FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

Cryoelectron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination.

Nucleosome chains fold and self-associate to form higher-order structures whose internal organization is unknown. Here, cryoelectron tomography (cryo-ET) of native human chromatin reveals intrinsic folding motifs such as (1) non-uniform nucleosome stacking, (2) intermittent parallel and perpendicular orientations of adjacent nucleosome planes, and (3) a regressive nucleosome chain path, which deviates from the direct zigzag topology seen in reconstituted nucleosomal arrays. By examining the self-associated structures, we observed prominent nucleosome stacking in cis and anti-parallel nucleosome interactions, which are consistent with partial nucleosome interdigitation in trans. Histone citrullination strongly inhibits nucleosome stacking and self-association with a modest effect on chromatin folding, whereas the reconstituted arrays undergo a dramatic unfolding into open zigzag chains induced by histone citrullination. This study sheds light on the internal structure of compact chromatin nanoparticles and suggests a mechanism for how epigenetic changes in chromatin folding are retained across both open and condensed forms.

Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.

The post-translational modification of key residues at the C-terminal domain of RNA polymerase II (RNAP2-CTD) coordinates transcription, splicing, and RNA processing by modulating its capacity to act as a landing platform for a variety of protein complexes. Here, we identify a new modification at the CTD, the deimination of arginine and its conversion to citrulline by peptidyl arginine deiminase 2 (PADI2), an enzyme that has been associated with several diseases, including cancer. We show that, among PADI family members, only PADI2 citrullinates R1810 (Cit1810) at repeat 31 of the CTD. Depletion of PADI2 or loss of R1810 results in accumulation of RNAP2 at transcription start sites, reduced gene expression, and inhibition of cell proliferation. Cit1810 is needed for interaction with the P-TEFb (positive transcription elongation factor b) kinase complex and for its recruitment to chromatin. In this way, CTD-Cit1810 favors RNAP2 pause release and efficient transcription in breast cancer cells.

The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis.

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

Candida albicans induces neutrophil extracellular traps and leucotoxic hypercitrullination via candidalysin.

The peptide toxin candidalysin, secreted by Candida albicans hyphae, promotes stimulation of neutrophil extracellular traps (NETs). However, candidalysin alone triggers a distinct mechanism for NET-like structures (NLS), which are more compact and less fibrous than canonical NETs. Candidalysin activates NADPH oxidase and calcium influx, with both processes contributing to morphological changes in neutrophils resulting in NLS formation. NLS are induced by leucotoxic hypercitrullination, which is governed by calcium-induced protein arginine deaminase 4 activation and initiation of intracellular signalling events in a dose- and time-dependent manner. However, activation of signalling by candidalysin does not suffice to trigger downstream events essential for NET formation, as demonstrated by lack of lamin A/C phosphorylation, an event required for activation of cyclin-dependent kinases that are crucial for NET release. Candidalysin-triggered NLS demonstrate anti-Candida activity, which is resistant to nuclease treatment and dependent on the deprivation of Zn2+ . This study reveals that C. albicans hyphae releasing candidalysin concurrently trigger canonical NETs and NLS, which together form a fibrous sticky network that entangles C. albicans hyphae and efficiently inhibits their growth.

Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration.

Citrullinated human and murine MOG35-55 display distinct biophysical and biochemical behavior.

The peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied extensively in its role as a key autoantigen in the neuroinflammatory autoimmune disease multiple sclerosis. Rodents and nonhuman primate species immunized with this peptide develop a neuroinflammatory condition called experimental autoimmune encephalomyelitis, often used as a model for multiple sclerosis. Over the last decade, the role of citrullination of this antigen in the disease onset and progression has come under increased scrutiny. We recently reported on the ability of these citrullinated MOG35-55 peptides to aggregate in an amyloid-like fashion, suggesting a new potential pathogenic mechanism underlying this disease. The immunodominant region of MOG is highly conserved between species, with the only difference between the murine and human protein, a polymorphism on position 42, which is serine in mice and proline for humans. Here, we show that the biophysical and biochemical behavior we previously observed for citrullinated murine MOG35-55 is fundamentally different for human and mouse MOG35-55. The citrullinated human peptides do not show amyloid-like behavior under the conditions where the murine peptides do. Moreover, we tested the ability of these peptides to stimulate lymphocytes derived from MOG immunized marmoset monkeys. While the citrullinated murine peptides did not produce a proliferative response, one of the citrullinated human peptides did. We postulate that this unexpected difference is caused by disparate antigen processing. Taken together, our results suggest that further study on the role of citrullination in MOG-induced experimental autoimmune encephalomyelitis is necessary.

