Peptidylarginine deiminase and protein citrullination in prion diseases

The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration.     

Insights into the mechanism of streptonigrin-induced protein arginine deiminase inactivation

Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the protein arginine deiminases (PADs 1–4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure–activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor.     

Bacterial and human peptidylarginine deiminases: targets for inhibiting the autoimmune response in rheumatoid arthritis?

Peptidylarginine deiminases (PADs) convert arginine within a peptide (peptidylarginine) into peptidylcitrulline. Citrullination by human PADs is important in normal physiology and inflammation. Porphyromonas gingivalis, a major pathogen in periodontitis, is the only prokaryote described to possess PAD. P. gingivalis infection may generate citrullinated peptides, which trigger anti-citrullinated peptide antibodies. In susceptible individuals, host protein citrullination by human PADs in the joint probably perpetuates antibody formation, paving the way for the development of chronic arthritis. Blockades of bacterial and human PADs may act as powerful novel therapies by inhibiting the generation of the antigens that trigger and sustain autoimmunity in rheumatoid arthritis.     

Recent advances in characterization of citrullination and its implication in human disease research: From method development to network integration

Post-translational modifications (PTM) of proteins increase the functional diversity of the proteome and have been implicated in the pathogenesis of numerous diseases. The most widely understood modifications include phosphorylation, methylation, acetylation, O-linked/N-linked glycosylation, and ubiquitination, all of which have been extensively studied and documented. Citrullination is a historically less explored, yet increasingly studied, protein PTM which has profound effects on protein conformation and protein-protein interactions. Dysregulation of protein citrullination has been associated with disease development and progression. Identification and characterization of citrullinated proteins is highly challenging, complicated by the low cellular abundance of citrullinated proteins, making it difficult to identify and quantify the extent of citrullination in samples, coupled with challenges associated with development of mass spectrometry (MS)-based methods, as the corresponding mass shift is relatively small, +0.984 Da, and identical to the mass shift of deamidation. The focus of this review is to discuss recent advancements of citrullination-specific MS approaches and integration of the potential methodology for improved citrullination identification and characterization. In addition, the association of citrullination in disease networks is also highlighted.     

N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis

Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0-35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ～50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine-treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.     

Myocardial citrullination in rheumatoid arthritis: a correlative histopathologic study

Introduction

The aim of this study was to explore the presence and localization of myocardial citrullination in samples from rheumatoid arthritis (RA) patients compared to rheumatic and non-rheumatic disease control groups.

Methods

Archived myocardial samples obtained during autopsy from 1995 to 2009 were assembled into four groups: RA; scleroderma; fatal myocarditis; and non-rheumatic disease controls. Samples were examined by immunohistochemistry (IHC) for the presence and localization of citrullination and peptidyl arginine deiminase enzymes (PADs) by a single cardiovascular pathologist blinded to disease group and clinical characteristics.

Results

Myocardial samples from seventeen RA patients were compared with those from fourteen controls, five fatal myocarditis patients, and ten scleroderma patients. Strong citrullination staining was detected exclusively in the myocardial interstitium in each of the groups. However, average and peak anti-citrulline staining was 59% and 44% higher, respectively, for the RA group compared to the combined non-RA groups (P < 0.05 for both comparisons). Myocardial fibrosis did not differ between the groups. In contrast to citrullination, PADs 1 to 3 and 6 were detected in cardiomyocytes (primarily PADs 1 and 3), resident inflammatory cells (primarily PADs 2 and 4), and, to a smaller extent, in endothelial cells and vascular smooth muscle cells. PAD staining did not co-localize with anti-citrulline staining in the interstitium and did not vary by disease state.

Conclusions

Staining for citrullination was higher in the myocardial interstitium of RA compared to other disease states, a finding that could link autoimmunity to the known increase in myocardial dysfunction and heart failure in RA.     

Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.     

Development of a fluorescent probe for detection of citrulline based on photo-induced electron transfer

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine residues to form citrulline residues. Aberrant citrullination of histones by one of the PAD isozymes, PAD4, is associated with various diseases, including rheumatoid arthritis, so high-throughput screening systems are needed to identify PAD4 inhibitors as chemical tools to investigate the role of PAD4, and as candidate therapeutic agents. Here, we utilized the addition-cyclization reaction between phenylglyoxal and citrulline under acidic conditions to design turn-on fluorescent probes for citrulline based on the donor-excited photoinduced electron transfer (d-PeT) mechanism. Among several derivatives of phenylglyoxal bearing a fluorescent moiety, we found that FGME enabled detection of citrulline without a neutralization process, and we used it to establish a simple methodology for turn-on fluorescence detection of citrulline.     

Identification and characterization of citrulline‐modified brain proteins by combining HCD and CID fragmentation

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high‐resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys‐C, and Glu‐C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher‐energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID‐triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.     

The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities

Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at + 1 and Tyr at + 3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from − 4 to + 4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.     

Peptidylarginine deiminases in citrullination,gene regulation,health and pathogenesis

Peptidylarginine deiminases are a family of enzymes that mediate post-translational modifications of protein arginine residues by deimination or demethylimination to produce citrulline. In vitro, the activity of PADs is dependent on calcium and reductive reagents carrying a free sulfhydryl group. The discovery that PAD4 can target both arginine and methyl-arginine for citrullination about 10 years ago renewed our interest in studying this family of enzymes in gene regulation and their physiological functions. The deregulation of PADs is involved in the etiology of multiple human diseases, including cancers and autoimmune disorders. There is a growing effort to develop isoform specific PAD inhibitors for disease treatment. However, the regulation of the activity of PADs in vivo remains largely elusive, and we expect that much will be learned about the role of these enzymes in a normal life cycle and under pathology conditions.     

