RESUMO
Anti-citrullinated protein autoantibodies (ACPA) are diagnostic for rheumatoid arthritis (RA). The antigens recognized by these autoantibodies are produced by protein arginine deiminases (PADs), particularly PAD4. However, it remains unknown why and how PAD4 causes this aberrant citrullination in RA. Here, we report that poly-perforin pores are present on freshly isolated neutrophils from RA patients, but not on healthy donor neutrophils. Neutrophils with perforin pores also contained intracellular citrullinated proteins in the region adjacent to the pores. This response was replicated in vitro by treating neutrophils with purified perforin, which generated intense dots of anti-perforin immunofluorescence, calcium influx, and intracellular citrullination. Extensive neutrophil killing in Felty's syndrome, an aggressive form of RA, correlated with particularly high ACPA, and PAD4 autoantibodies. In contrast, other forms of death, including NETosis, apoptosis, and pyroptosis, produced minimal citrullination. We conclude that neutrophil targeting by perforin leading to intracellular citrullination takes place in patients with RA.
Assuntos
Anticorpos Antiproteína Citrulinada , Artrite Reumatoide , Citrulinação , Neutrófilos , Perforina , Proteína-Arginina Desiminase do Tipo 4 , Humanos , Neutrófilos/metabolismo , Neutrófilos/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/imunologia , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Anticorpos Antiproteína Citrulinada/metabolismo , Anticorpos Antiproteína Citrulinada/imunologia , Perforina/metabolismo , Feminino , Masculino , Pessoa de Meia-Idade , Autoanticorpos/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Adulto , Síndrome de Felty/metabolismo , Síndrome de Felty/patologia , Armadilhas Extracelulares/metabolismo , Citrulina/metabolismo , IdosoRESUMO
PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.
Assuntos
Citrulinação , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Proteína-Arginina Desiminase do Tipo 4/química , Humanos , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/química , Conformação Proteica , Peptídeos/química , Peptídeos/metabolismo , Citrulina/química , Citrulina/metabolismo , Ligação Proteica , Sequência de AminoácidosRESUMO
Polycomb groups (PcGs) are transcriptional repressors, formed by a complex of several proteins, involved in multicellular development and cancer epigenetics. One of these proteins is the E3 ubiquitin-protein ligase RING1 (or RING1B), associated with the regulation of transcriptional repression and responsible for monoubiquitylation of the histone H2A. On the other hand, PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline, and it is also involved in the development of glioblastoma, among other types of cancers. In this work, we showed the association of PADI4 and RING1B in the nucleus and cytosol in several cancer cell lines by using immunofluorescence and proximity ligation assays. Furthermore, we demonstrated that binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that RING1B could bind to the active site of PADI4, as confirmed by protein-protein docking simulations. In vitro and in silico findings showed that binding to PADI4 occurred for the isolated fragments corresponding to both the N-terminal (residues 1-221) and C-terminal (residues 228-336) regions of RING1B. Binding to PADI4 was also hampered by GSK484, as shown by isothermal titration calorimetry (ITC) experiments for the sole N-terminal region, and by both NMR and ITC for the C-terminal one. The dissociation constants between PADI4 and any of the two isolated RING1B fragments were in the low micromolar range (~2-10 µM), as measured by fluorescence and ITC. The interaction between RING1B and PADI4 might imply citrullination of the former, leading to several biological consequences, as well as being of potential therapeutic relevance for improving cancer treatment with the generation of new antigens.
