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Paul R Thompson - One of the best experts on this subject based on the ideXlab platform.
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progesterone stimulates histone Citrullination to increase igfbp1 expression in uterine cells
Reproduction, 2021Co-Authors: Coleman H Young, Venkatesh V Nemmara, Paul R Thompson, Bryce Snow, Stanley Devore, Adithya Mohandass, Baskaran Thyagarajan, Amy M Navratil, Brian D CherringtonAbstract:Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone Citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates Citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting Citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone Citrullination and an increase in IGFBP1 expression.
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human cytomegalovirus induced host protein Citrullination is crucial for viral replication
Nature Communications, 2021Co-Authors: Gloria Griffante, Ari J Salinger, Paul R Thompson, Francesca Gugliesi, Selina Pasquero, Valentina Delloste, Matteo Biolatti, Santanu Mondal, Eranthie WeerapanaAbstract:Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated Citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 Citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 Citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of Citrullination in subverting cellular responses to viral infection.
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Human cytomegalovirus-induced host protein Citrullination is crucial for viral replication
'Springer Science and Business Media LLC', 2021Co-Authors: Gloria Griffante, Ari J Salinger, Paul R Thompson, Francesca Gugliesi, Selina Pasquero, Matteo Biolatti, Santanu Mondal, Eranthie Weerapana, Valentina Dell’oste, Robert J. LebbinkAbstract:Citrullination is a posttranslational modification of arginines. Here, the authors show that HCMV infection increases Citrullination of host and virus proteins to promote infection and that citrullinated interferon-inducible protein IFIT1 is impaired in RNA binding, as a potential mechanism of evasion
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colorectal cancer liver metastatic growth depends on pad4 driven Citrullination of the extracellular matrix
Nature Communications, 2018Co-Authors: Arseniy E Yuzhalin, Alex Gordonweeks, Marialaura Tognoli, Keaton Jones, Bostjan Markelc, Rebecca Konietzny, Roman Fischer, Aaron Muth, Eric Oneill, Paul R ThompsonAbstract:Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that Citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of Citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for Citrullination in metastatic growth is evident in murine models where inhibition of Citrullination substantially reduces liver metastatic burden. Additionally, Citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for Citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant Citrullination to drive the progression of CRC liver metastasis.
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Citrullination of nf κb p65 promotes its nuclear localization and tlr induced expression of il 1β and tnfα
Science immunology, 2017Co-Authors: Aaron Muth, Paul R Thompson, Mandar Bawadekar, Bo Sun, Nishant Dwivedi, Tyler J Bechtel, Janet L Paulsen, Miriam A ShelefAbstract:Many citrullinated proteins are known autoantigens in rheumatoid arthritis, a disease mediated by inflammatory cytokines, such as tumor necrosis factor-α (TNFα). Citrullinated proteins are generated by converting peptidylarginine to peptidylcitrulline, a process catalyzed by the peptidylarginine deiminases (PADs), including PAD1 to PAD4 and PAD6. Several major risk factors for rheumatoid arthritis are associated with heightened Citrullination. However, the physiological role of Citrullination in immune cells is poorly understood. We report that suppression of PAD activity attenuates Toll-like receptor-induced expression of interleukin-1β (IL-1β) and TNFα by neutrophils in vivo and in vitro but not their global transcription activity. Mechanistically, PAD4 directly citrullinates nuclear factor κB (NF-κB) p65 and enhances the interaction of p65 with importin α3, which brings p65 into the nucleus. The Citrullination-enhanced interaction of p65 with importin α3 and its nuclear translocation and transcriptional activity can be attributed to Citrullination of four arginine residues located in the Rel homology domain of p65. Furthermore, a rheumatoid arthritis-prone variant of PAD4, carrying three missense mutations, is more efficient in interacting with p65 and enhancing NF-κB activity. Together, these data not only demonstrate a critical role of Citrullination in an NF-κB-dependent expression of IL-1β and TNFα but also provide a molecular mechanism by which heightened Citrullination propagates inflammation in rheumatoid arthritis. Accordingly, attenuating p65-mediated production of IL-1β and TNFα by blocking the Citrullination of p65 has great therapeutic potential in rheumatoid arthritis.
