Cytokine Gene Regulation by Modification of Arginine Residues

描述(由申请人提供)：Th1和Th2亚群产生的细胞因子与感染性、过敏性和自身免疫性疾病的易感性有关。了解控制谱系特异性细胞因子表达的分子事件将为调节Th1/Th2反应提供有用的工具。Th2细胞产生IL-4受NFAT和NFAT辅助因子NIP45的调节。我们已经证明，NIP45的精氨酸甲基化促进了它与NFAT的相互作用，并增强了IL-4的转录。我们的数据将精氨酸甲基转移酶PRMT1定位在T细胞受体下游，这表明精氨酸甲基化可能是免疫受体信号通路中的一个重要修饰。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PAD4通过精氨酸残基的瓜氨酸化对NIP45甲基化进行负调控，从而阻止了NIP45被PRMT1甲基化的能力。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PAD4通过作用于NIP45和其他调节蛋白来控制Th细胞因子的表达。其具体目的是：1)确定PAD4拮抗NIP45诱导的Th细胞因子产生的机制。我们将(I)通过质谱学确定NIP45中的修饰精氨酸残基，(Ii)通过研究PAD4对NIP45/NFAT相互作用的影响来确定PAD4如何影响NIP45的活性，(Iii)确定NIP45中的瓜氨酸化是否起到防止甲基化以外的作用。2)研究PAD4活性的调节。我们将：(I)确定PAD4在Th细胞中的表达模式，(Ii)确定PAD4活性在Th细胞中是否受调节，(Iii)确定PAD4本身是否受精氨酸甲基化调节。3)分析PAD4基因缺陷小鼠的表型。我们将创建PAD4的第7-13外显子被loxP位点包围的小鼠，这样我们就可以使用CD4-CRE TG小鼠来消除T谱系中PAD4的表达。这些小鼠将使我们能够确定PAD4在Th细胞功能中的作用。

DESCRIPTION (provided by applicant): Cytokine production by the Th1 and Th2 subsets has been associated with susceptibility 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-4 transcription. 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-4 promoter activity. Therefore, we hypothesize that through actions on NIP45 and other regulatory proteins that PAD4 controls Th cell cytokine expression. The specific aims are: 1) Determine the mechanism by which PAD4 antagonizes NIP45-mediated induction 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) Analyze the 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.