3.4 Development of an Advanced Non-radioactive,
3 Results
First, it was investigated whether the reading domain HP1βinteracts with the H3K9 methylated peptides in the microplate assay. Biotinylated H3 peptides (residues 1 - 19) of all K9 methyla-tion states, were bound to the avidin coated wells. Unmethylated H3K9 and trimethylated H3K36 peptides were included as negative controls. The wells containing different peptides were incubated with GST-fused HP1βand binding of the reading domain was analyzed with a GST-specific antibody (Figure 59A).
Figure 59:Application of the HP1βββ protein as methyl lysine reader. (A) Interaction of GST-tagged HP1β with un-, mono-, di- and trimethylated H3K9 peptides detected with anti-GST antibody.
Trimethylated H3K36 peptide included as control. The signals were normalized to the trimethy-lated H3K9 peptide. (B) Interaction of GST-tagged HP1β protein with the same peptides used in A, detected with HP1β-specific antibody. The signals were normalized to the trimethylated H3K9 peptide.
Two independent experiments were performed. Both replicates were consistent. In this assay, the HP1βreading domain interacted strongly with the trimethylated H3K9 peptides (Figure 59A).
Approximately 1.7-fold weaker binding was observed with the dimethylated and 7.6-fold weaker with the monomethylated peptides. These results reflect the ratios of the binding constants of HP1β, shown in previous reports[176,177]. There, the binding constant of HP1βto H3K9me3 was around 6µM, to dimethylated H3K9 ~1.8-fold weaker and to monomethylated H3K9 ~7.3-fold weaker. The binding signals to the unmethylated H3K9 and trimethylated H3K36 peptides were both very weak (<5 %). The experiments were repeated under the same conditions as described above, but a primary antibody directed toward HP1β was used, instead of a GST-specific antibody. This was because in some experimental setups, the use of a reading domain-specific antibody might be more advantageous, for example if the PKMT itself is fused to a GST-tag (Figure 59B). The signals of HP1βbinding to the different peptides showed a pattern comparable to that observed with the GST-specific antibody. Although the signals were slightly increased, the ratio of monomethylated to unmethylated H3K9 peptides was almost the same as the ratio observed with the GST antibody. However, the ratios of trimethylated to dimethylated H3K9
we observed the same relative binding signals, suggesting a different binding specificity of the two primary antibodies. It seems that the HP1β-specific antibody exhibit a higher detection sensitivity towards HP1β bound to H3K9me1 compared to the GST-specific antibody, which is an advantage because it allows the detection of monomethylated H3K9. H3K9me1 is the methylation product of the first cycle of a PKMT, and this increases the sensitivity of the assay.
These results confirmed the suitability of reading domains to detect peptide methylation in a microplate assay approach.
To investigate the optimal concentration of the HP1β protein, un- and trimethylated H3K9 peptides were incubated with various concentration of the reading domain (Figure 60). The
Figure 60:Determination of optimal HP1βββ concentration. Interaction of unmethylated ([)and
trimethylated(v)H3K9 peptides with various concentrations of GST-tagged HP1β protein, detected with HP1β-specific antibody.
increasing concentration of HP1βled to an increase in the luminescence signal for the trimethy-lated peptide. However, at higher HP1β concentration, increased background (unmethylated H3K9 peptide) signal was noticed as well (Figure 60). All the following experiments were there-fore performed with 40 nM of reading domain.
Next, it was investigated if enzymatically methylated substrates could be detected in this ap-proach as well. An in vitromethylation of biotinylated H3 (1 - 19) peptide was performed with the recombinant SET domain of SUV39H1. The methylation reactions were carried out with 200 nM of SET-SUV39H1 to provide sufficient methylated peptide for the experiment. The unmethylated and trimethylated H3K9 peptides were used as controls and HP1βas reading do-main. For detecting the reading domain the HP1β-specific antibody was used (Figure 61). The signal of the SET-SUV39H1 methylated peptide was about 85 % of the synthetic trimethylated H3K9 control peptide and more than 7-fold higher than the signal observed with the unmethy-lated control peptide (Figure 61). This shows that a high level of methylation was obtained under these conditions and documents a very good dynamic range for the assay. A remarkable signal-to-noise (SN) ratio of 9.5 showed a very good reproducibility of the experiments. To fur-ther assess the quality of the assay, its Z-factor was calculated. This is an established statistical
3 Results
Figure 61:Analysis of SUV39H1 enzymatic activity using the HP1βββ protein as methyl reader. Un-methylated H3K9 peptide was Un-methylated by SUV39H1. UnUn-methylated and triUn-methylated H3K9 pep-tides were included as controls. The peppep-tides were incubated with the HP1βprotein and the inter-actions were detected with the HP1β-specific antibody. The signals were normalized to the synthetic H3K9me3 peptide.