Koichi Matsuda - One of the best experts on this subject based on the ideXlab platform.
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abstract lb 036 citrullinome analysis identifies the role of padi4 in regulating rna processing and tumor immunogenicity
Cancer Research, 2017Co-Authors: Chizu Tanikawa, Koichi MatsudaAbstract:Recent proteome analyses have provided a comprehensive overview of various posttranscriptional modifications (PTM); however, PTMs involving protein Citrullination remain unclear. Thus, we performed a proteomic analysis of citrullinated proteins and identified more than 100 PADI4 substrates, the majority of which are related to RNA processing and protein translation. Citrullination of RBMX (RNA binding motif protein, X-linked) inhibited the interaction with other hnRNP family members. RNA sequence analyses revealed altered splicing patterns in more than 300 genes in bone marrow cells in Padi4-/- mice. Approximately one-fifth of the PADI4 substrates contained an RG/RGG motif, and Citrullination by PADI4 competitively inhibited the methylation of the RGG motif in FET proteins (EWS and TAF15). In addition, PADI4-mediated Citrullination inhibited the aggregation of TAF15 proteins, which is a frequently-observed feature in neurodegenerative diseases, such as ALS. An antibody against citrullinated RGG motifs was observed in 11.5% (27 among 234) of serum samples from patients with HCV-related hepatocellular carcinoma, and female patients with high serum antibody exhibited poor prognosis. Our findings imply that PADI4-mediated Citrullination plays a key role in RNA processing, protein aggregation and tumor immunogenicity, and suggest that PADI4 activation has potential therapeutic implications for neurodegenerative disease and cancer immunotherapy. Citation Format: Chizu Tanikawa, Koichi Matsuda. Citrullinome analysis identifies the role of PADI4 in regulating RNA processing and tumor immunogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-036. doi:10.1158/1538-7445.AM2017-LB-036
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abstract 217 functional analysis of the protein Citrullination through p53 padi4 network in carcinogenesis
Cancer Research, 2011Co-Authors: Chizu Tanikawa, Koichi Matsuda, Yusuke NakamuraAbstract:Upon a wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However, its involvement in posttranslational modifications of proteins has not yet been well characterized. We recently reported the novel role of p53 in the regulation of protein Citrullination through the transactivation of peptidylarginine deiminase type 4 (PADI4). The PADI4 gene encodes an enzyme catalyzing the Citrullination of arginine residues in proteins, and ectopic expression of p53 or PADI4 induced protein Citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their Citrullination. Here we generated the Padi4-deficient mice and examined the role of protein Citrullination in vivo. When mice were exposed to X-ray (3 Gy), Padi4 expression was induced in thymus of irradiated wild type mice but not in p53-/- and Padi4-/- mice. Interestingly, TUNEL-positive cells in irradiated thymus were significantly decreased in Padi4-/- mice. Gene expression analysis using irradiated thymus tissues revealed several genes related to the immune response were regulated by Padi4. To further investigate the molecular mechanism whereby PADI4 regulates apoptotic pathway, we surveyed PADI4 substrates and found that various proteins such as core histones were citrullinated in response to DNA damage. These findings demonstrated the significance of PADI4-mediated protein Citrullination in the p53 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2011-217
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abstract 1102 regulation of protein Citrullination through p53 padi4 network in dna damage response
Cancer Research, 2010Co-Authors: Koichi Matsuda, Chizu Tanikawa, Yusuke NakamuraAbstract:Upon wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However its involvement in post-translational modifications of proteins has not been well characterized so far. We here report the novel role of p53 in the regulation of protein Citrullination. Citrullination (also referred as deimination) is one form of PTMs converting an arginine residue in protein to a citrulline residue. This reaction is mediated by a Ca2+-dependent enzyme called as peptidylarginine deiminase. Citrullination of proteins results in a loss of positive charge and causes a significant biochemical change. On the other hand, citrullinated peptides/proteins are recognized as a non-self protein and subsequently activate immune systems. Hence, protein Citrullination is now considered to play a pivotal role in various human diseases, but any roles of Citrullination in carcinogenesis were not reported so far. We found p53 transactivated peptidylarginine deiminase type 4 (PADI4) through intronic p53-binding site and ectopic expression of p53 or PADI4 induced protein Citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their Citrullination, indicating the regulation of protein Citrullination in a p53/PADI4 dependent manner. We identified that PADI4 citrullinated histone chaperon protein, nucleophosmin (NPM1) at arginine 197 residue in vivo under physiological condition. NPM1 was shown to continuously shuttle between the nucleus and cytoplasm and translocate from nucleoli to nucleoplasm as a response to DNA damage. Therefore, we investigated the effect of protein Citrullination on subcellular localization of NPM1. Immunocytochemical analysis revealed that Citrullination of NPM1 by PADI4 resulted in its translocation from nucleoli to nucleoplasm, while PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly knockdown of PADI4 attenuated p53-mediated growth inhibitory activity, demonstrating the significance of PADI4-mediated protein Citrullination in the p53 signaling pathway. Most of previous studies assessing enzymatic activities of PADIs were conducted under non-physiological condition using Ca ionophore or at higher Ca concentration. To our knowledge, this is the first report demonstrating the physiological regulatory mechanism of protein Citrullination. Thus, p53 that can directly or indirectly modulate biological property of various proteins through the protein Citrullination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1102.
