Dynamics of Arginine Methylation in Cellular Processes

背景资料。蛋白质中精氨酸残基的甲基化被称为蛋白质精氨酸N-甲基转移酶(PRMTs)，是一种修饰(PTM)，主要存在于染色质相关蛋白和RNA结合蛋白中，已被证明在许多重要的细胞过程中发挥调节作用。一旦蛋白水解性释放，游离二甲基精氨酸可以竞争性地抑制一氧化氮合酶，阻止它们在调节血管张力、疼痛、神经传递和对微生物的炎症反应中的功能。这种PTM最近已成为包括癌症、心血管疾病和炎症性肺部疾病在内的越来越多疾病的生物标记物。然而，与其他暂时性PTM不同的是，精氨酸甲基化是蛋白质上一个稳定的标记。在过去的几年里，我的实验室已经建立了研究精氨酸甲基化的技术，以便我们可以确定PRMT对细胞编程的贡献。最近，我们使用蛋白质组学技术观察到，经肿瘤坏死因子-α或脂多糖处理的培养的人肺上皮细胞的蛋白质与未经处理的细胞的蛋白质之间的精氨酸甲基化发生了巨大的变化。这些实验还揭示了炎症反应的蛋白质介质作为新的PRMT底物。研究计划目标。我们实验室的长期目标是了解PRMT对细胞增殖、分化和死亡的独立和联合作用。我们的短期目标之一，我们正在请求NSERC的支持，是在不同的炎症反应途径中建立PRMT特异性的精氨酸甲基化网络。科学的方法。我们将使培养的人类细胞株接受引起核因子-kappaB介导性炎症反应的治疗。来自处理和未处理的对照细胞的蛋白质将被收获，并被二甲精氨酸特异性抗体印迹，或用于蛋白质组分析，这是与UBC高通量生物学中心的Leonard Foster和Nichollas Scott博士正在进行的合作的一部分。我们的蛋白质组学方法利用了最近报道的甲基化多肽的浓缩策略，该策略在样品进行质谱分析之前使用亲水相互作用液相色谱。然后将比较来自对照组和治疗组的甲基化蛋白的相对定量，使我们能够观察PTM的变化，作为建立炎症中精氨酸甲基化网络的一种方式。下一步将是通过基于细胞的分析(通过siRNA降低特定PRMT的表达水平)或通过药物抑制具有小分子的培养细胞中的特定PRMT来确定特定的PRMT参与炎症途径。我们还将免疫共沉淀细胞中的甲基化蛋白，通过Western印迹和质谱学确定哪些PRMT与其底物相关联。这些底物随后将用体外甲基化试验进行测试，以证明PRMT底物的特异性。冲击力。精氨酸甲基化已被证明在重要的细胞过程中执行特定的功能，但这种PTM尚未在更广泛的细胞背景下进行研究。通过观察蛋白质组全球范围内精氨酸甲基化的变化，我们将确定其修饰可能在炎症事件中发挥关键作用的底物，并展示这种PTM如何参与各种信号通路。这项工作将提供对PRMT活动的整合如何有助于细胞编程并最终决定细胞命运的机械性洞察。我们的发现应该会影响癌症生物学、炎症和生物化学领域。

Background. The methylation of arginine residues within proteins by enzymes called protein arginine N-methyltransferases (PRMTs), is a modification (PTM) found predominantly in chromatin-associated and RNA-binding proteins, and has been shown to play regulatory roles in many important cellular processes. Once proteolytically liberated, free dimethylated arginines can competitively inhibit nitric oxide synthases and prevent their functions in regulating vascular tone, pain, neurotransmission, and the inflammatory response to microorganisms. This PTM has recently emerged as a biomarker for a growing list of diseases including cancer, cardiovascular, and inflammatory lung diseases. Unlike other transient PTMs, however, arginine methylation is a stable mark on proteins. For the past few years my laboratory has established techniques to study arginine methylation so that we may determine how PRMTs contribute to cellular programming. Recently, we were able to see using proteomic techniques large changes in arginine methylation between proteins from cultured human lung epithelial cells treated with tumor necrosis factor-alpha or lipopolysaccharide and proteins from untreated cells. These experiments also revealed protein mediators of the inflammatory response as new PRMT substrates. Research Program Objectives. The long-term objective of our lab is to understand the independent and combined contributions of PRMTs toward cell proliferation, differentiation, and death. One of our short-term goals, for which we are requesting NSERC support, is to establish PRMT-specific arginine methylation networks in different inflammatory response pathways. Scientific Approach. We will subject cultured human cell lines to treatments that cause an NF-kappaB-mediated inflammatory responses. Proteins from treated and untreated control cells will be harvested and either blotted with dimethylarginine-specific antibodies or used for proteomic analysis as part of an ongoing collaboration with Drs. Leonard Foster and Nichollas Scott at the UBC Centre for High-Throughput Biology. Our proteomic approach takes advantage of a recently reported enrichment strategy for methylated peptides using hydrophilic interaction liquid chromatography prior to sample analysis by mass spectrometry. Methylated proteins from control and treatment groups will then be compared for relative quantitation, allowing us to observe changes in PTMs as a way to establish arginine methylation networks in inflammation. The next step will be to determine specific PRMT involvement in inflammatory pathways either by employing cell-based assays in which specific PRMT expression levels will be decreased via siRNA, or by pharmacologically inhibiting specific PRMTs in cultured cells with small molecules. We will also co-immunoprecipitate methylated proteins from cells to determine which PRMTs associate with their substrates by Western blot and mass spectrometry. These substrates will then be tested with in vitro methylation assays to demonstrate PRMT substrate specificity. Impact. Arginine methylation has been demonstrated to perform specific functions in important cellular processes, but this PTM has yet to be studied in the broader context of the cell. By observing arginine methylation changes on the global scale of the proteome, we will identify substrates whose modifications may serve critical roles in inflammatory events, and demonstrate how this PTM participates in various signaling pathways. This work will provide mechanistic insight into how the integration of PRMT activities contributes to cellular programming and ultimately determines cell fate. Our findings should impact the fields of cancer biology, inflammation, and biochemistry.