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Tutorials: May 15, 2017 (Vol. 37, No. 10)

Monitor the Activity of Any Methyltransferase

New Universal Assay Platform Designed for High-Throughput Screening

  • Methylation is a key post-translational modification (PTM) of mammalian proteins and nucleic acids where unique enzymes known as methyltransferases (MTases) transfer methyl groups to a variety of nucleophiles (e.g., oxygen, carbohydrates, nucleic acids, small molecules, and carbon atoms on proteins).

    In proteins, lysine, arginine, and cysteine are the amino acid targets. In nucleic acids, cytosine is the target in DNA, and adenine in RNA.

    Methylation along with phosphorylation, acetylation, and ubiquitination, for example, are mechanisms used by organisms as epigenetic control of molecule function.

    PTMs are important in disease processes such as cancer, inflammation, metabolic abnormalities, and mental disorders. Methylation can lead to enhanced transcriptional activity of oncogenes or oncosuppressor proteins as well as abnormally altered cellular phenotypes.

    In humans, there are at least 208 S-adenosylmethionine (AdoMET or SAM)-dependent MTases. This class of enzymes makes up 0.9% of genes in the human genome, of which 30% are linked to disease states. Naturally, there is tremendous interest in the impact of methylation on disease development. And there is interest in developing modulators that affect enzyme activity, with potential in developing new drugs, and as such there is need for easier and more sensitive homogeneous methods to monitor the activity of various MTases.

    Current assays now include bioluminescence-, fluorescence-, colorimetric-, and radioactive-based assays (Table). Here we present the highlights of a new universal bioluminescent assay that makes monitoring MTase activity and screening for inhibitors/modulators of MTases easy.

    All of the methodologies for detecting methyltransferase activity listed here are universal. All but one are nonradioactive. The disadvantages are the main distinguishers of assay type.

  • Better Method—Screening for Modulators of Methyltransferases

    Since MTases alter the function of metabolites, a universal assay to monitor the activity of a broad range of these enzymes is highly desirable. To screen for new modulators across most classes of MTases, the ideal assay should be capable of monitoring enzyme activity regardless of the substrate type (protein, nucleic acid, or small molecule) or substrate complexity (peptide, protein, nucleic acid, oligonucleotide, or any small molecule).

    Finally, to be of most utility in screening, the assay should be robust, homogeneous, easy to use, sensitive to low amounts of enzyme and suitable to high-throughput formats in 96, 384, and 1536 multi-well plates.

    MTase-Glo™ Methyltransferase Assay possesses these important features, making it highly suitable for screening large compound libraries with high sensitivity. And because of its universality, the assay can be used with any MTase-substrate combination.

    The assay monitors formation of the reaction product S-adenosyl homocysteine (SAH) and can detect changes in activity of a broad range of MTases, including DNA, protein, RNA, and small molecule MTases. MTase-Glo Assay can be used for all classes of protein MTases (lysine and arginine) and with different types of substrates (peptides, large proteins, and even nucleosomes) to determine the specificity of these enzymes.

    The basis of MTase-Glo Assay is conversion of the universal reaction product, SAH, into ATP, which is subsequently converted to a luminescence read-out through a luciferase-luciferin reaction (Figure 1). The assay is sensitive to 30nM SAH, and the amount of light output is directly proportional to the amount of SAH produced, which we have confirmed by HPLC.

  • As shown in Figure 2, the assay can measure MTases such as lysine- and arginine-MTase. And the assay does so independently of the complexity of the substrate so that full-length histones, histone-derived peptides, histone octamers, as well as nucleosomes can be all used as substrates by the corresponding MTases.

    Similarly, the assay is capable of measuring the transfer of methyl groups to DNA (Figure 2) and RNA, by nucleic acid-specific MTases.

    Conclusion

    MTase-Glo Assay is bioluminescence-based and can be used to monitor the activities of MTases and their modulation by small molecules in a wide range of plate formats. The assay is well suited for high-throughput screening applications, is sensitive and robust as indicated by Z’-values >0.8, with minimal fluorescence interference during screening.

    The universality of the assay enables detection of MTases that use small molecules as substrates and provides tremendous advantages to the interrogation of multiple pathological indications.