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regulation of protein Citrullination through p53 padi4 network in dna damage response
Cancer Research, 2009Co-Authors: Chizu Tanikawa, Nobuaki Yoshida, Hidewaki Nakagawa, Koji Ueda, Yusuke Nakamura, Koichi MatsudaAbstract:Upon a wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However, its involvement in posttranslational modifications of proteins has not yet been well characterized. Here, we report the novel role of p53 in the regulation of protein Citrullination. p53 transactivated peptidylarginine deiminase type 4 ( PADI4 ) through an intronic p53-binding site. The PADI4 gene encodes an enzyme catalyzing the Citrullination of arginine residues in proteins, and ectopic expression of p53 or PADI4 induced protein Citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their Citrullination, indicating the regulation of protein Citrullination in a p53/PADI4-dependent manner. We found that PADI4 citrullinated the histone chaperone protein, nucleophosmin (NPM1), at the arginine 197 residue in vivo under physiologic conditions. Citrullination of NPM1 by PADI4 resulted in its translocation from the nucleoli to the nucleoplasm, whereas PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly, the knockdown of PADI4 attenuated p53-mediated growth-inhibitory activity, demonstrating the significance of PADI4-mediated protein Citrullination in the p53 signaling pathway.[Cancer Res 2009;69(22):8761–9]
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regulation of protein Citrullination through p53 padi4 network in dna damage response
Cancer Research, 2009Co-Authors: Chizu Tanikawa, Nobuaki Yoshida, Hidewaki Nakagawa, Koji Ueda, Yusuke Nakamura, Koichi MatsudaAbstract:Upon a wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However, its involvement in posttranslational modifications of proteins has not yet been well characterized. Here, we report the novel role of p53 in the regulation of protein Citrullination. p53 transactivated peptidylarginine deiminase type 4 (PADI4) through an intronic p53-binding site. The PADI4 gene encodes an enzyme catalyzing the Citrullination of arginine residues in proteins, and ectopic expression of p53 or PADI4 induced protein Citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their Citrullination, indicating the regulation of protein Citrullination in a p53/PADI4-dependent manner. We found that PADI4 citrullinated the histone chaperone protein, nucleophosmin (NPM1), at the arginine 197 residue in vivo under physiologic conditions. Citrullination of NPM1 by PADI4 resulted in its translocation from the nucleoli to the nucleoplasm, whereas PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly, the knockdown of PADI4 attenuated p53-mediated growth-inhibitory activity, demonstrating the significance of PADI4-mediated protein Citrullination in the p53 signaling pathway
Ger J M Pruijn - One of the best experts on this subject based on the ideXlab platform.
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peptidylarginine deiminase 2 is required for tumor necrosis factor alpha induced Citrullination and arthritis but not neutrophil extracellular trap formation
Journal of Autoimmunity, 2017Co-Authors: Mandar Bawadekar, Daeun Shim, Chad J Johnson, Thomas F Warner, Ryan Rebernick, Dres Damgaard, Claus Henrik Nielsen, Ger J M Pruijn, Jeniel E Nett, Miriam A ShelefAbstract:Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the Citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for Citrullination in inflamed lungs. PAD2 is important for Citrullination in healthy tissues and is present in NETs, but its role in Citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in Citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, Citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, Citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced Citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized Citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.
