The virtues and vices of protein citrullination

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.

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. 

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

The selected poster abstracts will be available soon

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.

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.

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.

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.

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.

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.