Genetic Engineering & Biotechnology News

MAY15 2017

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12 | MAY 15, 2017 | | Genetic Engineering & Biotechnology News Said A. Goueli 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, nu- cleic 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 or- ganisms as epigenetic control of molecule function. PTMs are important in disease processes such as cancer, inflammation, metabolic abnormalities, and mental disor- ders. Methylation can lead to enhanced transcriptional ac- tivity 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 in- terest in the impact of methylation on disease development. And there is interest in developing modulators that affect en- zyme 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-, fluores- cence-, colorimetric-, and radioactive-based assays (Table). Here we present the highlights of a new universal biolumi- nescent assay that makes monitoring MTase activity and screening for inhibitors/modulators of MTases easy. All of the methodologies for detecting methyltransferase ac- tivity 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 univer- sal assay to monitor the activity of a broad range of these enzymes is highly desirable. To screen for new modula- tors 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, oligo- nucleotide, 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 im- portant 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 pro- teins, and even nucleosomes) to determine the specificity of these enzymes. The basis of MTase-Glo Assay is conversion of the uni- versal reaction product, SAH, into ATP, which is subsequent- ly 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 modula- tion by small molecules in a wide range of plate formats. The assay is well suited for high-throughput screening applica- tions, 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 tremen- dous advantages to the interrogation of multiple pathologi- cal indications. Said A. Goueli, Ph.D. (, is a senior research fellow at Promega. Website: New Universal Assay Platform Designed for High-Throughput Screening Monitor the Activity of Any Methyltransferase Drug Discovery Tutorial Figure 2. Monitoring protein/DNA/RNA methyltransferase activities. Enzyme titration of: (A) protein lysine MTase (EHMT2-G9a); (B) protein arginine MTase (PRMT5/MEP50) using histone derived peptides; (C) protein lysine MTase (DOT1L) using core histones and oligonucleosomes as substrates; and (D) DNA MTase (DNMT3a) using DS-deoxyoligonucleotide. Table. Methyltransferase Activity Detection Methods at a Glance BIOLUMINESCENT Enzymatic detection of SAH with coupled conversion SAH into ATP, the most common enzymatic cofactor. Assay is very sensitive, easy to use, robust, homogeneous and amenable to HTS. Disadvantages: Requires purified enzyme; coupled enzyme systems typically require counter screens. FRE T Chromophore-based method, fluorescently labeled SAH competition with SAH produced during the methylation reaction. Binds to Terbium-labeled anti-SAH antibodies. Disadvantages: Fluorescence interference, negative signal, and antibody-dependent. Concentration of analytes and detection reagents have to be optimized with each substrate:SAM pair. Typically require counter screens. FLUORESCENCE Rely on the conversion of the SAH product into AMP and the competition of this product against fluorescently labeled AMP for binding to anti-AMP antibodies and detection is based on fluorescence polarization. Others generate fluorescently labeled product (resorufin) after multiple conversions and oxidation reactions. Disadvantages: Fluorescence interference, negative signal, and antibody-dependent. Concentration of analytes and detection reagents have to be optimized with each substrate:SAM pair. Sensitivity to high protein concentration causing interference with FP signal. Many steps that can affect the assay performance (e.g., reducing agents). Typically require counter screens. CO LO R I M E T R I C Depends upon generation of a colored product after multiple conversion steps and binding to OxiRed probe. Disadvantages: Fluorescence and color interference. Concentration of analytes and detection reagents have to be optimized with each substrate:SAM pair. Low sensitivity and multiple steps that are affected by assay conditions and substrate concentrations. Typically require counter screens. R A D I O AC T I V E Intracellular accumulation of 3 H-SAH Disadvantages: Nonhomogeneous; hazardous radioactivity; disposal costs; need to separate radiolabeled products from labeled substrates. Figure 1. (top) Schematic representation of the universal MTase-Glo Methyltransferase Assay. After the MTase reaction is complete, the MTase-Glo Reagent is added to convert SAH to ADP. The MTase-Glo Detection Solution is then added to convert ADP to ATP, which is detected via a luciferase reaction. Luminescence output, quantified using a GloMax luminometer, is proportional to the amount of SAH produced. Figures 1A and 1B. Titration of SAH using MTase-Glo Methyltransferase Assay. Luminescence output is proportional to the amount of SAH produced in the reaction with sensitivity down to 30 nM using solid white 384-well plates. B A A C B D

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