Assuntos
Ligação Proteica , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Humanos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citrulinação , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/genética , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/genéticaRESUMO
During investigating the role of peptidylarginine deiminase (PAD) enzymes in dilated cardiomyopathy (DCM), we observed unique spheroid formation in DCM-myofibroblasts that distinguished them from normal cardiac myofibroblasts. The present study aimed to assess the presence of PADs, the extracellular matrix (ECM), and citrullination in DCM spheroids using immunofluorescence staining and imaging techniques. The results revealed that spheroids derived from DCM-myofibroblasts displayed a more distinctive, tightly packed structure compared with those derived from human cardiac fibroblasts. DCM spheroids showed abundant protein expression of the PAD 2, 3, and 4 enzymes. Notably, increased Ki67 protein expression was associated with increased proliferation in DCM spheroids. Cytoskeletal proteins such as Col-1A, vimentin, α-SMA, and F-actin were highly abundant in DCM spheroids. Furthermore, DCM spheroids contained citrullinated cytoskeletal proteins, mainly citrullinated vimentin and citrullinated fibronectin. These observations supported the occurrence of PAD-mediated citrullination of ECM proteins in DCM spheroids. Collectively, these findings describe the distinctive features of DCM spheroids, representing the cellular characteristics of DCM myofibroblasts. Therefore, DCM spheroids can serve as an in vitro model for further investigations of disease morphology and therapeutic efficacy.
Assuntos
Citrulinação , Proteínas do Citoesqueleto , Miofibroblastos , Desiminases de Arginina em Proteínas , Humanos , Miofibroblastos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/análise , Desiminases de Arginina em Proteínas/metabolismo , Esferoides Celulares/metabolismo , Hidrolases/metabolismo , Células CultivadasRESUMO
In chronic obstructive pulmonary disease (COPD), inflammation gives rise to protease-mediated degradation of the key extracellular matrix protein, elastin, which causes irreversible loss of pulmonary function. Intervention against proteolysis has met with limited success in COPD, due in part to our incomplete understanding of the mechanisms that underlie disease pathogenesis. Peptidyl arginine deiminase (PAD) enzymes are a known modifier of proteolytic susceptibility, but their involvement in COPD in the lungs of affected individuals is underexplored. In this study, we showed that enzyme isotypes PAD2 and PAD4 are present in primary granules of neutrophils and that cells from people with COPD release increased levels of PADs when compared with neutrophils of healthy control subjects. By examining bronchoalveolar lavage and lung tissue samples of patients with COPD or matched smoking and nonsmoking counterparts with normal lung function, we reveal that COPD presents with markedly increased airway concentrations of PADs. Ex vivo, we established citrullinated elastin in the peripheral airways of people with COPD, and in vitro, elastin citrullination significantly enhanced its proteolytic degradation by serine and matrix metalloproteinases, including neutrophil elastase and matrix metalloprotease-12, respectively. These results provide a mechanism by which neutrophil-released PADs affect lung function decline, indicating promise for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.
Assuntos
Elastina , Neutrófilos , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4 , Proteólise , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Neutrófilos/imunologia , Elastina/metabolismo , Feminino , Masculino , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Pessoa de Meia-Idade , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Enfisema Pulmonar/imunologia , Idoso , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Citrulinação , Desiminases de Arginina em Proteínas/metabolismo , Elastase de Leucócito/metabolismo , Pulmão/imunologia , Pulmão/patologiaRESUMO
The human microbiome exists throughout the body, and it is essential for maintaining various physiological processes, including immunity, and dysbiotic events, which are associated with autoimmunity. Peptidylarginine deiminase (PAD) enzymes can citrullinate self-proteins related to rheumatoid arthritis (RA) that induce the production of anti-citrullinated protein antibodies (ACPAs) and lead to inflammation and joint damage. The present investigation was carried out to demonstrate the expression of homologs of PADs or arginine deiminases (ADs) and citrullinated proteins in members of the human microbiota. To achieve the objective, we used 17 microbial strains and specific polyclonal antibodies (pAbs) of the synthetic peptide derived from residues 100-200 of human PAD2 (anti-PAD2 pAb), and the recombinant fragment of amino acids 326 and 611 of human PAD4 (anti-PAD4 pAb), a human anti-citrulline pAb, and affinity ACPAs of an RA patient. Western blot (WB), enzyme-linked immunosorbent assay (ELISA), elution, and a test with Griess reagent were used. This is a cross-sectional case-control study on patients diagnosed with RA and control subjects. Inferential statistics were applied using the non-parametric Kruskal-Wallis test and Mann-Whitney U test generated in the SPSS program. Some members of phyla Firmicutes and Proteobacteria harbor homologs of PADs/ADs and citrullinated antigens that are reactive to the ACPAs of RA patients. Microbial citrullinome and homolog enzymes of PADs/ADs are extensive in the human microbiome and are involved in the production of ACPAs. Our findings suggest a molecular link between microorganisms of a dysbiotic microbiota and RA pathogenesis.
