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Overview

Theo Murphy meeting organised by Dr Maria Christophorou.

Citrullination is emerging as a fundamental regulatory mechanism in human physiology and pathophysiology, from immunity, neurobiology and embryonic development to rheumatology, neurodegeneration and cancer. Scientists from across these fields will come together for the first time to discuss the molecular and cellular biology of citrullination, approaches for its detection and modulation, future directions and implications for health and disease. 

Poster session and lightning talks

There will be an poster session on Monday 24 October at the meeting venue and the posters will be available to view for the duration of the meeting. There will also be the opportunity for poster presenters to give a short lightning talk on their poster during the meeting.

If you would like to apply to present a poster please submit your proposed title, abstract (not more than 200 words and in third person), author list, name of the proposed presenter and institution to the Scientific Programmes team no later than Monday 10 October 2022. Please include the text 'Poster abstract submission' in the email subject line and specify whether you would like to give a lightning talk or just present a poster. Please note that places are limited and posters are selected at the scientific organisers' discretion.

Attending this event

This meeting is intended for researchers in relevant fields, and is a residential meeting. This will be a residential meeting held at Eastwood Hall, Mansfield Road, Eastwood, Nottingham NG16 3SS.

  • Free to attend
  • Limited places, advance registration essential (more information about registration will be available soon)
  • This is an in-person meeting only

Enquiries: contact the Scientific Programmes team

Image credit: Johanna Grinat, Babraham Institute. Extracellular chromatin marked by citrullinated histone H3 (green). DNA in intact nuclei shown in blue. 

Organisers

Schedule


Chair

09:10-09:40
Is rheumatoid arthritis a disease of aberrant citrullination?

Abstract

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.

Speakers

09:40-09:50
Discussion
09:50-10:20
Role of PAD enzymes in development, reproduction, and cancer biology

Abstract

Peptidylarginine deiminases (PADs) convert arginine residues to citrulline in a process called citrullination or deamination. While the bulk of research on these enzymes has focused on their role in autoimmune disease, the author’s laboratory has focused more on the role of PADs in developmental biology, reproduction, and cancer. The research group found that PAD6 is primarily expressed in oocytes and early embryos and localizes to a poorly characterized structure called the cytoplasmic lattices. Analysis of PAD6 null mice found that PAD6 is required for the formation of these lattice structures and for the first cleavage division in developing embryos. The researchers also demonstrated that PAD2 and PAD4 play an important role in regulating estrogen signaling in breast cancer cells via citrullination of histones at target gene promoters. Further, the researchers showed that PAD2 facilitates estrogen receptor (ER)-DNA binding by directly interacting with ER at its binding sites and citrullinating histone H3 arginine 26, leading to a more open chromatin configuration. More recently, the research group has been investigating the phenotype of PAD2/PAD4 double KO mice. They found that, as opposed to single knockouts, deletion of both PAD2 and PAD4 suppresses male weanling weight, delays male puberty, and suppresses serum testosterone levels.

Speakers

10:20-10:30
Discussion
10:30-11:00
Break
11:00-11:30
PADI4 and hair follicle lineage progression

Abstract

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.

Speakers

11:30-11:40
Discussion
11:40-12:10
Citrullination in the regulation of cell fate

Abstract

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.

Speakers

12:10-12:20
Discussion
12:20-13:20
Lunch

Chair

13:20-13:50
Evolving Functions of Arginine Citrullination

Abstract

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.

Speakers

13:50-14:00
Discussion
14:00-14:30
Essential role of non-histone protein citrullination in maintaining the stemness of ovarian cancer cells

Abstract

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.

Speakers

14:30-14:40
Discussion
14:40-15:10
Break
15:10-15:40
Unravelling the role of citrullination in inflammatory disease and cancer

Abstract

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. 

Speakers

15:40-15:50
Discussion
15:50-16:20
Title of the talk will be available soon.

Abstract

Abstract will be available soon.

Speakers

16:20-16:30
Discussion
16:30-17:00
Lightning talks

Abstract

Poster presenters will be given the opportunity to apply to give a short talk.

The selected poster abstracts will be available soon

17:00-18:00
Poster session

Chair

09:00-09:30
Small molecule activation of protein citrullination

Abstract

Citrullination has been associated with various types of diseases including cancer and autoimmune disorders, while its role is not fully clarified especially in cancer. During past decades, a number of small molecules targeting protein citrullination have been reported, while most of them are inhibitors but not activators. The talk will focus on small molecule activation of protein citrullination, identification of new substrate of citrullination, as well as a rational screening and validation of small molecules activation of protein citrullination based on a natural product library. Discovery of small molecule targeting protein citrullination would be attractive not only to cue citrullination-dependent diseases, but also allow development of pharmacological approaches to investigate the role of citrullination in disease progression.

