Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4.

Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.

Do RA associated HLA-DR molecules bind citrullinated peptides or peptides from PAD4 to help the development of RA specific antibodies to citrullinated proteins?

PURPOSE: Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding a five amino acid basic motive, the shared epitope SE). Each HLA-DRB1 genotype defines a genotype specific risk of developing RA. RA is preceded by the emergence of anti citrullinated protein antibodies (ACPAs). Citrullin is a neutral version of arginin, a basic amino acid, formed after post translational modification by Peptidyl Arginyl Deiminases (PADs). HLA-DRB1 genes associated with RA are also associated with ACPAs. Two models might explain this association. Here we tested both models for prediction of HLA-DRB1 genotypic risks of developing RA. METHODS: We calculated the likelihoods for the 2 HLA-DR molecules encoded by 12 common HLA-DRB1 genotypes to bind at least one randomly chosen peptide from PAD4 or fibrinogen(native or citrullinatd) and compared them with the 12 respective HLA-DRB1genotypic risks of developing RA. RESULTS: HLA-DRB1 Genotypic risks of developing RA correlate with likelihoods of binding PAD4 peptides, not citrullinated Fibrinogen peptides. Thus, the molecular basis for the association of HLA-DR and ACPA positive RA is most likely the capability for RA associated HLA-DR molecules to bind peptides(s) from PAD4.

Isolated auto-citrullinated regions of PADI4 associate to the intact protein without altering their disordered conformation.

PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.

Peptidylarginine deiminase (PAD): A promising target for chronic diseases treatment.

In 1958, the presence of citrulline in the structure of the proteins was discovered for the first time. Several years later they found that Arginine converted to citrulline during a post-translational modification process by PAD enzyme. Each PAD is expressed in a certain tissue developing a series of diseases such as inflammation and cancers. Among these, PAD2 and PAD4 play a role in the development of rheumatoid arthritis (RA) by producing citrullinated autoantigens and increasing the production of inflammatory cytokines. PAD4 is also associated with the formation of NET structures and thrombosis. In the crystallographic structure, PAD has several calcium binding sites, and the active site of the enzyme consists of different amino acids. Various PAD inhibitors have been developed divided into pan-PAD and selective PAD inhibitors. F-amidine, Cl-amidine, and BB-Cl-amidine are some of pan-PAD inhibitors. AFM-30a and JBI589 are selective for PAD2 and PAD4, respectively. There is a need to evaluate the effectiveness of existing inhibitors more accurately in the coming years, as well as design and production of novel inhibitors targeting highly specific isoforms.

Immobilized PAD4 enzyme on magnetic nanoparticles for screening natural inhibitors from traditional Chinese medicines.

Dysregulation of peptidyl arginine deiminase 4 (PAD4) is involved in a variety of diseases including rheumatoid arthritis (RA) and Alzheimer's disease (AD), and it has emerged as potential and promising therapeutic target. However, no PAD4 inhibitor is ready for clinical use. Immobilized enzyme screening technology has gained increasing attention due to its low cost, reusability, easy separation from the reaction mixture, and resistance to changes in environmental conditions. In this study, PAD4 was immobilized on the magnetic nanoparticles (MNP) to prolong its activity stability, and a simple and rapid screening strategy of traditional Chinese medicine inhibitors based on immobilized PAD4 was established. The PAD4 enzyme was immobilized on magnetic nanoparticles (MNP) via Schiff base reaction using glutaraldehyde (GA) as crosslinking agent. Compared with free PAD4, the resulting MNP@GA@PAD4 exhibited an enhanced tolerance to temperature and storage stability, and its reusability was greatly improved with 66 % of initial enzyme activity after being recycled 10 times. The inhibitory activity of the immobilized PAD4 was assessed using two known PAD4 inhibitors GSK484 and BB-Cl-amidine. The semi-maximum inhibitory concentrations (IC50) of GSK484 and BB-Cl-amidine for MNP@GA@PAD4 were 1.00 and 0.97 µM, respectively, for free PAD4 were 0.64 and 0.85 µM, respectively. Finally, the MNP@GA@PAD4 was employed to rapid screen of natural PAD4 inhibitors from forty traditional Chinese medicines (TCMs). Under the same conditions, the controlled experiment was conducted with free PAD4. The screening results of TCMs inhibitors on MNP@GA@PAD4 and free PAD4 were similar, the alcohol extracts of Cinnamomi Cortex and Caryophylli Flos had significant inhibitory effects on PAD4 enzyme activity. The IC50 values of Cinnamomi Cortex extract for MNP@GA@PAD4 and free PAD4 were determined as 27 and 48 µg/mL, respectively. The IC50 values of Caryophylli Flos extracts for MNP@GA@PAD4 and free PAD4 were determined as 48 and 32 µg/mL, respectively. For the first time, this study proposed a method to immobilize PAD4 on magnetic materials, and developed a rapid, reusable and feasible strategy to screening natural PAD4 inhibitors from TCMs.

