Development of Activity-Based Proteomic Probes for Protein Citrullination.

Protein arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine to form peptidyl citrulline. This modification is increased in multiple inflammatory diseases and in certain cancers. PADs regulate a variety of signaling pathways including apoptosis, terminal differentiation, and transcriptional regulation. Activity-based protein profiling (ABPP) probes have been developed to understand the role of the PADs in vivo and to investigate the effect of protein citrullination in various pathological conditions. Furthermore, these ABPPs have been utilized as a platform for high-throughput inhibitor discovery. This review will showcase the development of ABPPs targeting the PADs. In addition, it provides a brief overview of PAD structure and function along with recent advances in PAD inhibitor development.

A Secreted Bacterial Peptidylarginine Deiminase Can Neutralize Human Innate Immune Defenses.

The keystone oral pathogen Porphyromonas gingivalis is associated with severe periodontitis. Intriguingly, this bacterium is known to secrete large amounts of an enzyme that converts peptidylarginine into citrulline residues. The present study was aimed at identifying possible functions of this citrullinating enzyme, named Porphyromonas peptidylarginine deiminase (PPAD), in the periodontal environment. The results show that PPAD is detectable in the gingiva of patients with periodontitis, and that it literally neutralizes human innate immune defenses at three distinct levels, namely bacterial phagocytosis, capture in neutrophil extracellular traps (NETs), and killing by the lysozyme-derived cationic antimicrobial peptide LP9. As shown by mass spectrometry, exposure of neutrophils to PPAD-proficient bacteria reduces the levels of neutrophil proteins involved in phagocytosis and the bactericidal histone H2. Further, PPAD is shown to citrullinate the histone H3, thereby facilitating the bacterial escape from NETs. Last, PPAD is shown to citrullinate LP9, thereby restricting its antimicrobial activity. The importance of PPAD for immune evasion is corroborated in the infection model Galleria mellonella, which only possesses an innate immune system. Together, the present observations show that PPAD-catalyzed protein citrullination defuses innate immune responses in the oral cavity, and that the citrullinating enzyme of P. gingivalis represents a new type of bacterial immune evasion factor.IMPORTANCE Bacterial pathogens do not only succeed in breaking the barriers that protect humans from infection, but they also manage to evade insults from the human immune system. The importance of the present study resides in the fact that protein citrullination is shown to represent a new bacterial mechanism for immune evasion. In particular, the oral pathogen P. gingivalis employs this mechanism to defuse innate immune responses by secreting a protein-citrullinating enzyme. Of note, this finding impacts not only the global health problem of periodontitis, but it also extends to the prevalent autoimmune disease rheumatoid arthritis, which has been strongly associated with periodontitis, PPAD activity, and loss of tolerance against citrullinated proteins, such as the histone H3.

There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis is one of the major periodontal agents and it has been recently hailed as a potential cause of the autoimmune disease rheumatoid arthritis. In particular, the peptidylarginine deiminase enzyme of P. gingivalis (PPAD) has been implicated in the citrullination of certain host proteins and the subsequent appearance of antibodies against citrullinated proteins, which might play a role in the etiology of rheumatoid arthritis. The aim of this study was to investigate the extracellular localization of PPAD in a large panel of clinical P. gingivalis isolates. Here we show that all isolates produced PPAD. In most cases PPAD was abundantly present in secreted outer membrane vesicles (OMVs) that are massively produced by P. gingivalis, and to minor extent in a soluble secreted state. Interestingly, a small subset of clinical isolates showed drastically reduced levels of the OMV-bound PPAD and secreted most of this enzyme in the soluble state. The latter phenotype is strictly associated with a lysine residue at position 373 in PPAD, implicating the more common glutamine residue at this position in PPAD association with OMVs. Further, one isolate displayed severely restricted vesiculation. Together, our findings show for the first time that neither the major association of PPAD with vesicles, nor P. gingivalis vesiculation per se, are needed for P. gingivalis interactions with the human host.

The Development of Benzimidazole-Based Clickable Probes for the Efficient Labeling of Cellular Protein Arginine Deiminases (PADs).

Citrullination is the post-translational hydrolysis of peptidyl-arginines to form peptidyl-citrulline, a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calcium-regulated enzymes. Aberrantly increased protein citrullination is associated with a slew of autoimmune diseases (e.g., rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis) and certain cancers. Given the clear link between increased PAD activity and human disease, the PADs are therapeutically relevant targets. Herein, we report the development of next generation cell permeable and "clickable" probes (BB-Cl-Yne and BB-F-Yne) for covalent labeling of the PADs both in vitro and in cell-based systems. Using advanced chemoproteomic technologies, we also report the off targets of both BB-Cl-Yne and BB-F-Yne. The probes are highly specific for the PADs, with relatively few off targets, especially BB-F-Yne, suggesting the preferential use of the fluoroacetamidine warhead in next generation irreversible PAD inhibitors. Notably, these compounds can be used in a variety of modalities, including the identification of off targets of the parent compounds and as activity-based protein profiling probes in target engagement assays to demonstrate the efficacy of PAD inhibitors.