Specificity and novel substrates of human protein glutamine methyltransferases

• Applicant: Professor Dr. Albert Jeltsch
• Subject Area: Biochemistry
• Term: from 2014 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263727319

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.

• DFG Programme: Research Grants