Abstract
Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.
Highlights
Neutrophils are critically important for our defense against bacterial pathogens[1,2]
Neutrophils subjected to membranolytic agents that cause a rapid and lethal influx of calcium undergo a rapid citrullination of multiple cellular proteins[23,52,53]
We found p47phox/NCF1 to be citrullinated at 6 arginine residues (R7, R70, R85, R90, R335, R336) and p67phox/NCF2 at 4 (R184, R188, R238, R328) (Table 1, Fig. S1)
Summary
Neutrophils are critically important for our defense against bacterial pathogens[1,2]. Further support for an important role of protein citrullination in RA pathogenesis comes from genetic association studies, which implicated first PADI429–33 and later PADI234 in the susceptibility to RA These genes encode for the Ca2+-dependent protein arginine deiminases (PAD) 4 and 2, respectively, which catalyze the conversion of arginine to citrulline in protein substrates[35,36,37,38]. We provide evidence that PAD4 in neutrophils associates with NCF1 and NCF2, translocates with them during NADPH oxidase activation, and is important in ROS production and bacterial killing. This association requires PAD4 to remain inactive (i.e., a non-enzymatic function); PAD4-mediated citrullination of NCF proteins dissociates the complexes and impairs ROS generation. Disruption of the NADPH oxidase-PAD4 complex by other means inhibits ROS production, leading to reduced killing of bacteria
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
