Abstract 1102: Regulation of protein citrullination through p53/PADI4 network in DNA damage response

Abstract

Abstract Upon wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However its involvement in post-translational modifications of proteins has not been well characterized so far. We here report the novel role of p53 in the regulation of protein citrullination. Citrullination (also referred as deimination) is one form of PTMs converting an arginine residue in protein to a citrulline residue. This reaction is mediated by a Ca2+-dependent enzyme called as peptidylarginine deiminase. Citrullination of proteins results in a loss of positive charge and causes a significant biochemical change. On the other hand, citrullinated peptides/proteins are recognized as a non-self protein and subsequently activate immune systems. Hence, protein citrullination is now considered to play a pivotal role in various human diseases, but any roles of citrullination in carcinogenesis were not reported so far. We found p53 transactivated peptidylarginine deiminase type 4 (PADI4) through intronic p53-binding site and ectopic expression of p53 or PADI4 induced protein citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their citrullination, indicating the regulation of protein citrullination in a p53/PADI4 dependent manner. We identified that PADI4 citrullinated histone chaperon protein, nucleophosmin (NPM1) at arginine 197 residue in vivo under physiological condition. NPM1 was shown to continuously shuttle between the nucleus and cytoplasm and translocate from nucleoli to nucleoplasm as a response to DNA damage. Therefore, we investigated the effect of protein citrullination on subcellular localization of NPM1. Immunocytochemical analysis revealed that citrullination of NPM1 by PADI4 resulted in its translocation from nucleoli to nucleoplasm, while PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly knockdown of PADI4 attenuated p53-mediated growth inhibitory activity, demonstrating the significance of PADI4-mediated protein citrullination in the p53 signaling pathway. Most of previous studies assessing enzymatic activities of PADIs were conducted under non-physiological condition using Ca ionophore or at higher Ca concentration. To our knowledge, this is the first report demonstrating the physiological regulatory mechanism of protein citrullination. Thus, p53 that can directly or indirectly modulate biological property of various proteins through the protein citrullination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1102.