Speakers

09:30-09:40
Discussion
09:40-10:10
A cyclic peptide toolkit to modulate PADI4 activity

Abstract

Peptidyl arginine deiminase IV (PADI4) catalyses the citrullination of a wide range of substrates, with roles in neutrophil extracellular trap formation, chemokine signalling and establishment of pluripotency. Aberrant activity is implicated in a range of pathologies, including rheumatoid arthritis, multiple sclerosis, and various cancers, with emerging evidence suggesting PADI4 inhibition has significant therapeutic potential. In this talk, I will describe recent work to develop a toolkit of potent, selective cyclic peptide modulators of PADI4. We employed the RaPID (Random non-standard Peptide Integrated Discovery) system to isolate binders of PADI4 from starting libraries of up to 1013 genetically-barcoded cyclic peptides. From three parallel screens designed to target different protein conformations we identified a series of nanomolar PADI4 peptide binders, with diverse activities. A lead peptide was developed as a cell permeable PADI4 inhibitor, whilst another non-inhibitory peptide was pursued as an affinity probe. Further peptides were identified as first-in-class activators of PADI4 to low calcium conditions both in vitro and in cells. Together, the peptides provide a powerful toolkit for further elucidation of the function of PADI4 in both normal development and disease, with potential to provide a basis for downstream drug development. 

Speakers

10:10-10:20
Discussion
10:20-10:50
Break
10:50-11:20
Lightning talks

Abstract

Poster presenters will be given the opportunity to apply to give a short talk.

The selected poster abstracts will be available soon

11:20-11:30
Discussion
11:30-12:00
Deimination in epidermal barrier and hair formation

Abstract

The epidermis is a barrier for preventing the skin penetration of pathogens, exogenous molecules, including allergens and toxins, and UV radiation. It also avoids the uncontrolled loss of body water and solutes. For many years, the authors are interested in the role of citrullination in the homeostasis of the epidermal barrier, as well as in skin diseases. They identified several epidermal targets of peptidyl-arginine deiminase (PAD) 1 and 3, including members of the S100-fused type protein family. The authors showed how deimination controls their fate in the upper epidermis, and how this process is regulated by the external environment. Their data also suggest that deimination is a key regulator of autophagy processes associated with epidermal keratinocyte differentiation. The authors recently demonstrated that deimination rate is reduced in the epidermis of patients affected by psoriasis, a common inflammatory skin condition, through the action of IL-22. Deimination is also important in hair follicle formation, particularly the trichohyalin-PAD3-transglutaminase pathway. Indeed, they demonstrated that mutations in PADI3 are responsible for two hair disorders, the uncombable hair syndrome and a particular form of alopecia exclusively affecting women of African ancestry, the Central Centrifugal Cicatricial Alopecia.

Speakers

12:00-12:10
Discussion
12:10-12:40
PAD4, histone citrullination and NETs in cardiovascular disease

Abstract

Neutrophil extracellular traps can play a pathological role in human disease, having been shown to contribute to the detrimental host response in sepsis and in thrombus formation in both arterial and venous thrombosis. Citrullinated histones, generated during NET formation by the enzyme peptidylarginine deaminase 4 (PAD4) serve as useful biomarkers of ongoing NET formation. Studies using mice fully deficient in PAD4 showed a protection from thrombus formation in a model of deep vein thrombosis, reduced coagulation and immunothrombosis in systemic infection, yet no increase in bacteremia in a model of polymicrobial sepsis. The authors have also seen that PAD4-/- mice maintain normal cardiac function in aging or after pressure overload injury, which they have recently shown to be neutrophil-dependent. In addition to the direct effect of NETs, PAD4 released on NETs can also modify proteins involved in regulation of blood clotting, including the VWF-cleaving enzyme ADAMTS13. PAD4 therefore provides a potential therapeutic target for NET-related diseases involving immunothrombosis as a driving mechanism.

Speakers

12:40-12:50
Discussion
12:50-13:50
Lunch

Chair

13:50-14:20
Global and site-specific mapping of the human citrullinome reveals broad scope of PADI4-induced citrullination

Abstract

Citrullination refers to the conversion of arginine into the non-essential amino acid citrulline. Despite its importance in physiology and disease, global identification of citrullinated proteins and modification sites has remained challenging. Here, the authors combined mass spectrometry-based proteomics with differentiation of leukemia cell line into neutrophil-like cells to reveal a comprehensive atlas of regulated citrullination sites. Collectively, the authors identified >13.000 citrullination sites within >3.500 proteins allowing deduction of general principles about the mechanistic and cell biological aspects of citrullination function. Using quantitative proteomics the authors monitored citrullination changes in response to the PADI4-specific inhibitor GSK484, providing site-specific regulation of thousands of PADI4 substrates, including signature histone marks and numerous non-histone events on transcriptional regulators and chromatin-related signalling effectors. Collectively, the authors describe systems attributes of the human citrullinome, reveal the existence of thousands citrullinated autoantigens in neutrophil cells, and provide a resource framework for investigating PADI4-specific functions and substrates for years to come.