Citrullination, a novel post-translational modification of elastin, is involved in COPD Pathogenesis.

Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. The current study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

BACKGROUND/AIMS: Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. METHODS: To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. RESULTS: All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. CONCLUSION: Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Vitamin B12 inhibits peptidylarginine deiminases and ameliorates rheumatoid arthritis in CAIA mice.

Rheumatoid arthritis is an autoimmune disease whose early onset correlates with dysregulated citrullination, a process catalyzed by peptidylarginine deiminase isoform 4 (PADI-4). Here, we report that PADI-4 is a novel target of vitamin B12, a water-soluble vitamin that serves as a cofactor in DNA synthesis and the metabolism of fatty and amino acids. Vitamin B12 preferentially inhibited PADI-4 over PADI-2 with comparable inhibitory activity to the reference compound Cl-amidine in enzymatic inhibition assays, and reduced total cellular citrullination levels including that of histone H3 citrullination mediated by PADI-4. We also demonstrated that hydroxocobalamin, a manufactured form of vitamin B12, significantly ameliorated the severity of collagen type II antibody induced arthritis (CAIA) in mice and diminished gene expression of the rheumatoid inflammatory factors and cytokines IL17A, TNFα, IL-6, COX-II and ANXA2, as well PADI-4. Therefore, the use of vitamin B12 to treat rheumatoid arthritis merits further study.

Neutrophils in Inflammatory Bone Diseases.

PURPOSE OF REVIEW: In this review, we summarize the current evidence that suggests that neutrophils play a key role in facilitating damage to local bone structures. RECENT FINDINGS: Neutrophil infiltration is a hallmark of inflammatory bone diseases such as rheumatoid arthritis (RA) and periodontitis disease (PD). Both of these human diseases are marked by an imbalance in bone homeostasis, favoring the degradation of local bone which ultimately leads to erosions. Osteoclasts, a multinucleated resident bone cell, are responsible for facilitating the turnover of bone and the bone damage observed in these diseases. The involvement of neutrophils and neutrophil extracellular trap formation have recently been implicated in exacerbating osteoclast function through direct and indirect mechanisms. We highlight a recent finding that NET proteins such as histones and elastase can generate non-canonical, inflammatory osteoclasts, and this process is mediated by post-translational modifications such as citrullination and carbamylation, both of which act as autoantigens in RA. It appears that NETs, autoantibodies, modified proteins, cytokines, and osteoclasts all ultimately contribute to local and permanent bone damage in RA and PD. However, more studies are needed to fully understand the role of neutrophils in inflammatory bone diseases.

The influence of estrogen on myocardial post-translational modifications and cardiac function in women.

The lifetime risk of heart failure (HF) is comparable in men and women; nevertheless, disparities exist in our understanding of how HF differs between sexes. Several differences in cardiac physiology exist between men and women including the propensity to develop specific HF phenotypes. Men are more likely to be diagnosed with HF failure with reduced ejection fraction, while women have a greater propensity to develop HF with preserved ejection fraction. The mechanisms responsible for these differences remain unclear. Post-translational modifications (PTMs) of myofilament proteins likely contribute to these sex-specific propensities. The role of PTMs in heart disease is an expanding field with immense potential therapeutic targets. However, numerous PTMs remain underexplored, particularly in the context of the female heart. Estrogen, a key gonadal hormone, cardioprotective in pre-menopausal women and its loss with menopause likely contributes to disease in aging women. However, how estrogen regulates PTMs to contribute to HF development is not fully clear. This review outlines key sex differences in HF along with characterizing the contributions of novel myocardial PTMs in cardiac physiology and their regulation by estrogen. Collectively, we highlight the necessity for further investigation into women's heart health and the distinctive mechanisms distinguishing women from men.

