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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Assay of Histone Methyltransferases Using Ac-Peptidyl-MCA asSubstratesNorikazu Nishino 1 , Tienabe K. Nsiama 1 , Hongfang Chi 1 , YasushiTakemoto 2 , Akihiro Ito 2 , and Minoru Yoshida 21 Kyushu Institute of Technology, Kitakyushu, Japan; 2 RIKEN, Wako, JapanIntroductionThe methylation of lysine side chain is often found at the N-terminal peptide segment(histone tail) of histone octamer as the post-translational modification [1]. The methylationis mediated by various histone methyltransferases (HMTs) such as G9a, Set9, and others[2,3]. Since the importance of modification of histone tails and other proteins bymethylation is recognized, the discovery of HMT inhibitors has been extensivelychallenging, though the screening method is limited by the conventional radioisotope (RI)detection and ELISA. In the present study, we successfully developed a high-throughputscreening procedure using fluorescent substrate of trypsin. The lysine residue in peptidylMCA (4-methylcoumarine-7-amide) can be methylated by HMT and then lose thesusceptibility toward tryptic activity. These actions of enzymes have been combined into asystem for convenient detection of HMT activity.Results and DiscussionAt the earliest stage, we prepared Boc-L-Lys(Me) n -MCA, where n = 1, 2, and 3, andsubjected to the action of trypsin and lysyl endopeptidase (LEP) to confirm the very poorsusceptibilities of Lys(Me) n toward these enzymes (Figure 1). In order to measure theactivity of HMTs, we used the fluorescent intensity of AMC (7-amino-4-methylcoumarin,ex. 390 nm, em. 460 nm) as an index. Boc-L-Lys-MCA was methylated by the action ofHMTs for an appropriate interval, then trypsin was added to hydrolyze the remainingsubstrate resulting into the release of AMC. Thus we could detect the HMT activity by thedecrease of fluorescence intensity.Fig. 1. Hydrolytic susceptibility of Boc-Lys(Me) n -MCA toward trypsin and LEP.In the next stage, we synthesized various Ac-peptidyl-MCA with Lys at the C-terminilearning from the amino acid sequences of histone tail peptide and some proteins which areknown as HMT substrates (Table 1). Since histone H3K4 and H3K9 are known as themethylation sites of Set9 and G9a, respectively, we examined the sequence specificity ofHMTs by employing these Ac-peptidyl-MCA. As a result we discovered thatAc-ARTKQTARK-MCA, the longest peptide is the most specific and susceptible substrateof G9a. However, Set9 methylated moderately histone tail-related Ac-peptidyl-MCA, butshowed higher activity to p53 and Estrogen Receptor α-sequences; Ac-KRSK-MCA fromERα was the best substrate of Set9 (data not shown). Thus, we confirmed the substratespecificities of HMTs regarding the aminoacid sequences. This assay system could beapplied for further understanding of various HMTs.480