Speakers

14:20-14:30
Discussion
14:30-15:00
Citrullination of neutrophil extracellular traps and autoimmunity

Abstract

Neutrophil extracellular traps (NETs) are fibrous networks of chromatin and antibacterial proteins that are extruded from neutrophils in order to capture pathogens and prevent them from spreading. Soon after their identification in 2004 it was suggested that NETs might contain antigens capable of inducing autoimmune responses. NETs contain many autoantigenic molecules such as DNA, histones and other intracellular proteins that are usually shielded from the immune system, but become exposed when NETs are formed. NET formation can be categorized based on the effects of different NET-inducing stimuli and the dependence on citrullination, which particularly involves PAD4-mediated citrullination of histone H3.

Using two distinct assays the authors investigated the reactivity of rheumatoid arthritis (RA) patient sera with NETs. Anti-NET antibodies were detected in 51% of the RA patients and this may at least in part be due to the frequent presence of anti-citrullinated protein antibodies (ACPA) in RA. Histone H3 citrullination appeared to be dependent on the NET-inducing stimulus and neutrophil protease activity reduced the levels of NET-associated citrullinated H3. Moreover, their data demonstrate that a large number of NET-associated autoepitopes can be removed by neutrophil proteases. The consequences for the autoimmune response against citrullinated proteins will be discussed.

Speakers

15:00-15:10
Discussion
15:10-15:30
Break
15:30-16:00
Insights into RA pathogenesis from the study of anti-PAD immune responses

Abstract

Immune responses to peptidylarginine deiminase (PAD) enzymes, proteins that catalyze the post-translational deamination of arginine residues in a process known as citrullination, have been discovered in subsets of patients with rheumatoid arthritis (RA). Among these, anti-PAD4 immune responses are the most widely studied and include both autoreactive CD4+ T cell and autoantibody responses. Anti-PAD4 antibodies are present in 25-40% of RA patients, are detectable in the pre-clinical phase prior to disease diagnosis, and associate with erosive joint damage. A subset of anti-PAD4 antibodies cross-react with the related enzyme PAD3 (termed anti-PAD3/4 antibodies), and associate with the most severe erosive joint disease and presence of interstitial lung disease. Anti-PAD3/4 antibodies possess the unique ability to activate PAD4 upon binding, resulting in the increased citrullination of physiologic substrates. Anti-PAD4 and PAD3/4 antibodies cloned from patients with RA are able to bind directly to the surface of monocytes, via recognition of PAD4 expressed on the cell surface, and trigger pro-inflammatory chemokine secretion and exacerbation of inflammatory arthritis in the collagen-induced arthritis mouse model. The ability of anti-PAD4 antibodies to augment citrullination and activate monocytes suggests that they may be direct pathogenic players in amplifying disease in patients with RA.

Speakers

16:00-16:10
Discussion
16:10-16:40
Protein citrullination is a novel therapeutic target for inflammatory diseases

Abstract

The Protein Arginine Deiminases (PADs) hydrolyse arginine residues to form citrulline. This post-translational modification, termed citrullination, is upregulated in several autoimmune disorders including Rheumatoid Arthritis (RA), lupus, and Alzheimer’s disease. While these enzymes are important regulators of gene transcription, the full spectrum of biological activities is relatively underexplored. The author will discuss his development of pan and isozyme specific PAD inhibitors and their use in vitro and in vivo. Also, he will describe his development of phenylglyoxal-based probes, which chemoselectively react with citrulline to facilitate the labelling and enrichment of the RA-associated citrullinome. Notably, citrullination modulates the activity of metabolic enzymes and serine protease inhibitors (SERPINs), including nicotinamide N-methyltransferase and numerous SERPINs (eg C1 inhibitor, anti-thrombin, and anti-plasmin). Inhibition of SERPIN activity is highly significant as it triggers increased protease activity, thereby activating multiple processes including tissue remodelling, altered blood coagulation, and the activation of the complement cascade. Finally, the author will describe their efforts to site-specifically incorporate citrulline into proteins.

Speakers

16:40-16:50
Discussion
16:50-17:10
Closing remarks and future directions

Speakers