Identification of autoantibodies against PsoP27 in synovial fluid derived from psoriatic arthritis and rheumatoid arthritis patients.

PsoP27 is an antigen expressed in psoriatic lesions. It plays an inflammatory role in psoriasis. This study objective was to characterize antibodies (Abs) against PsoP27 in patients with psoriatic arthritis (PsA) and rheumatoid arthritis (RA). Levels of Abs against native and citrullinated PsoP27 in PsA and RA patients' synovial fluid (SF) and sera were determined by ELISA. SF of osteoarthritis (OA) patients and sera of healthy donors were used as controls. Levels of Abs against PsoP27 were correlated with disease activity scores. Abs against native and citrullinated PsoP27 levels in SF of PsA (n = 48; 0.38 ± 0.03 and 0.44 ± 0.04, respectively) and RA (n = 22; 0.57 ± 0.1 and 0.62 ± 0.09, respectively) were significantly higher than in OA patients (n = 23; 0.14 ± 0.01 and 0.15 ± 0.01, respectively) (p < .0001). For both Abs, there were no significant differences between their level in PsA and RA patients. There was no difference in the level of Abs against citrullinated PsoP27 in SF of seronegative versus seropositive RA patients. Levels of Abs against both native and citrullinated PsoP27 in the SF and level of systemic C-reactive protein in PsA correlated positively, while in RA there were no significant correlations with disease activity scores. No differences in level of Abs against PsoP27 were found in the sera of all three study groups. Abs against native and citrullinated PsoP27 are present in PsA and RA SF but not in those of OA patients, suggesting a potential role of those Abs in inflammatory joint diseases.

Post-translational modifications such as citrullination are excellent targets for cancer therapy.

Under conditions of cellular stress, proteins can be post-translationally modified causing them to be recognized by the immune system. One such stress-induced post-translational modification (siPTM) is citrullination, the conversion of arginine residues to citrulline by peptidylarginine deiminase (PAD) enzymes. PAD enzymes are activated by millimolar concentrations of calcium which can occur during apoptosis, leading to precipitation of proteins, their subsequent uptake by B cells and stimulation of antibody responses. Detection of anti-citrullinated protein antibodies (ACPAs) is a diagnostic of rheumatoid arthritis (RA), where immune complexes stimulate inflammation around the joints. More recently, autophagy has been shown to play a role in the presentation of citrullinated peptides on MHC class II molecules to CD4+ helper T cells, suggesting that citrullination may be a way of alerting immune cells to cellular stress. Additionally, inflammation-induced IFNγ and concomitant MHC class II expression on target cells contributes to immune activation. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a pro-survival mechanism. Cancer cells also over express PAD enzymes and in light of this the hypothesis that citrullinated peptides stimulate CD4+ T cell responses that would recognize these siPTM's produced during autophagy has been investigated. The induction of potent citrullinated peptide-specific CD4 responses has been shown in both humans and HLA transgenic mouse models. Responses in mouse models resulted in potent anti-tumour responses against tumours expressing either constitutive or IFNγ-inducible MHC class II. The anti-tumour effect relied upon direct recognition of tumours by specific CD4 T cells suggesting that citrullinated peptides are attractive targets for cancer vaccines.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I.

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

Unveiling Drivers of Retinal Degeneration in RCS Rats: Functional, Morphological, and Molecular Insights.

Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, significantly contribute to adult blindness. The Royal College of Surgeons (RCS) rat is a well-established disease model for studying these dystrophies; however, molecular investigations remain limited. We conducted a comprehensive analysis of retinal degeneration in RCS rats, including an immunodeficient RCS (iRCS) sub-strain, using ocular coherence tomography, electroretinography, histology, and molecular dissection using transcriptomics and immunofluorescence. No significant differences in retinal degeneration progression were observed between the iRCS and immunocompetent RCS rats, suggesting a minimal role of adaptive immune responses in disease. Transcriptomic alterations were primarily in inflammatory signaling pathways, characterized by the strong upregulation of Tnfa, an inflammatory signaling molecule, and Nox1, a contributor to reactive oxygen species (ROS) generation. Additionally, a notable decrease in Alox15 expression was observed, pointing to a possible reduction in anti-inflammatory and pro-resolving lipid mediators. These findings were corroborated by immunostaining, which demonstrated increased photoreceptor lipid peroxidation (4HNE) and photoreceptor citrullination (CitH3) during retinal degeneration. Our work enhances the understanding of molecular changes associated with retinal degeneration in RCS rats and offers potential therapeutic targets within inflammatory and oxidative stress pathways for confirmatory research and development.

Insights into the study and origin of the citrullinome in rheumatoid arthritis.

The presence of autoantibodies and autoreactive T cells to citrullinated proteins and citrullinating enzymes in patients with rheumatoid arthritis (RA), together with the accumulation of citrullinated proteins in rheumatoid joints, provides substantial evidence that dysregulated citrullination is a hallmark feature of RA. However, understanding mechanisms that dysregulate citrullination in RA has important challenges. Citrullination is a normal process in immune and non-immune cells, which is likely activated by different conditions (eg, inflammation) with no pathogenic consequences. In a complex inflammatory environment such as the RA joint, unique strategies are therefore required to dissect specific mechanisms involved in the abnormal production of citrullinated proteins. Here, we will review current models of citrullination in RA and discuss critical components that, in our view, are relevant to understanding the accumulation of citrullinated proteins in the RA joint, collectively referred to as the RA citrullinome. In particular, we will focus on potential caveats in the study of citrullination in RA and will highlight methods to precisely detect citrullinated proteins in complex biological samples, which is a confirmatory approach to mechanistically link the RA citrullinome with unique pathogenic pathways in RA.

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.

Immune responses to citrullinated and homocitrullinated peptides in healthy donors are not restricted to the HLA SE shared allele and can be selected into the memory pool.

Citrullination and homocitrullination are stress induced post-translational modifications (siPTMs) which can be recognized by T cells. Peripheral blood mononuclear cells isolated from healthy donors and rheumatoid arthritis (RA) patients were stimulated with nine siPTM-peptides. CD45RA/CD45RO depletion was employed to determine if peptide-specific responses are naïve or memory. Human leucocyte antigen (HLA)-DP4 and HLA-DR4 transgenic mice were immunized with siPTM-peptides and immune responses were determined with ex vivo ELISpot assays. The majority (24 out of 25) of healthy donors showed CD4 T cell-specific proliferation to at least 1 siPTM-peptide, 19 to 2 siPTM-peptides, 14 to 3 siPTM-peptides, 9 to 4 siPTM-peptides, 6 to 5 siPTM-peptides and 4 to 6 siPTM-peptides. More donors responded to Vim28-49cit (68%) and Bip189-208cit (75%) compared with Vim415-433cit (33%). In RA patients, the presentation of citrullinated epitopes is associated with HLA-SE alleles; however, we witnessed responses in healthy donors who did not express the SE allele. The majority of responding T cells were effector memory cells with a Th1/cytotoxic phenotype. Responses to Vim28-49cit and Eno241-260cit originated in the memory pool, while the response to Vim415-433cit was naïve. In the HLA-DP4 and HLA-DR4 transgenic models, Vim28cit generated a memory response. Peptide-specific T cells were capable of Epstein-Barr virus transformed lymphoblastoid cell line recognition suggesting a link with stress due to infection. These results suggest siPTM-peptides are presented under conditions of cellular stress and inflammation and drive cytotoxic CD4 T cell responses that aid in the removal of stressed cells. The presentation of such siPTM-peptides is not restricted to HLA-SE in both humans and animal models.