Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys ¹⁴⁸ . Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

Protein citrullination is a post-translational modification (PTM) that is catalysed by the protein arginine deiminase (PAD) family of enzymes. This PTM involves the transformation of an arginine residue into citrulline. Protein citrullination is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation and DNA damage-induced apoptosis. Aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis (RA), lupus, atherosclerosis and ulcerative colitis. In RA, anti-citrullinated protein antibodies can be detected prior to disease onset and are thus a valuable diagnostic tool for RA. Notably, citrullinated proteins may serve more generally as biomarkers of specific disease states; however, the identification of citrullinated protein residues remains challenging owing to the small 1 Da mass change that occurs upon citrullination. Herein, we highlight the progress made so far in the development of pan-PAD and isozyme selective inhibitors as well as the identification of citrullinated proteins and the site-specific incorporation of citrulline into proteins. This article is part of the Theo Murphy meeting issue ‘The virtues and vices of protein citrullination’.

Introduction: MZB1 is an endoplasmic reticulum residential protein preferentially expressed in plasma cells, marginal zone and B1 B cells. Recent studies on murine B cells show that it interacts with the tail piece of IgM and IgA heavy chain and promotes the secretion of these two classes of immunoglobulin. However, its role in primary human B cells has yet to be determined and how its function is regulated is still unknown. The conversion of peptidylarginine to peptidylcitrulline, also known as citrullination, by peptidylarginine deiminases (PADs) can critically influence the function of proteins in immune cells, such as neutrophils and T cells; however, the role of PADs in B cells remains to be elucidated.Method: An unbiased analysis of human lung citrullinome was conducted to identify citrullinated proteins that are enriched in several chronic lung diseases, including rheumatoid arthritis-associated interstitial lung disease (RA-ILD), chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis, compared to healthy controls. Mass spectrometry, site-specific mutagenesis, and western blotting were used to confirm the citrullination of candidate proteins. Their citrullination was suppressed by pharmacological inhibition or genetic ablation of PAD2 and the impact of their citrullination on the function and differentiation of human B cells was examined with enzyme-linked immunosorbent assay, flow cytometry, and co-immunoprecipitation.Results: Citrullinated MZB1 was preferentially enriched in RA-ILD but not in other chronic lung diseases. MZB1 was a substrate of PAD2 and was citrullinated during the differentiation of human plasmablasts. Ablation or pharmacological inhibition of PAD2 in primary human B cells attenuated the secretion of IgM and IgA but not IgG or the differentiation of IgM or IgA-expressing plasmablasts, recapitulating the effect of ablating MZB1. Furthermore, the physical interaction between endogenous MZB1 and IgM/IgA was attenuated by pharmacological inhibition of PAD2.Discussion: Our data confirm the function of MZB1 in primary human plasmablasts and suggest that PAD2 promotes IgM/IgA secretion by citrullinating MZB1, thereby contributing to the pathogenesis of rheumatoid arthritis and RA-ILD.