Autophagy induces protein carbamylation in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Abstract

Autophagy is a homeostatic and physiological process that promotes the turnover of proteins and organelles damaged in conditions of cellular stress. We previously demonstrated that autophagy represents a key processing event creating a substrate for autoreactivity, which is involved in post-translational changes and generation of citrullinated peptides, recognized by the immune system in RA. In this study, we analysed whether autophagy is involved in other post-translational changes that can generate autoantigens, focusing on carbamylation processes. Carbamylation is a nonenzymatic post-translational modification, in which homocitrulline is generated by the reaction of cyanate with the primary amine of lysine residues; carbamylated peptides may accumulate during inflammation conditions. The role of autophagy in the generation of carbamylated proteins was evaluated in vitro in fibroblasts as well as in synoviocytes from RA patients, treated with 5 μM tunicamycin or 200 nM rapamycin; the correlation between autophagy and carbamylated proteins was analysed in mononuclear cells from 30 naïve early-active RA patients. Our results demonstrated that cells treated with tunicamycin or rapamycin showed a significant increase of carbamylated proteins. Immunoblotting and immunoprecipitation experiments identified vimentin as the main carbamylated protein. Furthermore, a correlation was found between autophagy and carbamylation levels in mononuclear cells of naïve RA patients. These data indicate that autophagy is able to induce in vitro carbamylation processes, and in vivo appears to be related to an increase in carbamylation during RA. These observations introduce a new pathogenetic mechanism of disease, which could contribute to more accurate monitoring of patients.