Identification of a new genetic variant (G231N, E232T, N235D) of peptidylarginine deiminase from P. gingivalis in advanced periodontitis.

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.

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.

Neutrophil extracellular traps in rheumatoid arthritis and periodontitis: Contribution of PADI4 gene polymorphisms.

AIM: We sought to investigate the release of neutrophil extracellular traps (NETs) in neutrophils from individuals with rheumatoid arthritis (RA) and controls and compare the presence of NETs in gingival tissues according to periodontal status. Also, the association between single nucleotide polymorphisms (SNPs) of the peptidyl arginine deaminase type 4 (PADI4) gene and the GTG haplotype with RA, periodontitis and NETs was evaluated in vitro. MATERIALS AND METHODS: Peripheral neutrophils were isolated by density gradient, and NET concentration was determined by the PicoGreen method. Immunofluorescence was studied to identify NETs by co-localization of myeloperoxidase (MPO)-citrullinated histone H3 (H3Cit). Genotyping for SNPs (PADI4_89; PADI4_90; PADI4_92; and PADI4_104) was performed in 87 individuals with RA and 111 controls. RESULTS: The release of NETs in vitro was significantly higher in individuals with RA and periodontitis and when stimulated with Porphyromonas gingivalis. Gingival tissues from subjects with RA and periodontitis revealed increased numbers of MPO-H3Cit-positive cells. Individuals with the GTG haplotype showed a higher release of NETs in vitro and worse periodontal parameters. CONCLUSIONS: The release of NETs by circulating neutrophils is associated with RA and periodontitis and is influenced by the presence of the GTG haplotype.

Peptidylarginine deiminase 2 citrullinates MZB1 and promotes the secretion of IgM and IgA.

Introduction: MZB1 is an endoplasmic reticulum residential protein preferentially expressed in plasma cells, marginal zone and B1 B cells. Recent studies on murine B cells show that it interacts with the tail piece of IgM and IgA heavy chain and promotes the secretion of these two classes of immunoglobulin. However, its role in primary human B cells has yet to be determined and how its function is regulated is still unknown. The conversion of peptidylarginine to peptidylcitrulline, also known as citrullination, by peptidylarginine deiminases (PADs) can critically influence the function of proteins in immune cells, such as neutrophils and T cells; however, the role of PADs in B cells remains to be elucidated. Method: An unbiased analysis of human lung citrullinome was conducted to identify citrullinated proteins that are enriched in several chronic lung diseases, including rheumatoid arthritis-associated interstitial lung disease (RA-ILD), chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis, compared to healthy controls. Mass spectrometry, site-specific mutagenesis, and western blotting were used to confirm the citrullination of candidate proteins. Their citrullination was suppressed by pharmacological inhibition or genetic ablation of PAD2 and the impact of their citrullination on the function and differentiation of human B cells was examined with enzyme-linked immunosorbent assay, flow cytometry, and co-immunoprecipitation. Results: Citrullinated MZB1 was preferentially enriched in RA-ILD but not in other chronic lung diseases. MZB1 was a substrate of PAD2 and was citrullinated during the differentiation of human plasmablasts. Ablation or pharmacological inhibition of PAD2 in primary human B cells attenuated the secretion of IgM and IgA but not IgG or the differentiation of IgM or IgA-expressing plasmablasts, recapitulating the effect of ablating MZB1. Furthermore, the physical interaction between endogenous MZB1 and IgM/IgA was attenuated by pharmacological inhibition of PAD2. Discussion: Our data confirm the function of MZB1 in primary human plasmablasts and suggest that PAD2 promotes IgM/IgA secretion by citrullinating MZB1, thereby contributing to the pathogenesis of rheumatoid arthritis and RA-ILD.

Citrullinated and MMP-degraded vimentin is associated with chronic pulmonary diseases and genetic variants in PADI3/PADI4 and CFH in postmenopausal women.

