Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Guitot, Karine; Scarabelli, Silvia; Drujon, Thierry; Bolbach, G.; Amoura, Mehdi; Burlina, Fabienne; Jeltsch, Albert; Sagan, Sandrine; Guianvarc’h, Dominique

doi:10.1016/j.ab.2014.04.006

Cited by 20 publications

(12 citation statements)

References 45 publications

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“…To establish the substrate efficiency of lysine-and K CP -containing histone peptides, we carried out enzyme kinetics analysis, employing the MALDI-TOF MS assays 22 . Both enzymes preferentially catalyze methylation of natural histone sequences, however, bulkier K CP -containing peptides still underwent favorable kinetics profiles (Table 1 and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Examining sterically demanding lysine analogs for histone lysine methyltransferase catalysis

Temimi

Tran

Teeuwen

et al. 2020

Sci Rep

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Methylation of lysine residues in histone proteins is catalyzed by S-adenosylmethionine (SAM)dependent histone lysine methyltransferases (KMts), a genuinely important class of epigenetic enzymes of biomedical interest. Here we report synthetic, mass spectrometric, nMR spectroscopic and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics studies on KMtcatalyzed methylation of histone peptides that contain lysine and its sterically demanding analogs. our synergistic experimental and computational work demonstrates that human KMts have a capacity to catalyze methylation of slightly bulkier lysine analogs, but lack the activity for analogs that possess larger aromatic side chains. overall, this study provides an important chemical insight into molecular requirements that contribute to efficient KMT catalysis and expands the substrate scope of KMTcatalyzed methylation reactions. Posttranslational modifications on histone proteins regulate the structure and function of human chromatin 1-3. Well-established examples include lysine acetylation, which is linked with the transcriptionally active region of human genome, and lysine methylation, which is associated with gene activation and suppression, depending on the histone sequence and methylation state 4,5. Histone lysine methylation is catalyzed by S-adenosylmethionine (SAM)-dependent histone lysine methyltransferases (KMTs), and can lead to a formation of monomethyllysine (Kme), dimethyllysine (Kme2) and trimethyllysine (Kme3) 6,7. It is generally believed that the methylation state depends on the constitution of the KMT active site (Fig. 1a) 8. With the exception of DOT1L, all members of KMT family possess the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain 9-11. Structural analyses of KMTs complexed with histone peptide/methylated peptide and S-adenosylhomocysteine product (SAH) revealed that the lysine side chain occupies a narrow, hydrophobic channel, typically comprised of side chains of several tyrosine and phenylalanine residues (Fig. 1b) 8. The positioning of the lysine's N ε amino group towards the electrophilic methyl group of the SAM cosubstrate results in an efficient methyl transfer via S N 2 reaction 12,13. Recent examinations of lysine analogs as substrates for human histone lysine methyltransferases revealed that KMTs possess a high degree of specificity for lysine residues. Enzymatic assays employing MALDI-TOF MS verified that human KMTs preferentially catalyze methylation of lysine residues with L-stereochemistry over D-stereochemistry 14. Combined experimental and computational studies on histone peptides that bear lysine analogs of different chain length revealed that lysine exhibits an optimal chain length for KMT-catalyzed methylation 15 , and that the enzymatic methylation is limited to N-nucleophiles 16. Members of KMTs were also found to catalyze methylation of the cysteine-derived γ-thialysine on intact histones and histone peptides 17,18. Substrate capturing studies using the genetically encoding photo-lysine showed th...

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Section: Resultsmentioning

confidence: 99%

Examining sterically demanding lysine analogs for histone lysine methyltransferase catalysis

Temimi

Tran

Teeuwen

et al. 2020

Sci Rep

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“…In common with the other MLL proteins, MLL4 has been associated with mono-, di-, and trimethylation of H3K4 in cellular studies ( Issaeva et al., 2007; Lee et al., 2013 ). We used MALDI-TOF mass spectrometry to investigate the methylated state of histone peptide following incubation with MLL4 ( Guitot et al., 2014 ). An unmodified histone peptide was incubated with an equimolar concentration of enzyme, and the reaction products were analyzed at specified time points ( Figure 6 C).…”

Section: Resultsmentioning

confidence: 99%

“…MALDI-TOF mass spectrometry was used to examine the reaction products of the methyltransferase reaction with the H3 peptide at specific time points, essentially as described by Guitot et al. (2014) .…”

Section: Methodsmentioning

confidence: 99%

Evolving Catalytic Properties of the MLL Family SET Domain

et al. 2015

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SummaryMethylation of histone H3 lysine-4 is a hallmark of chromatin associated with active gene expression. The activity of H3K4-specific modification enzymes, in higher eukaryotes the MLL (or KMT2) family, is tightly regulated. The MLL family has six members, each with a specialized function. All contain a catalytic SET domain that associates with a core multiprotein complex for activation. These SET domains segregate into three classes that correlate with the arrangement of targeting domains that populate the rest of the protein. Here we show that, unlike MLL1, the MLL4 SET domain retains significant activity without the core complex. We also present the crystal structure of an inactive MLL4-tagged SET domain construct and describe conformational changes that account for MLL4 intrinsic activity. Finally, our structure explains how the MLL SET domains are able to add multiple methyl groups to the target lysine, despite having the sequence characteristics of a classical monomethylase.

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“…To address these issues, we have developed a sensitive and fast assay to quantify in vitro E2/E3 enzyme activity using MALDI-TOF MS. It builds on our DUB MALDI-TOF assay ( Ritorto et al., 2014 ), which has enabled us to screen successfully for a number of selective DUB inhibitors ( Kategaya et al., 2017 , Magiera et al., 2017 , Weisberg et al., 2017 ), and adds to the increasing number of drug discovery assays utilizing label-free high-throughput MALDI-TOF MS. Apart from E2/E3 enzymes and DUBs ( Ritorto et al., 2014 ), high-throughput MALDI-TOF MS has now successfully been used for drug discovery screening of protein kinases ( Heap et al., 2017b ), protein phosphatases ( Winter et al., 2018 ), histone demethylases, and acetylcholinesterases ( Haslam et al., 2016 ), as well as histone lysine methyltransferases ( Guitot et al., 2014 ).…”

Section: Discussionmentioning

confidence: 99%

The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway

Cesare

Johnson

Barlow

et al. 2018

Cell Chemical Biology

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SummaryDue to their role in many diseases, enzymes of the ubiquitin system have recently become interesting drug targets. Despite efforts, primary screenings of compound libraries targeting E2 enzymes and E3 ligases have been strongly limited by the lack of robust and fast high-throughput assays. Here we report a label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry. The MALDI-TOF E2/E3 assay allows testing E2 enzymes and E3 ligases for their ubiquitin transfer activity, identifying E2/E3 active pairs, inhibitor potency and specificity and screening compound libraries in vitro without chemical or fluorescent probes. We demonstrate that the MALDI-TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared with standard biochemical assays.

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Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cited by 20 publications

References 45 publications

Examining sterically demanding lysine analogs for histone lysine methyltransferase catalysis

Examining sterically demanding lysine analogs for histone lysine methyltransferase catalysis

Evolving Catalytic Properties of the MLL Family SET Domain

The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway

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