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Description
Rheumatoid arthritis (RA) is one of the most widespread autoimmune conditions, affecting ~1% of population worldwide. The inflammation of the joints in RA is driven by autoantibodies against citrullinated epitopes (ACPA). Proteins citrullination is a posttranslational modification catalyzed by peptidylarginine deiminases (PAD1, PAD2, PAD3, PAD4 and PAD6) converting positively charged Arg residues to neutral citrulline residues. Although citrullination occurs both in physiological and pathological conditions, ACPA are found exclusively in RA patients and appear in circulation years before clinical onset of the disease. It is accepted that in subjects with specific genetic risk factors immunotolerance breakdown leading to ACPA generation happens on inflamed mucosal surfaces where excessive protein citrullination is carried out by PAD2 or PAD4. However, despite intense research on PADs it is still a mystery how citrullination happens in vivo. PADs activity is calcium-dependent and requires non-physiologically high concentration of Ca2+ (above 5 mM). Up to date, an intense search for PAD cofactors lowering their Ca2+ requirement failed. The groundbreaking character of the project is based on our seminal finding that a group of glycosaminoglycans (GAG), including heparin, heparan sulphate and chondroitin are PAD activators, which lower the Ca2+ requirement of these enzymes to physiological levels, explaining in vivo activity of PADs. The experimental results are fully supported by initial structural docking/modelling studies in silico showing the potential high-affinity heparin binding pockets formed by a cluster of positively charged residues within the PAD2 and PAD4 structure. The aim of the project is to identify and characterize the binding pocket(s) responsible for activation, which will allow targeted design of the novel PAD-specific, allosteric blockers with therapeutic potential, based on previously unidentified activation mechanism.
Summary of project results
Citrullination is a common posttranslational modification of proteins and peptides. The reaction is catalyzed by catalyzed by family of peptidyl arginine deiminase enzymes (PADs) converting arginine into citrulline is essential for many physiological processes including the formation of rigid structures such as myelin sheath, skin and hair, regulation of gene expression and innate immunity (Neutrophil Extracellular Traps formation). Aberrant citrullination has been observed in diseases of the nervous system, the skin and in rheumatoid arthritis (RA). To carry out reaction in vitro PADs require calcium concentration exceeding its concentration in pathophysiological fluids. Involvement in PADs cofactor allowing activation of the enzyme in vivo was postulated but unproven. The project was based on a novel and ground-braking finding that PAD4, the enzymes indicated in the pathogenesis of RA, specifically bind heparin and other glycosaminoglycans (GAGs), which serve as co-factors allowing enzyme activity at low calcium concentration. Project therefore aimed at the elucidation of the molecular mechanism of the PAD activation by GAGs in an attempt to describe molecular mechanisms governing PAD activity and open new prospects to develop therapeutics to treat RA in a future. Specifically, project aimed at: Kinetic characterization of the GAG- dependent activation of PAD4; analysis of PAD4 substrate specificity; analysis of the PAD4-GAG interaction in the biological context and analysis of the GAG content and PAD4 activating properties in the sera of RA patients.
