Cytokine Gene Regulation by Modification of Arginine Residues

Th1和Th2亚群产生的细胞因子与易感性有关 传染性、过敏性和自身免疫性疾病。理解控制的分子事件 谱系特异性细胞因子的表达将为调节Th1/Th2反应提供有用的工具。伊尔- 4 Th2细胞的产生受NFAT和NFAT辅助因子NIP45的调节。我们已经证明了 NIP45的精氨酸甲基化促进其与NFAT的相互作用，并增强IL-4的转录。 我们的数据将精氨酸甲基转移酶PRMT1定位在T细胞受体的下游， 提示精氨酸甲基化可能是免疫受体信号转导的重要修饰 小路。精氨酸甲基化被肽基精氨酸脱亚胺酶4(PAD4)的作用所抵消。 这五种PAD酶将蛋白质中的精氨酸残基转化为非典型氨基酸瓜氨酸。 PAD4主要在淋巴细胞中表达。我们发现NIP45甲基化是负性的 PAD4通过精氨酸残基的瓜氨酸化来调节，这阻止了NIP45被 由PRMT1甲基化。PAD4的表达显著降低了NIP45诱导的IL-4启动子活性。 因此，我们假设，通过对NIP45和其他调节蛋白的作用， PAD4控制Th细胞因子的表达。 1)确定PAD4拮抗NIP45介导的Th细胞诱导的机制 细胞因子的产生。我们将(I)按质量测定NIP45中的修饰精氨酸残留量 光谱分析，(Ii)通过研究PAD4的作用确定PAD4如何影响NIP45活性 关于NIP45/NFAT的相互作用，(Iii)确定NIP45内的瓜氨酸是否起到 除了防止甲基化以外的其他功能。 2)研究PAD4活性的调节。我们将：(I)确定PAD4表达模式 在Th细胞中，(Ii)确定PAD4活性在Th细胞中是否受到调节，(Iii)确定 PAD4本身受精氨酸甲基化的调节。 3)分析PAD4基因缺陷小鼠的表型。我们将创造小鼠，在这些小鼠中， PAD4的侧翼是loxP位点，因此我们可以使用 CD_4-Cre转基因小鼠。这些小鼠将使我们能够确定PAD4在Th细胞功能中的作用。

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.