Protein posttranslational modifications (PTMs) occur broadly in the human proteome, and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified prot Show more
Protein posttranslational modifications (PTMs) occur broadly in the human proteome, and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylation sites among histone and nonhistone proteins have been characterized; however, the list of readers associated with them is incomplete. This study introduces a modified yeast three-hybrid system (Y3H) to screen for methyllysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like the chromodomains of CBX1 or MPP8 as prey. We next conducted an unbiased screen using a library composed of human-specific open reading frames. It led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei. Our approach introduces a cost-effective method for screening reading domains binding to histone and nonhistone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the power of the Y3H assay which will allow for proteome-wide screens of PTM readers. Show less
New protein lysine methyltransferase (PKMT) assays are needed to facilitate screening for improved PKMT inhibitors, because PKMTs are mutated or overexpressed in several cancers. In cells, methylated Show more
New protein lysine methyltransferase (PKMT) assays are needed to facilitate screening for improved PKMT inhibitors, because PKMTs are mutated or overexpressed in several cancers. In cells, methylated lysine residues are recognized by reading domains such as the chromodomain of HP1β, which bind to target proteins in a lysine-methylation-specific manner. Herein we describe a sensitive, robust, and non-radioactive high-throughput PKMT assay that employs the HP1β chromodomain to detect the methylation of peptide substrates by the human SUV39H1 and SUV39H2 PKMTs. The assay has a very good dynamic range and high signal-to-noise ratio. It can be used to screen for PKMT inhibitors, as illustrated by analyzing the inhibition of SUV39H1 by chaetocin. The IC50 value of this inhibition was found to be 480 nM, which is close to its published value. Our data indicate that natural reading domains can be used as alternates to methyl-specific antibodies in PKMT assays. Reading domains can be produced recombinantly in E. coli at low cost and consistent quality, and they are accessible to protein design. Show less