We investigated the binding and activation of PAD4 by heparin. PAD4 showed specific binding to heparin, forming larger complexes in its presence. Heparin activated PAD4 with a dissociation constant (Kd) ~ 1 nM and reduced the calcium concentration needed for activation. MST and SPR confirmed a Kd around 8 nM. Heparin enabled full citrullination of histone H3 at lower calcium levels and stabilized PAD4. The effectiveness of heparin depended on its chain length and charge. Other glycosaminoglycans (GAGs) like dermatan sulphate and heparan sulphate also activated PAD4. The study concluded that heparin binding induces a shift in calcium affinity and conformational changes in PAD4, essential for its activation. Further we managed to profile the substrate specificity of PAD2 and PAD4 using the combinatory approach, creating libraries of peptides. PAD4 prefers certain amino acids with P2 Asp being crucial for its activity and selectivity. Based on this data, selective substrates and inhibitors for PAD2 and PAD4 were designed. These inhibitors were also converted into activity-based probes for visualizing PAD activity in cells. Cell-based studies progressed in two main directions. Experiments with isolated human neutrophils (PMNs) showed that heparin could trigger a PMN response and increase histone H3 citrullination. GAG-deficient cell lines were used to study PAD4 binding and activation through flow cytometry. PAD4 bound significantly more to wild-type CHO control cells compared to GAG-deficient CHO PgsB-650 and CHO PgsA-745 cells. Samples were acquired from two patient cohorts. PAD4 activation was measured in 268 RRA patients, with 13.1% deemed PAD4-positive. PAD4-positive individuals progressed to clinical arthritis faster than PAD4-negative ones. Further analysis showed that RA patients treated with methotrexate or biologics had lower serum PAD4-activating potential. A collaboration with the Federal University of Espirito Santo, Brazil, revealed lower heparin levels in RA patients compared to healthy volunteers, potentially due to their treatments. Additionally, cryo-electron microscopy revealed different binding modes for heparin oligomers depending on their size, with Dp12 binding near a flexible loop and Dp20 binding at the dimerization region. Mutagenesis showed that these regions are important but not solely responsible for heparin interaction and highlighted the necessity of dimerization for effective interaction.
Polish researchers: Project had significant impact on the performance of Polish researchers. It allowed implementation of modern techniques and combined laboratory experience of Polish Partners with clinical-focused work of the Norwegian Partner.
Young researchers: Project provided a significant support for young researchers. During the project 6 mentor-mentee relationships were established with scientists form Poland (5) and Norway (1). This support was realized not only on the financial level, but also by sharing the experience, allowing presentation of their work on the international stage, young researcher exchange between the partners and more, impacting the future career perspectives of involved researchers.
Collaboration between science and society: Project results have potential to impact the society in multiple ways. Firstly, the project generated significant interest from the science popularizers, who declared support in the dissemination of the project results to the general public. It must be noted, that rheumatoid arthritis is a widespread disease, with patients often affected throughout their life. Generation of basic knowledge in this subject may be met with the broad interest and this was visible during social media interaction through project channels, where several involved discussions with page followers happened. On the other hand, the impact of the project results in the medical field, in particular analysis of the PAD4 activation by RA patient sera has a great potential to improve rheumatoid arthritis diagnosis and Norwegian Partner is actively pursuing that possibility. Finally, the high-quality results generated throughout the project are prepared for the core publication, which is delayed due to the structural analysis, not planned originally in the project. We firmly believe, that these results will impact the PAD biology and citrullination field significantly, providing the explanation for the cryptic aspects of the PAD4 activity regulation. This is highly active field, with papers being published in Nature group journals regularly.
Enhanced collaboration between donor and beneficiary states: Although no new joint project proposals were applied yet, this is due to the proposal calendar. Project is highly prospective, with results indicating potential involvement of other GAG molecules, cartilage proteins and nucleic acids as potential PAD4 activators and these development will be pursued in the future in a collaborative effort.
Summary of bilateral results
The bilateral cooperation proceeded as planned in the contract, focusing on the implementation of research tasks and the exchange of expertise between the Polish and Norwegian partners. It was based on implementing research tasks and exchanging expertise between Polish and Norwegian partners. Although this collaboration has not yet resulted in joint publications, the project supported 17 researchers, exceeding the planned 7. Of these, 14 were from Poland (5 female, 9 male), and 3 were from Norway (2 female, 1 male), demonstrating active involvement from both sides. Although no new joint project proposals were applied yet, this is due to the proposal calendar. Project is highly prospective, with results indicating potential involvement of other GAG molecules, cartilage proteins and nucleic acids as potential PAD4 activators and these development will be pursued in the future in a collaborative effort. In December 2024, the principal investigator Tomasz Kantyka, PhD submitted a proposal"Characterization of PAD4 enzyme activation by biopolymers from glycosaminoglycan family" (Reg. No: 2023/51/B/NZ1/00958) under the Opus call. This project assumed the collaboration between Jagiellonian University in Krakow, University of Bergen and Wrocław University of Science and Technology, but not in a form of scientific consortium. The next joint proposal "Characterization of nucleic acids as the allosteric activators of PAD4" (Opus call) is in preparation and will be submitted in June 2024.