Specificity and novel substrates of human protein glutamine methyltransferases

蛋白质的修饰在生物学中起着关键作用。虽然一些修饰广泛分布并进行了详细研究（如丝氨酸、苏氨酸或酪氨酸的磷酸化，赖氨酸的乙酰化以及赖氨酸或精氨酸的甲基化），但谷氨酰胺的甲基化是一种罕见的修饰，没有进行非常深入的研究。目前，在人类蛋白质组中仅已知三种谷氨酰胺甲基化。这些修饰由三种已知的人谷氨酰胺甲基转移酶引入，即细胞溶质HemK2、线粒体HMPrmC和Fibrillarin酶。HemK2的缺失在小鼠中是致命的，表明这种酶具有非常重要的生理作用。这是这项工作的一个主要目标，以确定额外的基板HemK2可以解释致死敲除表型。达到这一目标的一种方法是确定酶的底物序列特异性。基于这些数据，我们将确定新的底物候选者，并在体外和体内研究它们的甲基化。我们已经证明，这种方法具有发现蛋白质甲基转移酶的新底物的巨大潜力，并且初始数据表明存在许多HemK2的新肽底物，并且在几种情况下和在人类细胞中证实了蛋白质水平的甲基化。此外，我们将应用蛋白质组学方法来研究HemK2的底物谱。为此，我们将从已经建立的HemK2敲低细胞系中制备蛋白质提取物，使用纯化的HemK2在体外对其进行甲基化，并鉴定甲基化底物。在此之后，我们将研究相应的甲基化是否也发生在细胞中的内源性蛋白质中。为了研究谷氨酰胺甲基化的新蛋白质底物的生物学作用，我们计划产生特异性结合谷氨酰胺甲基化蛋白质的抗体。这种方法已经取得了成功的一个例子。这些新型试剂将使我们能够研究不同细胞类型中底物蛋白的谷氨酰胺甲基化水平的差异以及细胞周期期间或细胞分化时谷氨酰胺甲基化的变化。此外，我们已经与研究我们最有希望的靶蛋白的小组建立了合作关系，并将进行特定的实验来研究谷氨酰胺甲基化是否影响其生物学功能。最后，我们计划克隆，表达和研究人Fibrillarin蛋白，最近已被证明甲基化组蛋白H2A中的一个谷氨酰胺残基。我们的研究结果将阐明蛋白质谷氨酰胺甲基转移酶的底物识别机制，新的谷氨酰胺甲基转移酶靶标的鉴定将大大提高我们对这种罕见且未充分研究的翻译后修饰类型的认识。

The modification of proteins plays a key role in biology. While some modifications are widely distributed and studied in great detail (such as the phosphorylation of serine, threonine or tyrosine, acetylation of lysine, and methylation of lysine or arginine), the methylation of glutamine is a rare modification that is not studied very intensively. Currently, only three glutamine methylations are known in the human proteome. These modifications are introduced by three known human glutamine methyltransferases, the cytosolic HemK2, the mitochondrial HMPrmC and the Fibrillarin enzymes. Deletion of HemK2 is lethal in mice, indicating a very important physiological role of this enzyme. It is one main goal of this work to identify additional substrates of HemK2 which may explain the lethal knock-out phenotype. One approach to reach this goal is to determine the substrate sequence specificities of the enzyme. Based on these data, we will identify novel substrate candidates and investigate their methylation in vitro and in vivo. We have already demonstrated that this approach has a great potential for discovery of novel substrates of protein methyltransferases and initial data indicate the presence of many novel peptide substrates of HemK2 and confirmed methylation at protein level in several cases and in human cells. In addition, we will apply a proteomics approach to study the substrate profile of HemK2. To this end, we will prepare protein extracts from already established HemK2 knock-down cell lines, methylate them in vitro using purified HemK2 and identify the methylated substrates. After this, we will investigate, if the corresponding methylations also occur with endogenous proteins in cells. To investigate the biological roles of the glutamine methylation of novel protein substrates, we plan to generate antibodies which specifically bind to glutamine methylated proteins. This approach was already successful for one example. These novel reagents will enable us to study differences in the level of glutamine methylation of the substrate proteins in different cell types and changes of glutamine methylation during the cell cycle or upon cellular differentiation. In addition, we have formed collaborations with groups working on our most promising target proteins, and will conduct specific experiments to investigate if the glutamine methylation affects their biological function. Finally, we plan to clone, express and investigate the human Fibrillarin protein, which very recently has been shown to methylate one glutamine residue in Histone H2A. Our results will shed light on the mechanism of substrate recognition of protein glutamine methyltransferases and the identification of novel glutamine methyltransferase targets will greatly advance our knowledge of this rare and not well studied type of post-translational modification.