Regulation of Signaling by Histidine Protein Methylation

摘要 共价翻译后蛋白修饰(PTM)有助于细胞生理学的各个方面，是一种 哺乳动物细胞中蛋白质功能多样性的主要来源。我们的首要目标是更好地理解 蛋白质甲基化信号在调节多种生物功能中的作用以及如何破坏 这些机制促成了癌症和其他疾病的病理。虽然最常被研究的 甲基化事件发生在赖氨酸和精氨酸残基上-谷氨酰胺、半胱氨酸和组氨酸残基也是 甲基化-尽管这些修饰事件被认为只发生在有限数量的蛋白质和 人们对催化这些化学反应的酶知之甚少。尤其是，虽然 通过甲基化修饰组氨酸残基被认为是蛋白质上的罕见事件，甲基化 组氨酸73处的肌动蛋白(肌动蛋白-H73me)是哺乳动物中的一种典型的修饰，已被鉴定出超过50 几年前。然而，肌动蛋白-H73me的功能是谜，而产生这种丰富的酶/S 修改事件未知。在初步工作中，我们已经确定SETD3是第一个已知的后生动物 蛋白质组氨酸甲基转移酶(PHMT)。SETD3，属于SET的一种较少研究的细胞质蛋白 结构域家族的酶，与肌肉功能和癌症的过程有关。然而，一个明确的 SETD3的功能未知。我们最初的数据确定SETD3是感染所需的关键宿主因子 由一大类肠道病毒引起。我们也有证据表明SETD3对平滑肌生理学的调节 在细胞内和体内。在人类中，除了肌动蛋白，几乎对分子、信号和蛋白都一无所知。 与组氨酸甲基化相关的生物学后果。我们的中心假设是组氨酸 肌动蛋白和其他蛋白质的甲基化被像SETD3这样的酶低估和显著 在信号转导、细胞生物学和疾病发病机制中的作用。 我们建议使用生化、细胞、遗传和蛋白质组学的方法来阐明分子， 组氨酸甲基化的生物学和病理功能，重点是围绕SETD3的生物学 和SETD3催化的肌动蛋白修饰。在目标1中，我们将进行实验以获得分子水平 通过SETD3了解肌动蛋白H73甲基化的后果。目标2的目标是调查 SETD3细胞功能。计划进行实验以确定SETD3‘S酶活性在肌动蛋白中的作用。 相关的细胞功能和癌细胞表型。我们还将探索其分子机制。 肌动蛋白与SETD3配对进行甲基化。目标3的目标是扩大我们对组氨酸的了解 人类中SETD3和肌动蛋白以外的甲基化信号。我们将使用计算、蛋白质组学和 鉴定和验证新的人类组氨酸甲基化蛋白和发现新的新的 组氨酸甲基转移酶。

Abstract Covalent post-translational protein modifications (PTMs) contribute to all aspects of cell physiology and are a primary source of protein functional diversity in mammalian cells. Our overarching goal is to better understand the role of protein methylation signaling in the regulation of diverse biological functions and how disruption in these mechanisms contributes to cancer and other disease pathologies. While the most commonly studied methylation events occur on lysine and arginine residues – glutamine, cysteine, and histidine residues are also methylated – though these modification events are thought to occur on only a limited number of proteins and relatively little is known about the enzymes that catalyze these chemical reactions. In particular, while modification of histidine residues by methylation is thought to be a rare event on proteins, the methylation of actin at histidine 73 (actin-H73me) is a canonical modification in mammals that was identified more than fifty years ago. However, the function of actin-H73me is enigmatic and the enzyme/s generating this abundant modification event are not known. In preliminary work we have identified SETD3 as the first known metazoan protein histidine methyltransferase (PHMT). SETD3, a little studied cytoplasmic protein that belongs to the SET domain family of enzymes, is implicated in processes involved in muscle function and cancer. However, a clear function for SETD3 is not known. Our initial data identified SETD3 as a critical host factor required for infection by a broad class of enteroviruses. We also have evidence for SETD3 regulation of smooth muscle physiology in cells and in vivo. In humans, beyond actin, virtually nothing is known about the molecular, signaling and biological consequences associated with histidine methylation. Our central hypothesis is that histidine methylation of actin and other proteins by enzymes such as SETD3 have an underappreciated and significant role in signal transduction, cell biology, and disease pathogenesis. We propose to use biochemical, cellular, genetic, and proteomic approaches to elucidate the molecular, biological and pathological functions of histidine methylation, with a focus on the biology surrounding SETD3 and SETD3-catalyzed modification of actin. In Aim 1 we will perform experiments to gain a molecular level understanding of the consequence of actin H73 methylation by SETD3. The goal of Aim 2 is to investigate SETD3 cellular functions. Experiments are planned to identify the role of SETD3’s enzymatic activity in actin- related cellular functions and cancer cell phenotypes. We will also explore the molecular mechanisms by which actin is paired with SETD3 for methylation. The goal of Aim 3 is to expand our knowledge of histidine methylation signaling in humans beyond that of SETD3 and actin. We will use computational, proteomic, and biochemical strategies to identify and validate novel human histidine methylated proteins and to discover new histidine methyltransferases.