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Citrullination and carbamylation in the pathophysiology of rheumatoid arthritis
Frontiers in Immunology, 2015Co-Authors: Ger J M PruijnAbstract:The discovery that Citrullination was crucial for the recognition of antigens by the most disease-specific class of autoantibodies in rheumatoid arthritis (RA) had a huge impact on studies aimed at understanding autoimmunity in this disease. In addition to the detailed characterization of anti-citrullinated protein antibodies, various studies have addressed the identity of citrullinated antigens. These investigations were facilitated by new methods to characterize these proteins, the analysis of protein Citrullination by peptidylarginine deiminases, the generation of a catalog of citrullinated proteins present in the inflamed joints of patients and the finding that the formation of extracellular traps is dependent on the activity of peptidylarginine deiminase activity. Recently, it was found that in addition to Citrullination also carbamylation, which results in chemically highly related modified proteins, yields antigens that are targeted by rheumatoid arthritis patient sera. Here, all of these aspects will be discussed, culminating in current ideas about the involvement of Citrullination and carbamylation in pathophysiological processes in autoimmunity, especially RA.
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demonstration of extracellular peptidylarginine deiminase pad activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for Citrullination of fibrinogen
Arthritis Research & Therapy, 2014Co-Authors: Dres Damgaard, Ger J M Pruijn, L Senolt, Michael Friberg Bruun Nielsen, Claus Henrik NielsenAbstract:Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hyperCitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for Citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined. Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for Citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the Citrullination process to occur. We present an assay with high specificity for PAD2 activity and show that Citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.
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methods for the detection of peptidylarginine deiminase pad activity and protein Citrullination
Molecular & Cellular Proteomics, 2014Co-Authors: Sanne M M Hensen, Ger J M PruijnAbstract:The post-translational conversion of peptidylarginine to peptidylcitrulline, a process also known as Citrullination, is catalyzed by the enzyme family of peptidylarginine deiminases (PADs) and has been demonstrated to be involved in many physiological processes, including the regulation of gene expression. In addition, Citrullination has been shown to be associated with several diseases, such as cancer, multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease. To get more insight into the role of PAD enzymes and Citrullination in both health and disease, experimental strategies to study PAD activity and to characterize citrullinated proteins in complex biological samples are crucial. Here, we describe the chemical, proteomic and antibody-based procedures that are currently available and discuss their applicability for the analysis of complex samples. The methods that have been developed can be used to provide more insight in the substrate specificity of PAD enzymes. Because the evidence that PADs play a pathophysiological role in the diseases mentioned above is increasing, they become attractive targets for therapeutic interventions. More knowledge of PAD specificity and the availability of reliable, high-throughput assays for PAD activity will facilitate the development of highly specific PAD inhibitors.
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local administration of glucocorticoids decreases synovial Citrullination in rheumatoid arthritis
Arthritis Research & Therapy, 2012Co-Authors: Dimitrios Makrygiannakis, Anthony P Nicholas, Ger J M Pruijn, Shankar Revu, Marianne Engstrom, Erik Af Klint, A I CatrinaAbstract:Introduction Protein Citrullination is present in the rheumatoid synovium, presumably contributing to the perpetuation of chronic inflammation, in the presence of specific autoimmunity. As a result, the present study examined the possibility that effective antirheumatic treatment will decrease the level of synovial Citrullination.
Claus Henrik Nielsen - One of the best experts on this subject based on the ideXlab platform.
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peptidylarginine deiminase 4 gene polymorphisms are associated with systemic lupus erythematosus and lupus nephritis
Scandinavian Journal of Rheumatology, 2019Co-Authors: Laura Massarenti, Dres Damgaard, Claus Henrik Nielsen, Christian Enevold, Niels Odum, Soren JacobsenAbstract:Objective: Peptidylarginine deiminase-4 (PAD4) is highly expressed by neutrophils and essential for Citrullination occurring during the formation of neutrophil extracellular traps, which have been ...
