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

Calcium Regulates the Nuclear Localization of Protein Arginine Deiminase 2

Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the post-translational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor target genes. However, PAD2 lacks a canonical nuclear localization sequence, and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Binding of calcium to PAD2 weakens this cytoplasmic interaction, which generates a pool of calcium-bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into the nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus

A Streamlined Data Analysis Pipeline for the Identification of Sites of Citrullination

Citrullination is an enzyme-catalyzed post-translational modification (PTM) that is essential for a host of biological processes, including gene regulation, programmed cell death, and organ development. While this PTM is required for normal cellular functions, aberrant citrullination is a hallmark of autoimmune disorders as well as cancer. Although aberrant citrullination is linked to human pathology, the exact role of citrullination in disease remains poorly characterized, in part because of the challenges associated with identifying the specific arginine residues that are citrullinated. Tandem mass spectrometry is the most precise method for uncovering sites of citrullination; however, due to the small mass shift (+0.984 Da) that results from citrullination, current database search algorithms commonly misannotate spectra, leading to a high number of false-positive assignments. To address this challenge, we developed an automated workflow to rigorously and rapidly mine proteomic data to unambiguously identify the sites of citrullination from complex peptide mixtures. The crux of this streamlined workflow is the ionFinder software program, which classifies citrullination sites with high confidence on the basis of the presence of diagnostic fragment ions. These diagnostic ions include the neutral loss of isocyanic acid, which is a dissociative event that is unique to citrulline residues. Using the ionFinder program, we have mapped the sites of autocitrullination on purified protein arginine deiminases (PAD1-4) and mapped the global citrullinome in a PAD2-overexpressing cell line. The ionFinder algorithm is a highly versatile, user-friendly, and open-source program that is agnostic to the type of instrument and mode of fragmentation that are used

The Rheumatoid Arthritis-Associated Citrullinome.

Increased protein citrullination is linked to various diseases including rheumatoid arthritis (RA), lupus, and cancer. Citrullinated autoantigens, a hallmark of RA, are recognized by anti-citrullinated protein antibodies (ACPAs) which are used to diagnose RA. ACPA-recognizing citrullinated enolase, vimentin, keratin, and filaggrin are also pathogenic. Here, we used a chemoproteomic approach to define the RA-associated citrullinome. The identified proteins include numerous serine protease inhibitors (Serpins), proteases and metabolic enzymes. We demonstrate that citrullination of antiplasmin, antithrombin, t-PAI, and C1 inhibitor (P1-Arg-containing Serpins) abolishes their ability to inhibit their cognate proteases. Citrullination of nicotinamide N-methyl transferase (NNMT) also abolished its methyltransferase activity. Overall, these data advance our understanding of the roles of citrullination in RA and suggest that extracellular protein arginine deiminase (PAD) activity can modulate protease activity with consequent effects on Serpin-regulated pathways. Moreover, our data suggest that inhibition of extracellular PAD activity will be therapeutically relevant

The Rheumatoid Arthritis-Associated Citrullinome.

Increased protein citrullination is linked to various diseases including rheumatoid arthritis (RA), lupus, and cancer. Citrullinated autoantigens, a hallmark of RA, are recognized by anti-citrullinated protein antibodies (ACPAs) which are used to diagnose RA. ACPA-recognizing citrullinated enolase, vimentin, keratin, and filaggrin are also pathogenic. Here, we used a chemoproteomic approach to define the RA-associated citrullinome. The identified proteins include numerous serine protease inhibitors (Serpins), proteases and metabolic enzymes. We demonstrate that citrullination of antiplasmin, antithrombin, t-PAI, and C1 inhibitor (P1-Arg-containing Serpins) abolishes their ability to inhibit their cognate proteases. Citrullination of nicotinamide N-methyl transferase (NNMT) also abolished its methyltransferase activity. Overall, these data advance our understanding of the roles of citrullination in RA and suggest that extracellular protein arginine deiminase (PAD) activity can modulate protease activity with consequent effects on Serpin-regulated pathways. Moreover, our data suggest that inhibition of extracellular PAD activity will be therapeutically relevant