Cytokine Gene Regulation by Modification of Arginine Residues

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Cytokine production by the Th1 and Th2 subsets have been associated with susceptiblity to infectious, allergic, and autoimmune diseases. Understanding the molecular events which control lineage-specific cytokine expression would provide useful tools to modulate the Th1/Th2 response. IL- 4 production by Th2 cells is regulated by NFAT and the NFAT cofactor, NIP45. We have shown that arginine methylation of NIP45 facilitates its interaction with NFAT and augments IL-4transcription. Our data positioned the arginine methyltransferase PRMT1 downstream of the T cell receptor, suggesting that arginine methylation may be an important modification in immune receptor signaling pathways. Arginine methylation is countered by the actions of peptidylarginine deiminase 4 (PAD4). The five PAD enzymes convert arginine residues within proteins into the atypical amino acid citrulline. PAD4 is expressed mainly in lymphocytes. We have found that NIP45 methylation is negatively regulated by PAD4 via citrullination of arginine residues, which prevents the ability of NIP45 to be methylated by PRMT1. PAD4 expression dramatically reduces NIP45-induced IL-4promoter activity. Therefore, we hypothesize that through actions on NIP45 and other regulatory proteins that PAD4 controls Th cell cytokine expression.The specific aimsare: 1) Determine the mechanism by which PAD4 antagonizes NIP45-mediatedinduction of Th cell cytokine production. We will (i) determine the modified arginine residues in NIP45 by mass spectrometry, (ii) determine how PAD4 influences NIP45 activity by studying the effects of PAD4 on the NIP45/NFAT interaction, (iii) determine whether citrullination within NIP45 serves a function other than preventing methylation. 2) Investigate the regulation of PAD4 activity. We will: (i) determine the PAD4 expression pattern in Th cells, (ii) determine whether PAD4 activity is regulated in Th cells, (iii) determine whether PAD4, itself, is regulated by arginine methylation. 3) Analyzethe phenotype of PAD4 deficient mice. We will create mice in which exons 7-13 of PAD4 are flanked by loxP sites so that we can ablate PAD4 expression in the T lineage using CD4-Cre Tg mice. These mice will allow us to determine the role of PAD4 in Th cell function.