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peptidylarginine deiminase 2 is required for tumor necrosis factor alpha induced Citrullination and arthritis but not neutrophil extracellular trap formation
Journal of Autoimmunity, 2017Co-Authors: Mandar Bawadekar, Daeun Shim, Chad J Johnson, Thomas F Warner, Ryan Rebernick, Dres Damgaard, Claus Henrik Nielsen, Ger J M Pruijn, Jeniel E Nett, Miriam A ShelefAbstract:Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the Citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for Citrullination in inflamed lungs. PAD2 is important for Citrullination in healthy tissues and is present in NETs, but its role in Citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in Citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, Citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, Citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced Citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized Citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.
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identification of Citrullination sites specific for peptidylarginine deiminase 2 pad2 and pad4 in fibrinogen from synovial fluid of patients with rheumatoid arthritis
Annals of the Rheumatic Diseases, 2017Co-Authors: Mandvi Sharma, Claus Henrik Nielsen, Dan Damgaard, L Senolt, Birte Svensson, A C B Jensen, Per HagglundAbstract:Background Protein Citrullination, i.e. conversion of arginine residues into citrulline residues, is a post-translational modification catalyzed by PAD, and is an important pathophysiological determinant in conditions such as Rheumatoid arthritis (RA). Identification of Citrullination sites on putative autoantigens is likely to enhance our understanding of PAD9s substrate specificity. Objectives Citrullinated fibrinogen is an autoantigen linked to the pathophysiology of RA.We have applied a novel MS-based proteomics approach to estimate the degree of Citrullination in synovial fluid (SF) from RA patients, and compare to fibrinogen citrullinated in vitro by PAD2/4, the most important PAD isoforms involved in RA. The estimated degree of Citrullination induced by the two isoforms is also compared to evaluate their relative impact. Methods Fibrinogen was citrullinated in vitro by PAD2/4 and Citrullination sites were identified by LC-MS/MS on a Q-exactive orbitrap following proteolytic digestion with Lys-C. These in vitro Citrullination profiles were compared to those observed in SF fibrinogen of four RA patients with varying DAS28 scores, CRP levels and leukocyte counts. DAS28 scores >5.1 and ≤2.4 correspond to moderate to severe and low activity of disease respectively. Patients with high inflammatory activity gave high CRP level and leukocyte count values. Results A total of 52 Citrullination sites were identified. Overall, PAD2 generated higher number of identified sites and higher degree of citrulline occupancy at given sites than PAD4. In fibrinogen from SF, 38 Citrullination sites were identified, of which 23 have not been previously reported. Several of these sites were identified in more than one patient, and were regarded as hotspots. Fibrinogen from patients with high DAS28 levels contained markedly more Citrullination sites and higher citrulline occupancy. Conclusions Study suggests that PAD2 citrullinates fibrinogen more efficiently than PAD4 and Citrullination of certain sites in fibrinogen from SF reflects disease activity. Identification of such sites may have diagnostic or prognostic value in RA and other inflammatory disorders. References D Makrygiannakis, E af Klint, I E Lundberg, R Lofberg, A-K Ulfgren, L Klareskog, A.I.C. Citrullination is an inflammation-dependent process. Ann. Rheum. Dis. 2006, 65 (9), 1219–1222. Foulquier,C.; Sebbag, M.; Clavel, C.; Chapuy-Regaud, S.; Al Badine, R.; Mechin, M. C.; Vincent, C.; Nachat, R.; Yamada, M.; Takahara, H. Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheumatoid arthritis synovium in close association with tissue inflammation. Arthritis Rheum. 2007, 56 (11), 3541–3553. Gestel, A.; Haagsma, C.; Riel, P. Validation of rheumatoid arthritis improvement criteria that include simplified joint counts. 1998, 41 (10), 1845–1850. Acknowledgements The Danish Council for Independent Research Natural Sciences (grant number 11–106246) and the Velux foundation. The Danish Research Foundation, Nordic Bioscience A/S and DTU are acknowledged for a joint PhD scholarship. LS was supported by the Ministry of Health of the Czech Republic (grant #00023728). Disclosure of Interest None declared
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demonstration of extracellular peptidylarginine deiminase pad activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for Citrullination of fibrinogen
Arthritis Research & Therapy, 2014Co-Authors: Dres Damgaard, Ger J M Pruijn, L Senolt, Michael Friberg Bruun Nielsen, Claus Henrik NielsenAbstract:Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hyperCitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for Citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined. Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for Citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the Citrullination process to occur. We present an assay with high specificity for PAD2 activity and show that Citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.