Citrullinated vimentin has been linked to several chronic and autoimmune diseases, but how citrullinated vimentin is associated with disease prevalence and genetic variants in a clinical setting remains unknown. The aim of this study was to obtain a better understanding of the genetic variants and pathologies associated with citrullinated and MMP-degraded vimentin. Patient Registry data, serum samples and genotypes were collected for a total of 4369 Danish post-menopausal women enrolled in the Prospective Epidemiologic and Risk Factor study (PERF). Circulating citrullinated and MMP-degraded vimentin (VICM) was measured. Genome-wide association studies (GWAS) and phenome wide association studies (PheWAS) with levels of VICM were performed. High levels of VICM were significantly associated with the prevalence of chronic pulmonary diseases and death from respiratory and cardiovascular diseases (CVD). GWAS identified 33 single nucleotide polymorphisms (SNPs) with a significant association with VICM. These variants were in the peptidylarginine deiminase 3/4 (PADI3/PADI4) and Complement Factor H (CFH)/KCNT2 gene loci on chromosome 1. Serum levels of VICM, a marker of citrullinated and MMP-degraded vimentin, were associated with chronic pulmonary diseases and genetic variance in PADI3/PADI4 and CFH/ KCNT2. This points to the potential for VICM to be used as an activity marker of both citrullination and inflammation, identifying responders to targeted treatment and patients likely to experience disease progression.

Peptidyl Arginine Deiminases in Chronic Diseases: A Focus on Rheumatoid Arthritis and Interstitial Lung Disease.

Protein citrullination is accomplished by a broad enzyme family named Peptidyl Arginine Deiminases (PADs), which makes this post-translational modification in many proteins that perform physiological and pathologic mechanisms in the body. Due to these modifications, citrullination has become a significant topic in the study of pathological processes. It has been related to some chronic and autoimmune diseases, including rheumatoid arthritis (RA), interstitial lung diseases (ILD), multiple sclerosis (MS), and certain types of cancer, among others. Antibody production against different targets, including filaggrin, vimentin, and collagen, results in an immune response if they are citrullinated, which triggers a continuous inflammatory process characteristic of autoimmune and certain chronic diseases. PAD coding genes (PADI1 to PADI4 and PADI6) harbor variations that can be important in these enzymes' folding, activity, function, and half-life. However, few studies have considered these genetic factors in the context of chronic diseases. Exploring PAD pathways and their role in autoimmune and chronic diseases is a major topic in developing new pharmacological targets and valuable biomarkers to improve diagnosis and prevention. The present review addresses and highlights genetic, molecular, biochemical, and physiopathological factors where PAD enzymes perform a major role in autoimmune and chronic diseases.

NET formation is a default epigenetic program controlled by PAD4 in apoptotic neutrophils.

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosis-induced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils.

Co-expression of PADI isoforms during progenitor differentiation enables functional diversity.

Protein isoforms, generated through alternative splicing or promoter usage, contribute to tissue function. Here, we characterize the expression of predicted Padi3α and Padi3ß isoforms in hair follicles and describe expression of Padi2ß, a hitherto unknown PADI2 isoform, in the oligodendrocyte lineage. Padi2ß transcription is initiated from a downstream intronic promoter, generating an N-terminally truncated, unstable, PADI2ß. By contrast to the established role of the canonical PADI2 (PADI2α) (Falcao et al. 2019 Cell Rep. 27, 1090-1102.e10. (doi:10.1016/j.celrep.2019.03.108)), PADI2ß inhibits oligodendrocyte differentiation, suggesting that PADI2 isoforms exert opposing effects on oligodendrocyte lineage progression. We localize Padi3α and Padi3ß to developing hair follicles and find that both transcripts are expressed at low levels in progenitor cells, only to increase in expression concomitant with differentiation. When expressed in vitro, PADI3α and PADI3ß are enriched in the cytoplasm and precipitate together. Whereas PADI3ß protein stability is low and PADI3ß fails to induce protein citrullination, we find that the enzymatic activity and protein stability of PADI3α is reduced in the presence of PADI3ß. We propose that PADI3ß modulates PADI3α activity by direct binding and heterodimer formation. Here, we establish expression and function of Padi2 and Padi3 isoforms, expanding on the mechanisms in place to regulate citrullination in complex tissues. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Protein citrullination: inhibition, identification and insertion.

Protein citrullination is a post-translational modification (PTM) that is catalysed by the protein arginine deiminase (PAD) family of enzymes. This PTM involves the transformation of an arginine residue into citrulline. Protein citrullination is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation and DNA damage-induced apoptosis. Aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis (RA), lupus, atherosclerosis and ulcerative colitis. In RA, anti-citrullinated protein antibodies can be detected prior to disease onset and are thus a valuable diagnostic tool for RA. Notably, citrullinated proteins may serve more generally as biomarkers of specific disease states; however, the identification of citrullinated protein residues remains challenging owing to the small 1 Da mass change that occurs upon citrullination. Herein, we highlight the progress made so far in the development of pan-PAD and isozyme selective inhibitors as well as the identification of citrullinated proteins and the site-specific incorporation of citrulline into proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Deimination in epidermal barrier and hair formation.

