In Vitro Histone Methyltransferase Assay
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INTRODUCTION Histone methyltransferases catalyze the addition of one or more methyl groups to a specific lysine or arginine residue within histones. Currently, there is a great deal of interest in histone methyltransferases, because mutations and misregulation of the genes encoding these proteins have been linked to various cancers and other diseases. Many genes encoding putative histone methyltransferases have been identified in eukaryotes, but the proteins they encode have not been functionally characterized. This protocol describes an in vitro assay for histone methyltransferase activity that uses bacterial cell extracts in which expression of a methyltransferase of interest is induced. In many cases, purification of the enzyme is unnecessary, making this experiment ideal for pilot studies. Bacterial cell extract containing the methyltransferase of interest is incubated with S-adenosyl-L-[ methyl - 3 H]-methionine and various histone substrates, many of which are commercially available. Incorporation of the methyl - 3 H can be measured easily by scintillation counting. The labeled substrate is visualized by SDS-polyacrylamide gel electrophoresis (PAGE) followed by fluorography. This allows the substrate specificity and activity of a histone methyltransferase of interest to be readily characterized.Protein methylation is crucial in epigenetics, and targeting the involved methyltransferases shows great potential for therapeutic intervention with several inhibitors in clinical trials for oncology indications. Therefore, characterization of protein methylation is essential for understanding the methyltransferase function and discovering chemical inhibitors and antagonists. While NMR has been used to measure methylation rates, isotopic labeling of protein or methyl donors can be costly and cannot characterize demethylation of proteins extracted from natural sources. Our method employs a four-quantum filter 1H-13C experiment that selectively detects methyl groups, providing a simple way to characterize methylation and demethylation features of methyltransferases and demethylases, respectively, without requiring isotopic labeling. In our experiments, we successfully observed the methylation of H3 under lysates from various cells and tissues from mice with cancerous growth, showing that H3 is mono- and dimethylated in all the tested lysates, but at varying rates and levels. This method could aid in discovering chemical inhibitors and antagonists against methyltransferases related to diseases by characterizing the methylation features of suspected tumors or areas of concern.
Demethylation
Protein methylation
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RNA methylation
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1. The ratio of the methyl donor, S-adenosylmethionine, to the co-product, S-adenosylhomocysteine (the methylation ratio) is known to control the activity of methyltransferases in tissues. Inactivation of the vitamin B12-dependent enzyme, methionine synthase, reduces the methylation ratio in rats and pigs in vivo. 2. We have determined the effect that such alterations have on neural protein ‘O’ and ‘N’ methyltransferases using an in vitro assay in rats, pigs and humans in the presence of the normal methylation ratio and the abnormal methylation ratios found experimentally in vivo in rats and pigs. 3. The methylation ratio found in the neural tissues of vitamin B12-inactivated pigs significantly inhibits the protein methyltransferases of pigs and humans. 4. By contrast, the altered methylation ratio found in vitamin B12-inactivated rats only marginally inhibits the equivalent rat methyltransferases. 5. This is consistent with the induction of a myelopathy by such treatment in pigs and humans, but not in the rat. 6. Dietary supplements of methionine given to vitamin B12-inactivated pigs have been shown to prevent the myelopathy in vivo by both elevating the neural S-adenosylmethionine level and resetting the methylation ratio. We find in our in vitro assay that these events reinstate the methyltransferase activity to near normal levels, thus explaining its protective effect in vivo.
Methionine synthase
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The observation of correlation between hypermethylation of rickettsial outer membrane protein B (OmpB) and the bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. Two protein lysine methyltransferases (rPKMT1 and rPKMT2) were found to methylate recombinant OmpB fragments through the incorporation of radioactively labeled methyl group from S‐adenosylmethionine (SAM). The two proteins share 45 % identity. LC/MS‐MS analyses showed both rPKMT1 and rPKMT2 methylated at numerous lysine residues of OmpB, but rPKMT1 catalyzed mono‐, di‐ and tri‐methylation of lysine, while rPKMT2 catalyzed exclusively trimethylation of lysine. We used X‐ray crystallography to investigate the structure of the lysine methyltransferases. The crystal structures of rPKMT1 and rPKMT2 and the respective complexes with SAM were determined. These are the first reported crystal structures of outer membrane protein methyltransferases. The crystal structures of rPKMT1 and rPKMT2 are superimposable. Both methyltransferases are dimeric, which was confirmed using dynamic light scattering. Each monomer consists of dimerization, SAM binding, middle, and C‐terminal domains. Comparison of the two structures revealed a number of structural insights of the unusual enzymatic methylation of OmpB.
Transferase
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O-methyltransferase
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Protein methylation
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Phosphoethanolamine methyltransferases add three methyl groups successively to their substrate to produce phosphocholine, an important precursor for phospholipid biosynthesis in diverse organisms. New work from Lee and Jez reveals critical domain movements that explain how multiple methylation reactions are uniquely coordinated by plant methyltransferases and provides insights into the evolution of this class of enzymes. As opposed to closely related family members, the intermediates in this pathway are likely shuttled between two tethered domains to ensure complete methylation.
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Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to containN G,N G-dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli werein vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, withS-adenosyl-l-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.
Protein methylation
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In plants,one of the most common modifications of secondary metabolites is methylation catalyzed by various methyltransferases. Recently,a new class of methyltransferases,the SABATH family of methyltransferases,was found to modify phytohormones and other small molecules.The SABATH methyltransferases share little sequence similarity with other well characterized methyltransferases.Arabidopsis has 24 members of the SABATH methyltransferase genes,and a subset of them has been shown to catalyze the formation of methyl esters with phytohormones and other small molecules.Physiological and genetic analyses show that methylation of phytohormones plays important roles in regulating various biological processes in plants,including stress responses,leaf development,and seed maturation/germination.In this review,we focus on phytohormone methylation by the SABATH family methyltransferases and the implication of these modifications in plant development.
O-methyltransferase
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Protein methylation is a post-translational modification with important roles in transcriptional regulation and other biological processes, but the enzyme–substrate relationship between the 68 known human protein methyltransferases and the thousands of reported methylation sites is poorly understood. Here, we propose a bioinformatic approach that integrates structural, biochemical, cellular, and proteomic data to identify novel cellular substrates of the lysine methyltransferase SMYD2. Of the 14 novel putative SMYD2 substrates identified by our approach, six were confirmed in cells by immunoprecipitation: MAPT, CCAR2, EEF2, NCOA3, STUB1, and UTP14A. Treatment with the selective SMYD2 inhibitor BAY-598 abrogated the methylation signal, indicating that methylation of these novel substrates was dependent on the catalytic activity of the enzyme. We believe that our integrative approach can be applied to other protein lysine methyltransferases, and help understand how lysine methylation participates in wider signaling processes.
Protein methylation
Immunoprecipitation
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