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Decoding isozyme-specific substrate recognition in protein arginine deiminases by in vitro 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. Here, we provide a systematic, isozyme-wide comparison of human PAD1–4 by combining controlled in vitro incubation of human cell lysates with recombinant enzymes, deep-coverage LC–MS/MS and advanced computational validation. Importantly, by incubating lysates under uniform, saturating enzyme and calcium conditions we aimed to isolate intrinsic substrate recognition potential from confounding factors such as isozyme expression levels, localization or intracellular calcium concentrations. We further leveraged deep-learning-based rescoring to elevate site-level confidence. With this approach we mapped ~30,000 citrullination sites across ~5,500 of proteins, enabling direct comparison of substrate repertoire, sequence motif, and structural context among PAD isozymes, setting the stage for mechanistic dissection of how binding-pocket homology and peripheral residues dictate specificity. Distinct isozyme-specific preferences persisted in time-course experiments (10 min–16 h), indicating sequence context rather than temporal dynamics drives specificity. Mutation analysis of eleven PAD4 variants revealed Q346, G403S, 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Sophia Laposchan

LAB HEAD: Chien-Yun Lee

PROVIDER: PXD071247 | Pride | 2026-04-22

REPOSITORIES: Pride

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			Action	DRS
	2024-10-16-decoys-contam-uniprotkb_human_AND_reviewed_true_AND_m_canonical_20428entries_2024_10_16.fasta.fas	Fasta
	YIG_129_L012_05_SoL_001_U01.raw	Raw
	YIG_129_L012_05_SoL_001_U02.raw	Raw
	YIG_129_L012_05_SoL_001_U03.raw	Raw
	YIG_129_L012_05_SoL_001_U04.raw	Raw

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Project description:Citrullination is a post-translational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations and while the modification has been linked to several diseases including rheumatoid arthritis and cancer, its physiological or pathophysiological roles remain largely unclear. In part this is owing to limitations in available methodology able to robustly enrich, detect and localize the modification. As a result, only few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ~70 million tandem mass spectra yielded ~13,000 candidate spectra which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetised ~2,200 citrullinated and 1,300 deamindated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundance proteins arguing that the development of specific enrichment methods would be required in order to study the full extent of cellular protein citrullination.

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

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