Assuntos
Anticorpos Antiproteína Citrulinada , Artrite Reumatoide , Citrulinação , Microbiota , Desiminases de Arginina em Proteínas , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anticorpos Antiproteína Citrulinada/imunologia , Anticorpos Antiproteína Citrulinada/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/microbiologia , Estudos de Casos e Controles , Citrulina/metabolismo , Estudos Transversais , Hidrolases/metabolismo , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/genéticaRESUMO
Rheumatoid arthritis (RA) is a chronic disease characterized by joint inflammation and severe disability. However, there is a lack of safe and effective drugs for treating RA. In our previous study, we discovered that myricetin (MC) and celecoxib have a synergistic effect in the treatment of RA. We conducted in vitro and in vivo experiments to further investigate the effects and mechanisms of action of MC. Our findings demonstrated that MC treatment effectively reduced the release of neutrophil extracellular traps (NETs) and alleviated the inflammatory response in RA. Mechanistic studies showed that MC prevents the entry of PADI4 and MPO into the cell nucleus, thereby protecting DNA from decondensation. In a rat arthritis model, MC improved histological changes in ankle joints and suppressed NET-related signaling factors. In conclusion, MC protects the ankle joints against arthritis by inhibiting MPO and PADI4, thereby reducing NET release. The pharmacological mechanism of MC in RA involves the inhibition of NET release.
Assuntos
Artrite Reumatoide , Modelos Animais de Doenças , Armadilhas Extracelulares , Flavonoides , Neutrófilos , Peroxidase , Proteína-Arginina Desiminase do Tipo 4 , Animais , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Artrite Reumatoide/tratamento farmacológico , Ratos , Flavonoides/farmacologia , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Humanos , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Artrite Experimental/tratamento farmacológico , Masculino , Ratos Sprague-Dawley , Celecoxib/farmacologia , Celecoxib/uso terapêutico , Células Cultivadas , Índice de Gravidade de DoençaRESUMO
Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.
Assuntos
Ácidos Aminossalicílicos , Artrite Reumatoide , Monócitos , Humanos , Desiminases de Arginina em Proteínas , Monócitos/metabolismo , Autoantígenos , Autoanticorpos , Fibrinogênio/metabolismo , Citrulina/metabolismoRESUMO
Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7â Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.
Assuntos
Domínio Catalítico , Proteína-Arginina Desiminase do Tipo 6 , Humanos , Cristalografia por Raios X , Proteína-Arginina Desiminase do Tipo 6/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/química , Desiminases de Arginina em Proteínas/genética , Conformação Proteica , Hidrolases/química , Hidrolases/metabolismo , Modelos Moleculares , Cálcio/metabolismoRESUMO
The post-translational modifications (PTMs) of proteins play a crucial role in increasing the functional diversity of proteins and are associated with the pathogenesis of various diseases. This review focuses on a less explored PTM called citrullination, which involves the conversion of arginine to citrulline. This process is catalyzed by peptidyl arginine deiminases (PADs). Different members of the PAD family have distinct tissue distribution patterns and functions. Citrullination is a post-translational modification of native proteins that can alter their structure and convert them into autoantigens; thus, it mediates the occurrence of autoimmune diseases. CD4+ T cells, including Th1, Th2, and Th17 cells, are important immune cells involved in mediating autoimmune diseases, allergic reactions, and tumor immunity. PADs can induce citrullination in CD4+ T cells, suggesting a role for citrullination in CD4+ T cell subset differentiation and function. Understanding the role of citrullination in CD4+ T cells may provide insights into immune-related diseases and inflammatory processes.
