimers confer effector-triggered immunity mediated by intracellular TNL receptors. In contrast, EDS1-PAD4 dimers have a broader role promoting basal immune responses that can be initiated inside cells by TNL- or CNL-type NLRs, and at the cell surface by LRR-receptor proteins. Characterizing the essential elements of these two EDS1 modules will help to connect intracellular and surface receptor signalling networks in the plant immune system. [Display omitted] [ABSTRACT FROM AUTHOR]

• Protein arginine deiminase type 4 (PAD4) is identified as being responsible for pathogenesis of rheumatoid arthritis. • PAD4 converts the arginine residue to citrulline in certain proteins, which subsequently act as autoantigens that stimulate antibody production. • We discovered a peptide with a better inhibitory activity against PAD4 compared to existing PAD4 inhibitors such as MTX and streptomycin. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC 50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC 50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors. [ABSTRACT FROM AUTHOR]

• PD1 alleviates severity of acute pancreatitis in three experimental mouse models. • PD1 inhibits NETs formation by downregulating PAD4 expression in neutrophils. • PD1 may be a therapeutic candidate for acute pancreatitis in clinical. Docosahexaenoic acid-derived protectin D1 (PD1) was identified critical in the resolution of inflammation in vivo, where it modulates the innate immune response and stimulates resolution. Acute pancreatitis (AP) is characterized by local pancreatic inflammation with mild forms whereas systemic inflammation with severe forms. Herein we investigate the impact of PD1 in murine models of pancreatitis. Three independent AP models, which induced in male mice via intraperitoneal injection of caerulein, L-arginine or pancreatic duct ligation, were used to confirm the protective effect of PD1. Infiltrations of neutrophils and macrophages in pancreas were detected by flow cytometry and immunohistochemistry. In vitro and in vivo neutrophil extracellular traps formation was detected by immunofluorescence staining. Expression of peptidylarginine deiminase 4 (PAD4) in activated neutrophils was evaluated by western blotting. Systemic treatment with PD1 reduced serum activities of amylase and lipase, blunted the concentrations of tumor necrosis factor-α and interleukin-6 in serum and protected against pancreas histologic damage in three AP models. PD1 also prolonged the survival in the pancreatic duct ligation model. Moreover, pancreatic infiltration of neutrophils and neutrophil CitH3 expression were reduced after PD1 administration. In vitro studies revealed PD1 decreased supernatant cell-free DNA and CitH3 levels and downregulated PAD4 expression in mouse bone-marrow derived neutrophils. However, in the caerulein mice pretreated with GSK484 hydrochloride, an inhibitor of PAD4, PD1 treatment showed no more protective effect. PD1 ameliorates AP by decreasing early infiltration of neutrophils into the pancreas and neutrophil extracellular traps formation through PAD4. These results supply the foundation to consider PD1 as a therapy for AP. [ABSTRACT FROM AUTHOR]

[Display omitted] • The effect of autocitrullination was studied by in vitro and in silico approaches. • PAD4 and citrullinated PAD4 show comparable activity toward different substrates. • Autocitrullination does not appear to play a role in the regulation of PAD4 activity. Protein arginine deiminase 4 (PAD4) is an enzyme capable of converting arginine (positively charged residue) into citrulline (neutral residue). PAD4 is a promiscuous enzyme since it citrullinates various substrates, including small peptides, large proteins and itself. The effect of autocitrullination on PAD4 activity remains controversial and inconclusive. We hypothesized that PAD4 autocitrullination may influence the activity of PAD4 by indirectly altering its binding to substrate. We employed mass spectrometry analysis to study the process of autocitrullination. The kinetics of citrullination of PAD4 and citrullinated PAD4 (citPAD4) towards substrates of different sizes (0.17–15.4 kDa), i.e. free arginine, a peptidyl substrate, and histone H3, were studied by colorimetric assay and Western blotting. Molecular dynamics (MD) simulations were performed to investigate structural dynamic and binding properties of PAD4/citPAD4 in the absence and presence of substrates. We observed that 23/27 arginine residues in PAD4 (85 %) can be citrullinated, including R372, R374 and R639 located near the substrate binding pocket. PAD4 and citPAD4 expressed comparable enzymatic activities towards different substrates. In agreement with experimental results, MD simulations indicated that autocitrullination does not change the shape of the substrate binding pocket and PAD4/citPAD4 exhibited comparable binding free energy with a H3-derived peptidyl substrate (6-TARKS-10). While the effect of autocitrullination on PAD4 activity thus far remained unclear and controversial, here we have demonstrated that autocitrullination does not affect the activity of PAD4. Thus, the regulation of PAD4 activity is probably not controlled by autocitrullination but likely by other mechanisms that need further investigation. [ABSTRACT FROM AUTHOR]