Peptidylarginine deiminases (PADs) transform a protein arginine residue into the non-standard amino acid citrulline. This calcium-dependent post-translational modification of proteins is called citrullination or deimination. As described in this special issue, PADs play a role in various physiological processes, and PAD deregulations are involved in many human diseases. Three PADs are expressed in the epidermis, where their roles begin to be deciphered. PAD1 and PAD3 are involved in keratinocyte differentiation, particularly in the epidermal barrier function, keratins, filaggrin and filaggrin-related proteins being the most abundant deiminated epidermal proteins. Reduced amounts of deiminated proteins and PAD1 expression may be involved in the pathogenesis of psoriasis and atopic dermatitis, two very frequent and chronic skin inflammatory diseases. The trichohyalin/PAD3/transglutaminase three pathway is important for hair shaft formation. Mutations of the PADI3 gene, leading to a decreased activity or abnormal localization of the corresponding isotype, are the cause of a rare hair disorder called uncombable hair syndrome, and are associated with the central centrifugal cicatricial alopecia, a frequent alopecia mainly affecting women of African ancestry. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Citrullination of matrisomal proteins in health and diseases.

Proteins once translated are subjected to post-translational modifications (PTMs) that can critically modify their characteristics. Citrullination is a unique type of PTM that is catalysed by peptidylarginine deiminase (PAD) enzymes, which regulate a multitude of physiological functions such as apoptosis, gene expression and immune response by altering the structure and function of cellular proteins. However, emerging data have unravelled compelling evidence to support that PAD-mediated citrullination is not exclusive to cellular proteins; rather citrullination of extracellular matrix (ECM) proteins also plays a major contributing role in various physiological/pathological conditions. Here, we discuss putative mechanisms for citrullination-induced alterations in the function of ECM proteins. Further, we put emphasis on influential roles of ECM citrullination in various pathological scenarios to underscore the clinical potential of its manipulation in human diseases. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Small molecule activates citrullination through targeting PAD2.

Citrullination is a post-translational modification catalysed by peptidyl arginine deiminase (PAD) enzymes, and dysregulation of protein citrullination is involved in various pathological disorders. During the past decade, a panel of citrullination inhibitors has been developed, while small molecules activating citrullination have rarely been reported so far. In this study, we screened citrullination activator using an antibody against citrullinated histone H3 (cit-H3), and a natural compound demethoxycurcumin (DMC) significantly activated citrullination. The requirement of PAD2 for DMC-activated citrullination was confirmed by a loss of function assay. Notably, DMC directly engaged with PAD2, and showed binding selectivity among PAD family enzymes. Point mutation assay indicated that residue E352 is essential for DMC targeting PAD2. Consistently, DMC induced typical phenotypes of cells with dysregulation of PAD2 activity, including citrullination-associated cell apoptosis and DNA damage. Overall, our study not only presents a strategy for rationally screening citrullination activators, but also provides a chemical approach for activating protein citrullination. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Porphyromonas gingivalis Peptidyl Arginine Deiminase (PPAD) in the Context of the Feed-Forward Loop of Inflammation in Periodontitis.

Periodontitis is a widespread chronic inflammatory disease caused by a changed dysbiotic oral microbiome. Although multiple species and risk factors are associated with periodontitis, Porphyromonas gingivalis has been identified as a keystone pathogen. The immune-modulatory function of P. gingivalis is well characterized, but the mechanism by which this bacterium secretes peptidyl arginine deiminase (PPAD), a protein/peptide citrullinating enzyme, thus contributing to the infinite feed-forward loop of inflammation, is not fully understood. To determine the functional role of citrullination in periodontitis, neutrophils were stimulated by P. gingivalis bearing wild-type PPAD and by a PPAD mutant strain lacking an active enzyme. Flow cytometry showed that PPAD contributed to prolonged neutrophil survival upon bacterial stimulation, accompanied by the secretion of aberrant IL-6 and TNF-α. To further assess the complex mechanism by which citrullination sustains a chronic inflammatory state, the ROS production and phagocytic activity of neutrophils were evaluated. Flow cytometry and colony formation assays showed that PPAD obstructs the resolution of inflammation by promoting neutrophil survival and the release of pro-inflammatory cytokines, while enhancing the resilience of the bacteria to phagocytosis.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Inhibition of protein arginine deiminase II suppresses retinoblastoma in orthotopic transplantation in mice.