parameter to judge the overall quality of a high-throughput screening assay. With the results observed in the experiments, a Z-factor of 0.65 was received. In larger high-throughput assay systems, Z-factors >0.7 are considered as very good and factors >0.5 are acceptable[178]. To investigate if this assay can be used to screen for PKMT inhibitors, the fungal toxin chaetocin, which was reported to inhibit the methyltransferase activity of SUV39H1, was tested[173]. The SET-SUV39H1 enzyme was pre-incubated with DMSO and different concentrations of chaetocin for 15 min. Afterwards, biotinylated H3 (1 - 19) peptides were methylated with the pre-incubated enzymes (20 nM final concentration of SET-SUV39H1), in methylation buffer containing unla-beled SAM. The methylation reactions were transferred to the avidin coated plate and handled as described above. For detection of the reading domain, the HP1β-specific antibody was used (Figure 62).
Figure 62:Analysis of SUV39H1 enzymatic activity in presence of various concentrations of chaetocin using the HP1βββ protein as methyl reader. The signal of the methylated peptide ob-served after methylation without inhibitor was set as 100 %. The signal obtained with unmethylatd
With increasing concentration of chaetocin, a stronger inhibition of the methyltransferase ac-tivity of SUV39H1 was observed (Figure 62). The data were analyzed by least-squares fitting method to an equation, which described a simple enzyme inhibition reaction. The calculated IC50 value of the inhibition of SUV39H1 by chaetocin for our data is 480 nM, which matches with the published IC50 of 600 nM[173].
Afterwards, the methylation of these samples was further verified by mass spectrometric. The H3 (1 - 19) peptides were methylated by SUV39H1 with various concentration of chaetocin. As control, we included H3 peptide methylated by SUV39H1 in the absence of inhibitor (Figure 63).
Figure 63:MALDI MS analysis of the H3K9 peptide methylation samples incubated with SUV39H1 in presence of various concentrations of chaetocin as shown in Figure 62. The peptide sequence is ARTKQTARKSTGGKAPRKQ-K(Biot)-NH2 and its theoretical molecular weight in the unmethylated state is 2423 kDa. The theoretical masses of the methylated peptides are indicated.
The mass spectrum of the control sample indicated all three methylation levels of H3K9, al-though the highest peak was observed for the unmethylated state (2423.4 kDa). The signals of
3 Results
mono- (2437.4 kDa) and dimethylated (2451.4 kDa) H3K9 were weaker and the trimethyled form of H3K9 (2465.4 kDa) was very weak, but still detectable (Figure 63). However, with increas-ing concentrations of chaetocin the MALDI analysis showed strongly decreased levels of H3K9 methylation. This is validating the results of the ELISA assay. In summary, it was shown that reading domains can be used for high-throughput PKMT inhibitor screens instead of antibodies and that the results obtained by the novel assay were confirmed by mass spectrometry.
4 Discussion
PTMs, such as protein methylation, are important regulators of cellular processes. Methylation of histone proteins regulates chromatin structure, thereby affecting functions, such as transcrip-tional regulation and DNA damage response[30–33]. Additionally, methylation of non-histone proteins controls many other protein functions and properties, such as protein stability, activ-ity, protein-protein interactions and cellular localization[43,44,46,47]. The enzymes responsible for transferring the methyl group to protein substrates play important biological roles. This is indicated by the finding that abnormal expression or aberrant methyltransferase activity is often associated with various diseases and cancer types[129,130,141]. In the recent years, numer-ous reports discovered many novel protein methyltransferase substrates and the number of new protein methylation sites is growing rapidly[97,102]. The identification of such novel substrates is important for a complete understanding of the function of the methyltransferase enzymes and the role of protein methylation in various signaling functions.
In this work, an established method to characterize the substrate specificity profile of three different protein methyltransferases (PMTs) was used[179,180]. Based on the identified substrate recognition motif of the enzymes, several novel substrates were discovered and their methylation was confirmedin vivoand in vitro.