Abstract: Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes cartilage and bone destruction. The mechanisms leading to RA are unknown. There is currently no reliable cure for RA. Early treatment can reduce inflammation, joint damage and bone destruction. Thus, early diagnosis of RA is critical. However, definitive diagnosis of RA can be difficult. Immunologic tests that can be performed for the diagnosis of RA include detection of anti citrullinated protein antibodies (ACPAs). However, one third of RA patients have no ACPAs. To identify new autoantibodies in RA, we used the sera of RA patients to screen protein arrays containing 8000 human proteins. We found and validated two major autoantigens: PAD4 (peptidyl arginine deiminase 4) and BRAF (v raf murine sarcoma viral oncogene homolog B1) catalytic domain. We identified peptide targets of anti PAD4 and BRAF autoantibodies. We observed that anti PAD4 are inhibitory whereas anti BRAF stimulate BRAF activity. Anti PAD4 and anti BRAF antibodies may be used to diagnose RA, particularly in the absence of anti citrullinated protein antibodies. [Copyright &y& Elsevier]

Neutrophil extracellular traps (NETs) are incriminated in several immune and inflammatory diseases including ulcerative colitis (UC). Analysis of colonic tissues for NETs-related markers in UC carries prognostic and therapeutic implications. This work aims to evaluate the immunohistochemical (IHC) expression of NETs-associated-protein arginine deaminase 4 (PAD4) in colonic biopsies from UC patients in comparison to normal colon (NC). Association between PAD4 expression level and histopathologic grade, patient's therapeutic response and other clinicopathological prognostic predictors in UC are determined. This cohort study included biopsies from 42 UC patients and 11 NC controls. Clinicopathological data including patient's age at diagnosis, gender, presenting symptoms, anatomical disease extent, extra-intestinal manifestations, type and response to therapy and surgical interventions were recorded and tabulated. Histopathological grading of disease activity and associated epithelial changes were assessed. PAD4 immunostaining was conducted using Horseradish Peroxidase technique and scored semiquantitatively considering intensity and percentage of nuclear staining of lamina propria inflammatory cells. Appropriate statistical tests were applied. Anti-PAD4 was localized mainly in the nuclei of lamina propria infiltrating neutrophils. It was expressed more significantly in UC (95.2 %) compared to NC (p 0.001). Increased PAD4 expression level was significantly associated with increasing histopathologic grade, anatomical disease extent, lacking response to therapy and subjection to radical surgery (p:0.001, = 0.038, 0.046, 0.046 respectively). Age, gender, presenting symptoms, extra-intestinal manifestations and epithelial changes showed insignificant associations. This study characterizes a subset of UC patients with high histopathological grade of activity, pancolonic involvement, strong/moderate PAD4 expression levels and who are unresponsive to routine medical therapeutic regimens rendering them candidates for radical surgery. In conjunction with histopathological grading, IHC evaluation of PAD4 in UC is recommended to guide patient's selection for targeted therapy using the novel-discovered selective PAD4 inhibitors. [ABSTRACT FROM AUTHOR]