Chemotherapies are used for treating retinoblastoma; however, numerous patients suffer from recurrence or symptoms due to chemotherapy, which emphasizes the need for alternative therapeutic strategies. The present study demonstrated that protein arginine deiminase Ⅱ (PADI2) was highly expressed in human and mouse retinoblastoma tissues due to the overexpression of E2 factor (E2F). By inhibiting PADI2 activity, the expression of phosphorylated AKT was reduced, and cleaved poly (ADP­ribose) polymerase level was increased, leading to induced apoptosis. Similar results were obtained in orthotopic mouse models with reduced tumor volumes. In addition, BB­Cl­amidine showed low toxicity in vivo. These results suggested that PADI2 inhibition has potential clinical translation. Furthermore, the present study highlights the potential of epigenetic approaches to target RB1­deficient mutations at the molecular level. The current findings provide new insights into the importance of retinoblastoma intervention by managing PADI2 activity according to the treatment of specific inhibitors and depletion approaches in vitro and in orthotopic mouse models.

PADs and NETs in digestive system: From physiology to pathology.

Peptidylarginine deiminases (PADs) are the only enzyme class known to deiminate arginine residues into citrulline in proteins, a process known as citrullination. This is an important post-translational modification that functions in several physiological and pathological processes. Neutrophil extracellular traps (NETs) are generated by NETosis, a novel cell death in neutrophils and a double-edged sword in inflammation. Excessive activation of PADs and NETs is critically implicated in their transformation from a physiological to a pathological state. Herein, we review the physiological and pathological functions of PADs and NETs, in particular, the involvement of PAD2 and PAD4 in the digestive system, from inflammatory to oncological diseases, along with related therapeutic prospects.

ROLE OF PEPTIDYLARGININE DEIMINASE AND NEUTROPHIL EXTRACELLULAR TRAPS IN INJURIES: FUTURE NOVEL DIAGNOSTICS AND THERAPEUTIC TARGETS.

ABSTRACT: Injuries lead to an early systemic inflammatory state with innate immune system activation. Neutrophil extracellular traps (NETs) are a complex of chromatin and proteins released from the activated neutrophils. Although initially described as a response to bacterial infections, NETs have also been identified in the sterile postinjury inflammatory state. Peptidylarginine deiminases (PADs) are a group of isoenzymes that catalyze the conversion of arginine to citrulline, termed citrullination or deimination. PAD2 and PAD4 have been demonstrated to play a role in NET formation through citrullinated histone 3. PAD2 and PAD4 have a variety of substrates with variable organ distribution. Preclinical and clinical studies have evaluated the role of PADs and NETs in major trauma, hemorrhage, burns, and traumatic brain injury. Neutrophil extracellular trap formation and PAD activation have been shown to contribute to the postinjury inflammatory state leading to a detrimental effect on organ systems. This review describes our current understanding of the role of PAD and NET formation following injury and burn. This is a new field of study, and the emerging data appear promising for the future development of targeted biomarkers and therapies in trauma.

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.

Inducement of ER Stress by PAD Inhibitor BB-Cl-Amidine to Effectively Kill AML Cells.

OBJECTIVE: Acute myeloid leukemia (AML) is a highly heterogeneous and recurrent hematological malignancy. Despite the emergence of novel chemotherapy drugs, AML patients' complete remission (CR) remains unsatisfactory. Consequently, it is imperative to discover new therapeutic targets or medications to treat AML. Such epigenetic changes like DNA methylation and histone modification play vital roles in AML. Peptidylarginine deminase (PAD) is a protein family of histone demethylases, among which the PAD2 and PAD4 expression have been demonstrated to be elevated in AML patients, thus suggesting a potential role of PADs in the development or maintenance of AML and the potential for the identification of novel therapeutic targets. METHODS: AML cells were treated in vitro with the pan-PAD inhibitor BB-Cl-Amidine (BB-Cl-A). The AML cell lines were effectively induced into apoptosis by BB-Cl-A. However, the PAD4-specific inhibitor GSK484 did not. RESULTS: PAD2 played a significant role in AML. Furthermore, we found that BB-Cl-A could activate the endoplasmic reticulum (ER) stress response, as evidenced by an increase in phosphorylated PERK (p-PERK) and eIF2α (p-eIF2α). As a result of the ER stress activation, the BB-Cl-A effectively induced apoptosis in the AML cells. CONCLUSION: Our findings indicated that PAD2 plays a role in ER homeostasis maintenance and apoptosis prevention. Therefore, targeting PAD2 with BB-Cl-A could represent a novel therapeutic strategy for treating AML.