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.

Dr I-Cheng Ho, Brigham and Women’s Hospital, Harvard Medical School, US

Dr I-Cheng Ho, Brigham and Women’s Hospital, Harvard Medical School, US

Dr. I-Cheng Ho graduated from Taipei Medical University in 1986 and earned his PhD degree at University of Michigan in 1991 under the supervision of Dr. Jeffrey Leiden. He subsequently completed his postdoctoral training at the laboratory of Dr. Laurie Glimcher at Harvard School of Public Healthy in 1997. He received his clinical training at Taipei Veterans General Hospital, University of Michigan Hospital, and Brigham and Women's Hospital. He is currently a member of American Society of Clinical Investigation, an associate professor of Harvard Medical School, a board-certified rheumatologist at Brigham and Women's Hospital. His clinical interest is in rheumatoid arthritis and his current research focus is on the physiological and pathological roles of protein citrullination, particularly in the setting of autoimmune diseases. His past and current research supports include funding from Rheumatology Research Foundation, National Health Research Institutes of Taiwan, Department of Defense, and National Institutes of Health. 

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.

Dr Venizelos Papayannopoulos, The Francis Crick Institute, UK

Dr Venizelos Papayannopoulos, The Francis Crick Institute, UK

Veni did his undergraduate studies at Rutgers University, USA (1998) working with Ken Irvine on the mechanisms that establish tissue barriers during Drosophila development. He then moved to San Francisco to pursue a PhD with Wendell Lim at the University of California San Francisco (2004), studying protein-lipid signaling in the regulation of actin dynamics. For his post-doctoral work, he joined the lab of Arturo Zychlinsky in Berlin to investigate the molecular mechanisms driving the formation of neutrophil extracellular traps before becoming a group leader at the MRC National Institute for Medical Research and later the Francis Crick Institute in 2012.

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.

Dr Maria Genander, Karolinska Institutet, Sweden

Dr Maria Genander, Karolinska Institutet, Sweden

Maria’s scientific interests are centred around stem cell biology and the link to cancer. After elucidating mechanisms of intestinal stem cell proliferation during her PhD at Karolinska Institutet, Maria moved to Rockefeller University for her postdoctoral work in 2011, focusing on BMP-mediated hair follicle stem cell quiescence under the guidance of Elaine Fuchs. Maria returned to Karolinska Institutet and established her independent group in 2016, using the skin and esophageal epithelium as model systems to understand stem and tumour cell behaviour. In her talk, she will discuss some of the work that the lab is doing with regards to delineating the function of PADI4 in the hair follicle stem cell linage.

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.

Dr Maria Christophorou, Babraham Institute, UK

Dr Maria Christophorou, Babraham Institute, UK

Maria is a biomedical scientist interested in understanding how cell signaling inputs are translated into the epigenetic changes that underlie cell fate. She studied biology at MIT as a Fulbright Scholar and obtained a PhD in cancer biology from UCSF. As a postdoctoral fellow in epigenetics at the Gurdon Institute, University of Cambridge, she identified a novel role for protein citrullination in the establishment of pluripotency and described a mechanism by which it mediates chromatin decondensation. She is currently a Principal Investigator at the Babraham Institute in Cambridge, UK. The work of her team continues to focus primarily on protein citrullination, with the aim of understanding how it is regulated, the molecular mechanisms through which it modulates cell physiology and how its deregulation impacts on disease development. Maria’s work has been funded by fellowships from EMBO, HFSP, Wellcome Trust and Royal Society. She was awarded the Wellcome-Beit Prize in 2015. 

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.

Dr Priyanka Sharma, Montpellier Cancer Research Institute (IRCM), France

Dr Priyanka Sharma, Montpellier Cancer Research Institute (IRCM), France

Priyanka Sharma (ORCID 0000-0003-3890-3590) is a biomedical scientist, who recently (November 2020) joined the French National Institute of Health and Medical Research, working at the Montpellier cancer research centre, France. She completed her Ph.D. in biomedical science in India followed by her postdoctoral training at Institute Pasteur, Paris, France, and the Centre for Genomic Regulation, Barcelona, Spain with availed fellowships from Novartis and Marie Curie foundations. Her young team is focused to understand the molecular mechanisms to understand the transcriptional and epigenetic plasticity in cancer. 

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.

Dr Xuesen Zhang, Nanjing Medical University, China

Dr Xuesen Zhang, Nanjing Medical University, China

Dr Xuesen Zhang, a Professor in the Department of Biochemistry and Molecular Biology at Nanjing Medical University. He received his Bachelor’s degree and Master’s degree from Dalian Medical University in 1999 and 2002 respectively. Upon completion of his doctorate in Chinese Academy of Sciences in 2005, Dr Zhang accepted his postdoctoral training at the University of Iowa. In 2007, he joined Professor Scott Coonrod at Cornell University, and received more comprehensive postdoctoral training in the study of citrullination and breast cancer. In 2013, he was appointed as a Principal Investigator in Nanjing Medical University. Dr Zhang’s research has mainly focused on the role of non-histone protein citrullination in regulating tumor cell signaling pathways. He was awarded Susan G Komen for the Cure postdoctoral fellowship in 2010, and the Distinguished Professor of Jiangsu Province, China in 2016.

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. 

Professor Nicholas La Thangue FRSE, FMedSci, Department of Oncology, Oxford University, UK

Professor Nicholas La Thangue FRSE, FMedSci, Department of Oncology, Oxford University, UK

Nick is Professor of Cancer Biology in the Oncology Department at Oxford University. He is a translational cancer biologist with a deep interest in developing novel therapeutics. He is a Fellow of the Royal Society of Edinburgh, the Academy of Medical Sciences, the European Academy of Cancer Science, the Lister Institute and Professorial Fellow of Linacre College Oxford. He has over 300 publications and is an inventor on numerous patents.

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.

Professor Brian Cherrington, University of Wyoming, USA

Professor Brian Cherrington, University of Wyoming, USA

Brian Cherrington completed his undergraduate degree at Washington University in St Louis and then received a MSc and PhD in reproductive endocrinology from Colorado State University.  In his first postdoctoral research at the University of California, San Diego, he investigated gene programs in anterior pituitary gonadotrope cells. He then completed a second postdoctoral research at Cornell University investigating the function of peptidylarginine deiminase (PAD) enzymes in the mammary gland and breast cancer.  He joined the department of Zoology and Physiology at the University of Wyoming in 2011 and is currently an Associate Professor.  His lab studies the function of PAD enzymes and citrullinated proteins in gonadotrope cells and the female reproductive tract.

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