The virtues and vices of protein citrullination

Rheumatoid arthritis (RA), an autoimmune disease with a worldwide incidence up to 0.24%, is characterized by the presence of anti-citrullinated protein antibodies. Citrullination is a unique form of post-translational modification of proteins. Its physiological roles are still not fully understood. Several major environmental and genetic risk factors of RA, such as cigarette smoking and the C1858T single nucleotide polymorphism of the PTPN22 gene, are associated with local or systemic hypercitrullination. Emerging data has demonstrated that hypercitrullination not only expands the pool of citrullinated antigens but also modulates the function of innate and adaptive immune cells, thereby directly or indirectly contributing to the pathogenesis of RA. The functional impacts and mechanisms of action of citrullination in neutrophils and T lymphocytes will be reviewed and discussed. In addition, new evidence demonstrating a critical role of citrullination in regulating the function of B lymphocytes will be presented. The audience is expected to gain a global view of how aberrant citrullination participates in various steps towards the development of RA.

Neutrophils, are key innate immune antimicrobial effector cells that were once only appreciated for their antimicrobial properties. In addition, neutrophil tune inflammation by producing their own cytokines during infection or via the release of large web-like extracellular chromatin structures called neutrophil extracellular traps (NETs) that activate macrophages, monocytes and dendritic cells. NET-mediated macrophage activation drives the early phases of atherogenesis. Protein citrullination has gained attention in NET studies due to the role of protein arginine deiminase 4 (PAD4) in histone citrullination, a process that is thought to be required for chromatin decondensation. The talk will explore how histones, DNA, citrullination, and chromatin fragmentation synergize downstream of NET formation to drive inflammation below the histone cytotoxicity threshold. The synergy between histones and DNA is critical for sub-lethal histone-mediated signaling and relies on distinct roles for citrullinated histones and DNA. Histones bind and activate TLR4, whereas DNA recruits TLR4 to histone-containing endosomes. Citrullination is dispensable for NETosis but potentiates histone-mediated signaling. Consistently, chromatin blockade or PAD4 deficiency reduces atherosclerosis. Additional pathologic functions of extracellular histones as regulators of neutrophil populations during severe infections will also be discussed.

Stem and progenitor cell state transitions are closely controlled to enable tissue formation. How PADIs and citrullination contribute to lineage progression is largely unknown. The researchers find that PADI4 is dynamically regulated during hair follicle formation, and that PADI4 mRNA is enriched in a subset of progenitor cells in the hair bulb. Loss of PADI4 in vivo results in increased progenitor proliferation and retarded commitment to the hair shaft lineage, suggesting that PADI4 acts to balance progenitor proliferation and commitment to differentiation. Characterization of the PADI4-dependent citrullinome reveals protein targets linked to mRNA processing and translational control, and in vivo OP-Puro incorporation confirm that translation rates are increased in the absence of PADI4. Mechanistically, lack of PADI4 leads to phosphorylation of AKT and S6 whereas paradoxically, activity of the downstream translational initiator 4E-BP1 is reduced, suggesting a PADI4-dependent rewiring of the translational machinery. Collectively, the researchers' findings suggest a role for PADI4 in orchestrating hair follicle lineage progression through fine tuning of the translational landscape.

The discovery that somatic cells can be reprogrammed to a pluripotent state and instructed to differentiate into various cell types promises to revolutionise regenerative medicine. However, in vitro reprogramming is inefficient and we lack a clear mechanistic understanding of the reprogramming process and rational approaches to enhance its efficiency. The author discovered that the citrullinating enzyme peptidylarginine deiminase IV (PADI4) regulates the establishment of pluripotency during mammalian pre-implantation development and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. In the course of reprogramming, citrullination of histone H3 is an early event that precedes the expression of pluripotency markers and the establishment of iPS colonies. Inhibition of H3 citrullination via pharmacological or genetic perturbation of PADI4 inhibits reprogramming. In addition, the researchers find that deletion of Padi4 biases lineage commitment of embryonic stem cells during differentiation. The author will discuss their unpublished work investigating how PADI4 mediates the integration of cell signalling inputs and their translation into the epigenetic and transcriptional changes that underlie cell identity transitions during embryonic development and cell reprogramming.

