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Cell Chem Biol. 2016 Sep 22;23(9):1049-1055. doi: 10.1016/j.chembiol.2016.07.013. Epub 2016 Aug 25.

Approaches and Guidelines for the Identification of Novel Substrates of Protein Lysine Methyltransferases.

Author information

1
Faculty of Chemistry, Institute of Biochemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
2
Faculty of Chemistry, Institute of Biochemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany. Electronic address: albert.jeltsch@ibc.uni-stuttgart.de.

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.

KEYWORDS:

enzyme specificity; lysine methylation; post-translational modification; protein lysine methyltransferase; proteome

PMID:
27569752
DOI:
10.1016/j.chembiol.2016.07.013
[Indexed for MEDLINE]
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