Decoding isozyme-specific substrate recognition in protein arginine deiminases by proteome-wide citrullination mapping

Abstract

Protein arginine deiminases (PADs) convert arginine to citrulline, altering protein structure and function. Of the five human isozymes, PAD1-4 are catalytically active with distinct tissue-specificities, yet isozyme-specific substrate recognition remains poorly defined. We profiled PAD1-4 substrate landscapes via mass spectrometry, identifying [~]30,000 citrullination sites across [~]5,500 proteins. Only 14% of sites were shared among all four, reflecting distinct sequence preferences: PAD1-2 showed broad specificity, whereas PAD3-4 favored arginines flanked by acidic or glycine residues. These preferences persisted over 10 min-16 h, indicating sequence context rather than temporal dynamics drives specificity. Mutation analysis of eleven PAD4 variants revealed Q346, G403, R639, and H640 as key determinants distinguishing substrate recognition from that of PAD2. This work provides the most comprehensive PADs substrate atlas to date, defining isozyme-specific motifs and molecular determinants, and guiding development of selective inhibitors and probes to interrogate citrullination mechanisms in health and disease.