Protein citrullination (deimination) is the post-translational modification of arginine to the non-coded amino acid citrulline, catalyzed by the peptidyl arginine deiminases (PADIs) enzyme family. Of the five PADIs, PADI2 is widely expressed, and it regulates essential normal and pathological cellular processes that impact multiple cancers. How PADI2 has evolved in mammals to gain its fundamental function is not fully understood. By performing a systematic evolutionary analysis, the group of researchers identified positively selected residues in PADI2, the majority of them are structurally exposed mainly on the N-terminal and middle domains of PADI2. Their data suggests that these residues have roles in maintaining the PADI2 interactions with cognate proteins. In particular, the researchers demonstrate experimentally that the positively selected leucine 162 (L162) encompassing loop in the middle domain participates in the interaction of PADI2 with the positive-transcription elongation factor (P-TEFb) complex, which is essential for the active transcription and cellular proliferation. Thus, the author will discuss this work on PADI2 evolution that revealed the function of the PADI2 non-catalytic domain in modulating transcription. This knowledge could be key to understanding the PADI2 function across several cancers as well as pinpointing potential targetable regions of the protein.

There is growing evidence from basic to clinical studies that supports the crucial roles of PADIs-catalyzed protein citrullination associated with the onset and progression of cancers. In their previous studies, the authors have demonstrated that PADI2-catalyzed MEK1 citrullination activates ERK1/2 phosphorylation, which promotes IGF2BP1-mediated Sox2 mRNA stability, a key stemness-related factor, in endometrial cancer, thus enhancing a tumor-initiating cell phenotype. This novel finding indicated that PADIs-mediated kinases citrullination in tumours can affect the tumorigenesis thorough targeting cancer stem cells. Interestingly, in their recent study, the authors found that PADI1 enzymatic activity was also required to maintain the stemness characteristics and malignancy of ovarian cancer cells. Specifically, PADI1-catalyzed AKT2 citrullination facilitates its phosphorylation, which activates transcription factor CEBPB to promote the transcription of stemness genes. Meanwhile, the five-year survival rate of patients with high PADI1 expression in primary ovarian cancer was significantly lower than that of patients with low PADI1 expression, further highlighting the crucial role of PADI1 in the pathogenesis of ovarian cancer. Exploring the mechanism of drug resistance in ovarian cancer and seeking new effective therapeutic potential targets is of great significance to improve the survival rate of these patients. This study may help establish PADI1 as a novel therapeutic target.

Increasing evidence suggests that citrullination is a key process involved in inflammatory disease and cancer. The aim of this study is to understand the role of citrullination by identifying key citrullinated targets and relate this information to the disease context.  E2F is a family of master transcription regulators involved in regulating diverse cellular fates. In previous studies the authors have found that the E2F1 subunit is regulated through post-translational events, which includes citrullination, mediated by peptidyl arginine deiminase (PAD) 4. Subsequently, they used genome-wide analysis to study gene expression changes dependent on PAD4 and E2F1 and identified an unexpected level of control on RNA processing together with citrullinated RNA binding proteins that mediate the events.  Notably, important differences were apparent when either inflammatory or cancer cells were compared to normal cells. The results suggest that PAD4 has widespread effects on gene expression mediated through the E2F pathway and candidate RNA binding proteins, and that gene expression is affected at multiple levels, including transcription and RNA processing. 

Some of the first research in the peptidylarginine deiminase (PAD) biology field characterized these enzymes in the female reproductive tract and anterior pituitary gland. Anterior pituitary gland gonadotrope cells synthesize and secrete luteinizing hormone (LH) and follicle stimulating hormone (FSH), which are required for follicle development and ovulation in all female mammals. In mice, PAD2 expression is elevated in gonadotrope cells during the estrus phase of the estrous cycle, where it citrullinates histones. The research data shows that histone citrullination suppresses the expression of DiGeorge Syndrome Critical Region 8 (DGCR8) microprocessor complex subunit, a riboprotein required for canonical miRNA biogenesis. PAD mediated changes in DGCR8 expression in gonadotropes during the estrous cycle lead to fluctuations in miRNA biogenesis, which modulates female reproduction. The authors are also investigating the function of PADs and citrullinated proteins in the human cervicovaginal (CV) mucosa. Women have a 3-fold higher incidence of rheumatoid arthritis (RA) compared to men, and risk factors for RA include age of menarche, parity, and the postpartum period. RA can be clinically diagnosed by the presence of anti-citrullinated protein antibodies (ACPA) in blood. While studies support that ACPA form at mucosal sites, this work has focused on the lung, gingiva, or gut mucosa, none of which explains the sex differences underlying the increased prevalence of RA in women. The authors' work shows that fluid from the healthy CV mucosa contains active PAD enzymes, cit-proteins, and ACPA that fluctuate across the menstrual cycle. Using mass spectrometry, the authors identified the CV mucosa citrullinome, which differs between samples from healthy women and those at risk for and with RA. The research data suggests that PADs and cit-proteins are normally released at the CV mucosa during times of mucosal tissue disruption and inflammation; yet, as this process repeats with each menstrual cycle, during pregnancy and postpartum, it may increase the likelihood of RA in women.

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