Protein citrullination: inhibition, identification and insertion

Abstract

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'.

Keywords: citrullination; deimination; inhibitor; neutrophil extracellular trap formation; rheumatoid arthritis.

Figure 1.

Protein citrullination and catalytic mechanism. (a) PAD catalysed hydrolysis of peptidyl-arginine to generate peptidyl-citrulline. (b) PAD4 uses a reverse protonation mechanism to convert arginine into citrulline. (Online version in colour.)

Figure 2.

Structures of PAD inhibitors and mechanism of inactivation by haloacetamidine containing inhibitors. (a) Structures of F-amidine, chloro (Cl)-amidine, tetrazole Cl-amidine and biphenyl-benzimidazole (BB)-Cl-amidine, respectively. Structures of PADs specific inhibitors including threonine aspartate F-amidine (TDFA) for PAD4, SM26 for PAD1 and AFM30a for PAD2. (b) Mechanisms by which haloacetamidine inhibitors irreversibly inhibit the PADs. (c) Structures of the PAD4 selective inhibitors GSK484, BMS-P5 and JBI-589. (Online version in colour.)

Figure 3.

PAD2 undergoes a series of calcium dependent conformational changes. Wild-type PAD2 structures soaked with 0 mM (apoenzyme (a), PDB: 4N20) and 10 mM CaCl₂ (b), PDB: 4N2B) and the PAD2 F221/222A mutant soaked in 10 mM CaCl₂ (holoenzyme (c), PDB: 4N2C). (Online version in colour.)

Figure 4.

Development of phenylglyoxal-based probes to visualize protein citrullination. (a) Chemical structures of rhodamine-phenylglyoxal (Rh-PG) and biotin-phenylglyoxal (biotin-PG). (b) Phenylglyoxal fragment preferentially reacts with citrulline (over arginine) at acidic pH to form an imidazalone ring structure. (Online version in colour.)