Assuntos
Linfócitos T CD4-Positivos , Citrulinação , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Citrulina/metabolismo , Arginina/metabolismoRESUMO
Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis.
Assuntos
Periodontite Crônica , Porphyromonas gingivalis , Humanos , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , Peptídeos , Periodonto/metabolismo , Periodontite Crônica/genéticaRESUMO
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.
Assuntos
Hidrolases , Processamento de Proteína Pós-Traducional , Camundongos , Animais , Desiminases de Arginina em Proteínas/metabolismo , Proteína-Arginina Desiminase do Tipo 4/genética , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Hidrolases/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Macrófagos/metabolismoRESUMO
Introduction: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens as well as which peptidylarginine deiminases (PADs) are required for their production remain incompletely defined. Here, we investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins bound by APCAs, and if their formation requires PAD2 or PAD4. Methods: Thioglycolate-induced peritoneal macrophages from wild-type, PAD2-/-, and PAD4-/- mice or human peripheral blood-derived M1 macrophages were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (citH4) antibody or sera from ACPA+ or ACPA- rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, and quantified. Results: We found that ionomycin and monosodium urate crystals reliably induced murine citH4+ METs, which were reduced in the absence of PAD2 and lost in the absence of PAD4. Also, IgG from ACPA+, but not ACPA-, rheumatoid arthritis sera bound to murine METs, and in the absence of PAD2 or PAD4, ACPA-bound METs were lost. Finally, ionomycin induced human METs that are citH4+ and ACPA-bound. Discussion: Thus, METs may contribute to the pool of citrullinated antigens bound by ACPAs in a PAD2- and PAD4-dependent manner, providing new insights into the targets of immune tolerance loss in rheumatoid arthritis.
Assuntos
Ácidos Aminossalicílicos , Artrite Reumatoide , Armadilhas Extracelulares , Humanos , Camundongos , Animais , Desiminases de Arginina em Proteínas/metabolismo , Autoanticorpos , Proteína-Arginina Desiminase do Tipo 4 , Ionomicina/metabolismo , Histonas/metabolismo , Macrófagos/metabolismoRESUMO
BACKGROUND: Peptidyl (protein) arginine deiminases (PADs) provide the transformation of peptidyl arginine to peptidyl citrulline in the presence of calcium with posttranslational modification. The dysregulated PAD activity plays an important role on too many diseases including also the cancer. In this study, it has been aimed to determine the potential cytotoxic and apoptotic activity of chlorine-amidine (Cl-amidine) which is a PAD inhibitor and whose effectiveness has been shown in vitro and in vivo studies recently on human glioblastoma cell line Uppsala 87 malignant glioma (U-87 MG) forming an in vitro model for the glioblastoma multiforme (GBM) which is the most aggressive and has the highest mortality among the brain tumors. METHODS: In the study, the antiproliferative and apoptotic effects of Cl-amidine on GBM cancer model were investigated. The antiproliferative effects of Cl-amidine on U-87 MG cells were determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate method at the 24th and 48th hours. The apoptotic effects were analyzed by Annexin V and Propidium iodide staining, caspase-3 activation, and mitochondrial membrane polarization (5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide) methods in the flow cytometry. RESULTS: It has been determined that Cl-amidine exhibits notable antiproliferative properties on U-87 MG cell line in a time and concentration-dependent manner, as determined through the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Assessment of apoptotic effects via Annexin V and Propidium iodide staining and 5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide methods has revealed significant efficacy, particularly following a 24-hour exposure period. It has been observed that Cl-amidine induces apoptosis in cells by enhancing mitochondrial depolarization, independently of caspase-3 activation. Furthermore, regarding its impact on healthy cells, it has been demonstrated that Cl-amidine shows lower cytotoxic effects when compared to carmustine, an important therapeutic agent for glioblastoma. CONCLUSION: The findings of this study have shown that Cl-amidine exhibits significant potential as an anticancer agent in the treatment of GBM. This conclusion is based on its noteworthy antiproliferative and apoptotic effects observed in U-87 MG cells, as well as its reduced cytotoxicity toward healthy cells in comparison to existing treatments. We propose that the antineoplastic properties of Cl-amidine should be further investigated through a broader spectrum of cancer cell types. Moreover, we believe that investigating the synergistic interactions of Cl-amidine with single or combination therapies holds promise for the discovery of novel anticancer agents.