Chizu Tanikawa - One of the best experts on this subject based on the ideXlab platform.
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Citrullination of rgg motifs in fet proteins by pad4 regulates protein aggregation and als susceptibility
Cell Reports, 2018Co-Authors: Chizu Tanikawa, Koji Ueda, Akari Suzuki, Aritoshi Iida, Ryoichi Nakamura, Naoki Atsuta, Genki Tohnai, Gen Sobue, Naomi Saichi, Yukihide MomozawaAbstract:Summary Recent proteome analyses have provided a comprehensive overview of various posttranslational modifications (PTMs); however, PTMs involving protein Citrullination remain unclear. We performed a proteomic analysis of citrullinated proteins, and we identified more than 100 PAD4 (peptidyl arginine deiminase 4) substrates. Approximately one-fifth of the PAD4 substrates contained an RG/RGG motif, and PAD4 competitively inhibited the methylation of the RGG motif in FET proteins (FUS, EWS, and TAF15) and hnRNPA1, which are causative genes for ALS (amyotrophic lateral sclerosis). PAD4-mediated Citrullination significantly inhibited the aggregation of FET proteins, a frequently observed feature in neurodegenerative diseases. FUS protein levels in arsenic-induced stress granules were significantly increased in Padi4 −/− mouse embryonic fibroblasts (MEFs). Moreover, rs2240335 was associated with low expression of PADI4 in the brain and a high risk of ALS (p = 0.0381 and odds ratio of 1.072). Our findings suggest that PAD4-mediated RGG Citrullination plays a key role in protein solubility and ALS pathogenesis.
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abstract lb 036 citrullinome analysis identifies the role of padi4 in regulating rna processing and tumor immunogenicity
Cancer Research, 2017Co-Authors: Chizu Tanikawa, Koichi MatsudaAbstract:Recent proteome analyses have provided a comprehensive overview of various posttranscriptional modifications (PTM); however, PTMs involving protein Citrullination remain unclear. Thus, we performed a proteomic analysis of citrullinated proteins and identified more than 100 PADI4 substrates, the majority of which are related to RNA processing and protein translation. Citrullination of RBMX (RNA binding motif protein, X-linked) inhibited the interaction with other hnRNP family members. RNA sequence analyses revealed altered splicing patterns in more than 300 genes in bone marrow cells in Padi4-/- mice. Approximately one-fifth of the PADI4 substrates contained an RG/RGG motif, and Citrullination by PADI4 competitively inhibited the methylation of the RGG motif in FET proteins (EWS and TAF15). In addition, PADI4-mediated Citrullination inhibited the aggregation of TAF15 proteins, which is a frequently-observed feature in neurodegenerative diseases, such as ALS. An antibody against citrullinated RGG motifs was observed in 11.5% (27 among 234) of serum samples from patients with HCV-related hepatocellular carcinoma, and female patients with high serum antibody exhibited poor prognosis. Our findings imply that PADI4-mediated Citrullination plays a key role in RNA processing, protein aggregation and tumor immunogenicity, and suggest that PADI4 activation has potential therapeutic implications for neurodegenerative disease and cancer immunotherapy. Citation Format: Chizu Tanikawa, Koichi Matsuda. Citrullinome analysis identifies the role of PADI4 in regulating RNA processing and tumor immunogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-036. doi:10.1158/1538-7445.AM2017-LB-036
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abstract 217 functional analysis of the protein Citrullination through p53 padi4 network in carcinogenesis
Cancer Research, 2011Co-Authors: Chizu Tanikawa, Koichi Matsuda, Yusuke NakamuraAbstract:Upon a wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However, its involvement in posttranslational modifications of proteins has not yet been well characterized. We recently reported the novel role of p53 in the regulation of protein Citrullination through the transactivation of peptidylarginine deiminase type 4 (PADI4). The PADI4 gene encodes an enzyme catalyzing the Citrullination of arginine residues in proteins, and ectopic expression of p53 or PADI4 induced protein Citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their Citrullination. Here we generated the Padi4-deficient mice and examined the role of protein Citrullination in vivo. When mice were exposed to X-ray (3 Gy), Padi4 expression was induced in thymus of irradiated wild type mice but not in p53-/- and Padi4-/- mice. Interestingly, TUNEL-positive cells in irradiated thymus were significantly decreased in Padi4-/- mice. Gene expression analysis using irradiated thymus tissues revealed several genes related to the immune response were regulated by Padi4. To further investigate the molecular mechanism whereby PADI4 regulates apoptotic pathway, we surveyed PADI4 substrates and found that various proteins such as core histones were citrullinated in response to DNA damage. These findings demonstrated the significance of PADI4-mediated protein Citrullination in the p53 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2011-217
