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Review
. 2019:420:233-251.
doi: 10.1007/82_2018_132.

Development of Activity-Based Proteomic Probes for Protein Citrullination

Venkatesh V Nemmara  1   2 , Paul R Thompson  3   4
Affiliations
Review

Development of Activity-Based Proteomic Probes for Protein Citrullination

Venkatesh V Nemmara et al. Curr Top Microbiol Immunol. 2019.

Abstract

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.

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Figures

Fig. 1
Fig. 1
a PAD-catalyzed hydrolysis of peptidyl arginine to peptidyl citrulline. b Backbone conformation of PAD2 showing both the apo and holo forms that is generated upon calcium binding. The structural change due to calcium binding is clearly evident in the catalytic domain (green), which harbors the catalytic cysteine C647 (shown in red in the catalytic domain). c Crystal structure of PAD4 C654A protomer bound to the substrate BAA (PDB code 1WDA). d Co-crystal structure of BB-F-amidine (5a) bounds to PAD4 (PDB code 5N0 M). e Proposed catalytic mechanism for PAD4
Fig. 2
Fig. 2
a Structure of reversible PAD4 inhibitors. The IC50 values for each compound with PAD4 are shown underneath. These compounds are >15-fold selective for PAD4. b Structure of irreversible PAD inhibitors. The generic warhead on the irreversible inhibitors is colored in pink. The second-order rate constants for PAD inactivation by covalent inhibitors are shown in the table
Fig. 3
Fig. 3
a Schematic representation depicting the use of clickable ABPPs. b Chemical structures of RFA and RCA. c Schematic overview of the fluorescence polarization assay using RFA as an ABPP for high-throughput inhibitor discovery. d Structures of first-generation ‘clickable’ PAD-targeted ABPPs. Labeled probes are subjected to click-chemistry using fluorescein reporters through a post-inactivation strategy. e Structures of second-generation ‘clickable’ PAD-targeted ABPPs. Probe-labeled proteins are subjected to click-chemistry using TAMRA azide reporter through a post-inactivation strategy
Fig. 4
Fig. 4
a Chemical structures of Rh-PG and BPG and the chemoselective reaction of Rh-PG with citrulline under acidic conditions. b Schematic representation of the strategy for the chemical derivatization of peptidyl citrulline using Rh-PG and biotin PG
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