title: Approaches and Guidelines for the Identification of Novel Substrates of Protein Lysine Methyltransferases.
abstract: Protein lysine methylation is emerging as a general post-translational modification (PTM) with essential functions regulating protein stability, activity, and protein-protein interactions. One of the outstanding challenges in this field is linking protein lysine methyltransferases (PKMTs) with specific substrates and lysine methylation events in a systematic manner. Inability to validate reported PKMT substrates delayed progress in the field and cast unnecessary doubt about protein lysine methylation as a truly general PTM. Here, we aim to provide a concise guide to help avoid some of the most common pitfalls in studies searching for new PKMT substrates and propose a set of seven basic biochemical rules: (1) include positive controls; (2) use target lysine mutations of substrate proteins as negative controls; (3) use inactive enzyme variants as negative controls; (4) report quantitative methylation data; (5) consider PKMT specificity; (6) validate methyl lysine antibodies; and (7) connect cellular and in vitro results. We explain the logic behind them and discuss how they should be implemented in the experimental work.
authors: Faculty of Chemistry, Institute of Biochemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
authors: Srikanth
authors: S
authors: Kudithipudi
authors: Faculty of Chemistry, Institute of Biochemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany. Electronic address: albert.jeltsch@ibc.uni-stuttgart.de.
authors: Albert
authors: A
authors: Jeltsch
mesh_terms: D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008745:Methylation; D008958:Models, Molecular; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine
keywords: enzyme specificity; lysine methylation; post-translational modification; protein lysine methyltransferase; proteome