Cytokine Gene Regulation by Modification of Arginine Residues

Th 1和Th 2亚群的细胞因子产生与以下易感性相关： 传染性、过敏性和自身免疫性疾病。了解控制着 谱系特异性细胞因子表达将提供有用的工具来调节Th 1/Th 2应答。白介素- Th 2细胞的4产生受NFAT和NFAT辅因子NIP 45调节。我们已经证明 NIP 45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PRMT 1定位在T细胞受体的下游， 表明精氨酸甲基化可能是免疫受体信号传导中的重要修饰 途径。精氨酸甲基化被肽基精氨酸脱亚胺酶4（PAD 4）的作用抵消。 五种PAD酶将蛋白质内的精氨酸残基转化为非典型氨基酸瓜氨酸。 PAD 4主要在淋巴细胞中表达。我们已经发现NIP 45甲基化是负性的。 由PAD 4通过精氨酸残基的瓜氨酸调节，这阻止了NIP 45的能力， 甲基化的PRMT 1。PAD 4的表达显著降低NIP 45诱导的IL-4启动子活性。 因此，我们假设通过对NIP 45和其他调节蛋白的作用， PAD 4控制Th细胞细胞因子的表达。 1)确定PAD 4拮抗NIP 45介导的Th细胞诱导的机制 细胞因子产生。我们将（i）以质量计测定NIP 45中修饰的精氨酸残基 （ii）通过研究PAD 4的作用来确定PAD 4如何影响NIP 45活性 （iii）确定NIP 45内的瓜氨酸是否作为NIP 45/NFAT相互作用的抑制剂， 除了防止甲基化之外。 2)研究PAD 4活性的调节。我们将：（i）确定PAD 4表达模式 在Th细胞中，（ii）确定PAD 4活性是否在Th细胞中被调节，（iii）确定是否 PAD 4本身受精氨酸甲基化调控。 3)分析PAD 4缺陷小鼠的表型。我们将创建小鼠，其中外显子7-13 PAD 4的侧翼是loxP位点，因此我们可以使用免疫抑制剂来消除T谱系中的PAD 4表达。 CD 4-Cre Tg小鼠。这些小鼠将使我们能够确定PAD 4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.