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abstract 1102 regulation of protein Citrullination through p53 padi4 network in dna damage response
Cancer Research, 2010Co-Authors: Koichi Matsuda, Chizu Tanikawa, Yusuke NakamuraAbstract:Upon wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However its involvement in post-translational modifications of proteins has not been well characterized so far. We here report the novel role of p53 in the regulation of protein Citrullination. Citrullination (also referred as deimination) is one form of PTMs converting an arginine residue in protein to a citrulline residue. This reaction is mediated by a Ca2+-dependent enzyme called as peptidylarginine deiminase. Citrullination of proteins results in a loss of positive charge and causes a significant biochemical change. On the other hand, citrullinated peptides/proteins are recognized as a non-self protein and subsequently activate immune systems. Hence, protein Citrullination is now considered to play a pivotal role in various human diseases, but any roles of Citrullination in carcinogenesis were not reported so far. We found p53 transactivated peptidylarginine deiminase type 4 (PADI4) through intronic p53-binding site and ectopic expression of p53 or PADI4 induced protein Citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their Citrullination, indicating the regulation of protein Citrullination in a p53/PADI4 dependent manner. We identified that PADI4 citrullinated histone chaperon protein, nucleophosmin (NPM1) at arginine 197 residue in vivo under physiological condition. NPM1 was shown to continuously shuttle between the nucleus and cytoplasm and translocate from nucleoli to nucleoplasm as a response to DNA damage. Therefore, we investigated the effect of protein Citrullination on subcellular localization of NPM1. Immunocytochemical analysis revealed that Citrullination of NPM1 by PADI4 resulted in its translocation from nucleoli to nucleoplasm, while PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly knockdown of PADI4 attenuated p53-mediated growth inhibitory activity, demonstrating the significance of PADI4-mediated protein Citrullination in the p53 signaling pathway. Most of previous studies assessing enzymatic activities of PADIs were conducted under non-physiological condition using Ca ionophore or at higher Ca concentration. To our knowledge, this is the first report demonstrating the physiological regulatory mechanism of protein Citrullination. Thus, p53 that can directly or indirectly modulate biological property of various proteins through the protein Citrullination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1102.
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regulation of protein Citrullination through p53 padi4 network in dna damage response
Cancer Research, 2009Co-Authors: Chizu Tanikawa, Nobuaki Yoshida, Hidewaki Nakagawa, Koji Ueda, Yusuke Nakamura, Koichi MatsudaAbstract:Upon a wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However, its involvement in posttranslational modifications of proteins has not yet been well characterized. Here, we report the novel role of p53 in the regulation of protein Citrullination. p53 transactivated peptidylarginine deiminase type 4 ( PADI4 ) through an intronic p53-binding site. The PADI4 gene encodes an enzyme catalyzing the Citrullination of arginine residues in proteins, and ectopic expression of p53 or PADI4 induced protein Citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their Citrullination, indicating the regulation of protein Citrullination in a p53/PADI4-dependent manner. We found that PADI4 citrullinated the histone chaperone protein, nucleophosmin (NPM1), at the arginine 197 residue in vivo under physiologic conditions. Citrullination of NPM1 by PADI4 resulted in its translocation from the nucleoli to the nucleoplasm, whereas PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly, the knockdown of PADI4 attenuated p53-mediated growth-inhibitory activity, demonstrating the significance of PADI4-mediated protein Citrullination in the p53 signaling pathway.[Cancer Res 2009;69(22):8761–9]