Peptidylarginine deiminases 4 as a promising target in drug discovery.

Peptidylarginine deaminase 4 (PAD4) is a crucial post-translational modifying enzyme catalyzing the conversion of arginine into citrulline residues, and mediating the formation of neutrophil extracellular traps (NETs). PAD4 plays a vital role in the occurrence and development of cardiovascular diseases, autoimmune diseases, and various tumors. Therefore, PAD4 is considered as a promising drug target for disease diagnosis and treatment. More and more efforts are devoted to developing highly efficient and selective PAD4 inhibitors via high-throughput screening, structure-based drug design and structure-activity relationship study. This article outlined the physiological and pathological functions of PAD4, and corresponding representative small molecule inhibitors reported in recent years.

Autocitrullination of PAD4 does not alter its enzymatic activity: In vitro and in silico studies.

BACKGROUND: Protein arginine deiminase 4 (PAD4) is an enzyme capable of converting arginine (positively charged residue) into citrulline (neutral residue). PAD4 is a promiscuous enzyme since it citrullinates various substrates, including small peptides, large proteins and itself. The effect of autocitrullination on PAD4 activity remains controversial and inconclusive. We hypothesized that PAD4 autocitrullination may influence the activity of PAD4 by indirectly altering its binding to substrate. METHODS: We employed mass spectrometry analysis to study the process of autocitrullination. The kinetics of citrullination of PAD4 and citrullinated PAD4 (citPAD4) towards substrates of different sizes (0.17-15.4 kDa), i.e. free arginine, a peptidyl substrate, and histone H3, were studied by colorimetric assay and Western blotting. Molecular dynamics (MD) simulations were performed to investigate structural dynamic and binding properties of PAD4/citPAD4 in the absence and presence of substrates. RESULTS: We observed that 23/27 arginine residues in PAD4 (85 %) can be citrullinated, including R372, R374 and R639 located near the substrate binding pocket. PAD4 and citPAD4 expressed comparable enzymatic activities towards different substrates. In agreement with experimental results, MD simulations indicated that autocitrullination does not change the shape of the substrate binding pocket and PAD4/citPAD4 exhibited comparable binding free energy with a H3-derived peptidyl substrate (6-TARKS-10). CONCLUSION: While the effect of autocitrullination on PAD4 activity thus far remained unclear and controversial, here we have demonstrated that autocitrullination does not affect the activity of PAD4. Thus, the regulation of PAD4 activity is probably not controlled by autocitrullination but likely by other mechanisms that need further investigation.

Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination.

Aspergillus fumigatus (A. fumigatus) is an environmental fungus and a human pathogen. Neutrophils are critical effector cells during the fungal infections, and neutropenia is a risk factor for the development of pulmonary aspergillosis. Neutrophil extracellular traps (NETs) are released by neutrophils in response to A. fumigatus and inhibit the conidial germination. In this work, we observed that the receptors TLR2, TLR4, and Dectin-1 were dispensable for the A. fumigatus induced NET release. In contrast CD11b/CD18 was critical for the NET release in response to A. fumigatus conidia, and this required the CD11b I-domain-mediated recognition, whereas the blockade of the CD11b lectin domain did not affect the A. fumigatus induced NET release. A. fumigatus induced NET release relied on the activity of spleen tyrosine kinase (Syk), Src family kinase(s), and class IA PI3 kinase δ. Although A. fumigatus promoted histone citrullination, this process was dispensable for the NET release in response to A. fumigatus conidia. The A. fumigatus induced NET release required the reactive oxygen species generation by the NOX2 complex, in a downstream pathway requiring CD11b/CD18, Src kinase family activity, Syk and PI3K class IA δ. Our findings thus reveal the signaling pathways involved in the formation of NETs in response to A. fumigatus.

Autoantibodies to protein-arginine deiminase (PAD) 4 in rheumatoid arthritis: immunological and clinical significance, and potential for precision medicine.

Introduction: The protein-arginine deiminase (PAD) 4 enzyme plays an important role in the pathogenesis of rheumatoid arthritis (RA) and also represents an antigenic target. Anti-PAD4 antibodies can be present in RA and are associated with specific clinical features. Areas covered: This review aims to analyze the current knowledge and recent findings on anti-PAD4 antibodies in RA and their clinical and immunological significance. Expert opinion: Anti-PAD4 antibodies are not currently used in clinical practice for the management of RA. Nevertheless, there is growing evidence of their relevance in RA, and of their potential utility to improve diagnosis, patient stratification, and prognosis.