PTMs act as an added layer of regulation for protein-protein interactions and protein stability with consequential effects on downstream signaling pathways including gene transcription, cell growth, and apoptosis. Citrullination or deimination is one such PTM in which the calcium-dependent Protein Arginine Deiminases (PADs 1-4 and 6) are responsible for the conversion of peptidyl-arginine into peptidyl-citrulline. This modification converts the positive charge of the guanidinium group and replaces it with a neutral ureido group. Although the physiological effects of citrullination are still unclear, this PTM is known to regulate a number of processes including NET formation and cellular differentiation. A recent report by the Coonrod group identified Histone H3R26 as an in vivo target of PAD2 in multiple cell lines. Citrullination at this site is promoted by 17?-estradiol (E2) stimulation of estrogen receptor ? (ER?). Downstream effects include chromatin decondensation and transcriptional activation of more than 200 genes under the control of ER?(1). Interestingly, unlike PAD4, PAD2 does not contain a canonical nuclear localization signal (NLS, 56-PPAKKKST-63) and we hypothesize that transport into the nucleus is facilitated by protein-protein interactions. Crystallographic structures of PAD2 show several potential docking sites, including two N-terminal immunoglobulin-like domains and the phenylalanine (FF) finger motif described below(2).
Aim 1 focuses on the unique FF-motif as preliminary data suggests it regulates the cellular activity of PAD2 by acting as a docking site for an unidentified regulatory protein(s). To address this hypothesis, we will take a combinatorial and integrated approach that involves biochemical and proteomic experiments designed to identify these novel PBPs.
In Aim 2, we will address a second hypothesis that focuses on the notion that 'readers'of protein citrullination exist to interpret protein citrullination and facilitate physiological resposes to citrullination. In total, these studies will increase our understanding of the mechanisms that regulate PAD activity. As a consequence, we expect that our studies will identify the conditions under which these enzymes are activated in cells thereby enhancing our understanding of the physiological roles of protein citrullination. We also expect that these studies will help identify the precise molecular structure of the enzyme that should be targeted for inhibition, thereby, facilitating future efforts to develop chemical probes and therapeutics targeting these enzymes.
Mounting evidence indicates that dysregulated Protein Arginine Deiminase (PAD) activity is linked to the promotion of numerous cancers and inflammatory diseases, including rheumatoid arthritis, ulcerative colitis, and breast cancer. The proposed studies are focused on understanding how enzyme activity is regulated.
|Lewallen, Daniel M; Bicker, Kevin L; Subramanian, Venkataraman et al. (2015) Chemical Proteomic Platform To Identify Citrullinated Proteins. ACS Chem Biol 10:2520-8|
|Slade, Daniel J; Fang, Pengfei; Dreyton, Christina J et al. (2015) Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design. ACS Chem Biol 10:1043-53|
|Dreyton, Christina J; Knuckley, Bryan; Jones, Justin E et al. (2014) Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism. Biochemistry 53:4426-33|
|Dreyton, Christina J; Anderson, Erin D; Subramanian, Venkataraman et al. (2014) Insights into the mechanism of streptonigrin-induced protein arginine deiminase inactivation. Bioorg Med Chem 22:1362-9|