Assuntos
Antineoplásicos , Glioblastoma , Nitrofenóis , Ornitina/análogos & derivados , Humanos , Cloro , Glioblastoma/metabolismo , Anexina A5 , Benzeno , Carbocianinas/farmacologia , Caspase 3/metabolismo , Iodetos/metabolismo , Iodetos/farmacologia , Propídio , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Amidinas/farmacologia , Arginina/metabolismo , ApoptoseRESUMO
Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.
Assuntos
Artrite Reumatoide , Citrulinação , Citrulina , Proteína-Arginina Desiminase do Tipo 4 , Humanos , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Citrulina/metabolismo , Células HL-60 , Proteômica/métodos , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/genética , Especificidade por Substrato , Líquido Sinovial/metabolismoRESUMO
Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.
Assuntos
Proteína-Arginina Desiminase do Tipo 4 , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Proteína-Arginina Desiminase do Tipo 4/química , Humanos , Domínio Catalítico , Microscopia Crioeletrônica , Modelos Moleculares , Anticorpos/química , Anticorpos/imunologia , Anticorpos/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/tratamento farmacológico , Hidrolases/metabolismo , Hidrolases/química , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/químicaRESUMO
Rheumatoid arthritis is an autoimmune disease whose early onset correlates with dysregulated citrullination, a process catalyzed by peptidylarginine deiminase isoform 4 (PADI-4). Here, we report that PADI-4 is a novel target of vitamin B12, a water-soluble vitamin that serves as a cofactor in DNA synthesis and the metabolism of fatty and amino acids. Vitamin B12 preferentially inhibited PADI-4 over PADI-2 with comparable inhibitory activity to the reference compound Cl-amidine in enzymatic inhibition assays, and reduced total cellular citrullination levels including that of histone H3 citrullination mediated by PADI-4. We also demonstrated that hydroxocobalamin, a manufactured form of vitamin B12, significantly ameliorated the severity of collagen type II antibody induced arthritis (CAIA) in mice and diminished gene expression of the rheumatoid inflammatory factors and cytokines IL17A, TNFα, IL-6, COX-II and ANXA2, as well PADI-4. Therefore, the use of vitamin B12 to treat rheumatoid arthritis merits further study.
Assuntos
Artrite Reumatoide , Vitamina B 12 , Camundongos , Animais , Desiminases de Arginina em Proteínas/metabolismo , Hidrolases/metabolismo , Proteína-Arginina Desiminase do Tipo 4 , Citrulina/metabolismo , Anticorpos , ColágenoRESUMO
UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.
Assuntos
Desoxirribonuclease I , Medicamentos de Ervas Chinesas , Armadilhas Extracelulares , Raios Ultravioleta , Animais , Masculino , Camundongos , Calcimicina/farmacologia , Desoxirribonuclease I/farmacologia , Desoxirribonuclease I/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/efeitos da radiação , Histonas/metabolismo , Peróxido de Hidrogênio/metabolismo , Inflamação/metabolismo , Camundongos Endogâmicos ICR , Neutrófilos/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
BACKGROUND/AIMS: Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. METHODS: To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. RESULTS: All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. CONCLUSION: Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.