Microbial pathways to subvert host immunity generate citrullinated neoantigens targeted in rheumatoid arthritis

The specific association between antibodies to citrullinated proteins and rheumatoid arthritis (RA) has centered interest on understanding why citrullinated proteins become immunogenic in this disease, which is believed to inform the origins of autoimmunity in RA. Since citrullination is a physiologic post-translational modification (PTM), one theory is that conditions promoting abnormal citrullination are initiators of self-reactive immune responses to citrullinated proteins in RA. Foremost candidates that dysregulate the normal balance of citrullination are microbial agents, which can exploit citrullination as an effector mechanism to subvert host antimicrobial activities and maximize their progeny. Here, we will use the host-pathogen interface as a unifying model to link microbe-induced citrullination and the loss of immunological tolerance to citrullinated antigens in RA.     

Proteomics of post-translational modifications in colorectal cancer: Discovery of new biomarkers

Colorectal cancer (CRC) is one of the costliest health problems and ranks second in cancer-related mortality in developed countries. With the aid of proteomics, many protein biomarkers for the diagnosis, prognosis, and precise management of CRC have been identified. Furthermore, some protein biomarkers exhibit structural diversity after modifications. Post-translational modifications (PTMs), most of which are catalyzed by a variety of enzymes, extensively increase protein diversity and are involved in many complex and dynamic cellular processes through the regulation of protein function. Accumulating evidence suggests that abnormal PTM events are associated with a variety of human diseases, such as CRC, thus highlighting the need for studying PTMs to discover both the molecular mechanisms and therapeutic targets of CRC. In this review, we begin with a brief overview of the importance of protein PTMs, discuss the general strategies for proteomic profiling of several key PTMs (including phosphorylation, acetylation, glycosylation, ubiquitination, methylation, and citrullination), shift the emphasis to describing the specific methods used for delineating the global landscapes of each of these PTMs, and summarize the recent applications of these methods to explore the potential roles of the PTMs in CRC. Finally, we discuss the current status of PTM research on CRC and provide future perspectives on how PTM regulation can play an essential role in translational medicine for early diagnosis, prognosis stratification, and therapeutic intervention in CRC.     

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.     

Citrulline-modified phage display: a novel high-throughput discovery approach for the identification of citrulline-containing ligands

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology.     

Identification of Novel Site‐Specific Alterations in the Modification Level of Myelin Basic Protein Isolated from Mouse Brain at Different Ages Using Capillary Electrophoresis–Mass Spectrometry

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. MBP is subjected to extensive posttranslational modifications (PTMs) that are known to be crucial for the regulation of these interactions. Here, we report capillary electrophoresis–mass spectrometric (CE–MS) analysis for the separation and identification of MBP peptides that incorporate the same PTM at different sites, creating multiple localization variants, and the ability to analyze challenging modifications such as asparagine and glutamine deamidation, isomerization, and arginine citrullination. Moreover, we observed site‐specific alterations in the modification level of MBP purified from brain of mice of different age. In total, we identified 40 modifications at 33 different sites, which include both previously reported and seven novel modifications. The identified modifications include Nα‐terminal acetylation, mono‐ and dimethylation, phosphorylation, oxidation, deamidation, and citrullination. Notably, some new sites of arginine methylation overlap with the sites of citrullination. Our results highlight the need for sensitive and efficient techniques for a comprehensive analysis of PTMs.     

ABAP: antibody-based assay for peptidylarginine deiminase activity

Members of the family of peptidylarginine deiminases (PADs, EC 3.5.3.15) catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies produced by rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expression levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Here we describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method is based on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that are exclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP, is very sensitive and can be applied to monitor PAD activity in biological samples.     

Discovery of peptidylarginine deiminase-4 substrates by protein array: antagonistic citrullination and methylation of human ribosomal protein S2

Peptidylarginine deiminase (PAD) catalyzes the posttranslational citrullination of selected proteins in a calcium dependent manner. The PAD4 isoform has been implicated in multiple sclerosis, rheumatoid arthritis, some types of cancer, and plays a role in gene regulation. However, the substrate selectivity of PAD4 is not well defined, nor is the impact of citrullination on many other pathways. Here, a high-density protein array is used as a primary screen to identify 40 previously unreported PAD4 substrates, 10 of which are selected and verified in a cell lysate-based secondary assay. One of the most prominent hits, human 40S ribosomal protein S2 (RPS2), is characterized in detail. PAD4 citrullinates the Arg-Gly repeat region of RPS2, which is also an established site for Arg methylation by protein arginine methyltransferase 3 (PRMT3). As in other systems, crosstalk is observed; citrullination and methylation modifications are found to be antagonistic to each other, suggesting a conserved posttranslational regulatory strategy. Both PAD4 and PRMT3 are found to co-sediment with the free 40S ribosomal subunit fraction from cell extracts. These findings are consistent with participation of citrullination in the regulation of RPS2 and ribosome assembly. This application of protein arrays to reveal new PAD4 substrates suggests a role for citrullination in a number of different cellular pathways.     

The Plant PTM Viewer,a central resource for exploring plant protein modifications

Post‐translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site‐specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry‐based PTM data to remain at pace with future PTM plant studies.