Protein Methyltransferases Research Articles

Structure and function of DNA methyltransferases.

In prokaryotes, the major role of DNA methylation is to protect host DNA against degradation by restriction enzymes. In eukaryotes, DNA methylation has been implicated in the control of several cellular processes, including differentiation, gene regulation, and embryonic development. Structural work on HhaI DNA methyltransferase demonstrates that the substrate nucleotide is completely flipped out of the helix during the modification reaction and has provided much insight into the enzymatic properties of S-adenosyl-L-methionine (SAM)-dependent DNA-modifying enzymes. Structural comparison of three enzymes, HhaI C5-cytosine methyltransferase, TaqI N6-adenine methyltransferase, and catechol O-methyltransferase, reveals a striking similarity in protein folding and indicates that many SAM-dependent methyltransferases have a common catalytic-domain structure. This feature permits the prediction of tertiary structure for other DNA, RNA, protein, and small-molecule methyltransferases from their amino acid sequences, including the eukaryotic CpG methyltransferases.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) N-methyltransferase (protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco. With limited internal amino acid sequence information obtained from HPLC-purified peptic polypeptides from Rubisco LSMT, a full-length cDNA clone was isolated utilizing polymerase chain reaction-based technology and conventional bacteriophage library screening. The 1802 bp cDNA of Rubisco LSMT encodes a 489 amino acid polypeptide with a predicted molecular mass of ca. 55 kDa. A derived N-terminal amino acid sequence with features common to chloroplast transit peptides was identified. The deduced sequence of Rubisco LSMT did not exhibit regions of significant homology with other protein methyltransferases. Southern blot analysis of pea genomic DNA indicated a low gene copy number of Rubisco LSMT in pea. Northern analysis revealed a single mRNA species of about 1.8 kb encoding for Rubisco LSMT which was predominately located in leaf tissue. Illumination of etiolated pea seedlings showed that the accumulation of Rubisco LSMT mRNA is light-dependent. Maximum accumulation of Rubisco LSMT transcripts occurred during the initial phase of light-induced leaf development which preceded the maximum accumulation of rbcS and rbcL mRNA. Transcript levels of Rubisco LSMT in mature light-grown tissue were similar to transcript levels in etiolated tissues indicating that the light-dependent accumulation of Rubisco LSMT mRNA is transient. This is the first reported DNA and amino acid sequence for a protein methylase III enzyme.

Staging and follow up of patients with lymphoproliferative disorder.

<h3>SUMMARY</h3> Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4⁺ T cell differentiation assays revealing the remarkable potential of individual probes to alter multiple T cell subpopulations with important implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity.

Prenylated protein methyltransferases do not distinguish between farnesylated and geranylgeranylated substrates.

Proteins that are post-translationally modified by prenylation can be either farnesylated (C-15) or geranylgeranylated (C-20) by separate prenyltransferase enzymes. Prenylated proteins are also methylated at their C-terminal residue by S-adenosylmethionine-linked methylation. In this paper we show that the methylation of farnesylated and geranyl-geranylated substrates can be accounted for by the presence of a single enzyme. It is demonstrated that the Km and Vmax. values for the retinal rod outer segment methyltransferase, measured with small molecule farnesylated and geranylgeranylated substrates, are identical. These substrates mutually inhibit each other's methylation, with KI values being equal to their Km values. The Km for S-adenosylmethionine was measured to be the same with either farnesylated or geranylgeranylated substrates. Competitive inhibitors of the methyltransferase containing either a geranylgeranyl or a farnesyl group equally block the methylation of synthetic geranylgeranylated and farnesylated substrates of the enzyme. Importantly, these inhibitors are also equipotent at inhibiting the methylation of the physiological substrates of the rod outer segment methyltransferase. These substrates are both farnesylated and geranylgeranylated. One of these substrates had previously been identified as the farnesylated gamma subunit of transducin. Therefore it appears that the same enzymic activity can methylate both farnesylated and geranylgeranylated substrates.

Effects of sinefungin andS-adenosylhomocysteine on DNA and protein methyltransferases fromStreptomycesand other bacteria

Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S. incarnatus. It is structurally related to S-adenosyl-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells. In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA and protein MTases) and on other bacterial DNA-MTases. Protein MTases are resistant to sinefungin, whereas DNA-MTases are inhibited. Adenine MTases however, seem more sensitive to this analogue than cytosine MTases.

Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria

Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S. incarnatus. It is structurally related to S-adenosyl-methionine (SAM) and S- adenosyl- l-homocysteine (SAH). Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells. In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA) and protein MTases) and on other bacterial DNA-MTases. Protein Mtases are resistant to sinefungin, whereas DNA-MTases are inhibited. Adenine MTases however, seem more sensitive to this analogue than cytosine MTases.

Studies on protein methyltransferase in human cerebrospinal fluid

Protein methyltransferases, rich in most mammalian brains, were studied in human cerebrospinal fluid (CSF). Among several well-characterized groups of methyltransferases, protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23) was found in significant amounts in human CSF samples. Both myelin basic protein (MBP) -specific and histone-specific protein methylase I activities were observed, the latter being generally higher in most CSF. S-Adenosyl-L-homocysteine, a potent product inhibitor for the methyltransferase, inhibited approximately 90% of MBP-specific protein methylase I activity at a concentration of 1 mM. The optimum pH of the MBP-specific protein methylase I was found to be around 7.2. Identity of exogenously added MBP as the methylated substrate for CSF enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An amino acid analysis of the [methyl-3H]protein hydrolysate showed two major radioactive peaks cochromatographing with monomethyl- and dimethyl (symmetric)-arginine. Human CSF contained relatively high endogenous protein methylase I activity (activity measured without added substrate protein): The endogenous substrate can be immunoprecipitated by antibody raised against calf brain MBP. Finally, CSF from several neurological patients were analyzed for protein methylase I, and the results are presented.

In situ hybridization of Drosophila polytene chromosomes with digoxigenin-dUTP-labeled probes.

Bicoid directs pattern formation in the developing Drosophila embryo, and does so by performing two seemingly unrelated tasks; it activates transcription and represses translation. To understand how Bicoid carries out this dual role, we sought to identify Bicoid-ancillary proteins that might mediate Bicoid's function in transcription or translation. We used a customized version of the two-hybrid method and found two Bicoid-interacting proteins, Bin1 and Bin3, both of which interact with Bicoid in vitro. Bin1 is similar to a human protein (SAP18) involved in transcription regulation, and Bin3, described in this paper, is similar to a family of protein methyltransferases that modify RNA-binding proteins. Given that Bicoid's role as a translation regulator requires RNA binding, we suggest that the Bicoid-interacting methyltransferase might be important for that role. The custom two-hybrid method we used, in which Bicoid is bound to DNA via its own DNA binding domain, rather than via a fusion-protein tether, should be generally applicable to other DNA binding proteins.

Protein O-carboxylmethyltransferase in spermatozoa from normal and infertile men

In confirmation of several reports, suspensions of normal washed human spermatozoa exposed to a nonionic detergent exhibited considerable activity of the enzyme protein O-carboxylmethyltransferase (PCM), which catalyzes the methyl esterification of carboxyl groups of dicarboxylic amino acid residues in proteins. Various methods for assay of human spermatozoal PCM levels were evaluated, and some properties of the enzyme were studied. Normal human spermatozoa appear to be devoid of other types of protein methyltransferases that catalyze N-methylations of arginyl or lysyl residues in proteins. Spermatozoal PCM levels in infertile patients with motile sperm cells tended to be somewhat higher than those of normal control subjects, especially in those instances where the spermatozoal populations contained abnormally high proportions of immature forms of spermatozoa. Although totally immotile spermatozoa obtained from certain patients exhibited very low PCM activities (as recently reported by other investigators), in this study no invariant relationship between zero motility indexes and spermatozoal PCM was observed. These results are discussed in light of various current hypotheses regarding the functions of PCM in animal cells.

Protein methylation in animal cells I. Purification and properties of [formula omitted]N-methyltransferase from Krebs II ascites cells

1. A protein methylase which specifically transfers methyl groups from S- adenosyl- l-methionine to arginine residues of histones has been substantially purified from Krebs II ascites cells. The purified enzyme was obtained free of contamination by other protein methyl transferases specific for carboxyl and lysine residues. This latter activity copurified with the present enzyme until advanced stages of purification. 2. The purified enzyme does not require any divalent cation for maximum activity. It is inhibited by ionic strength, N-ethylmaleimide and S- adenosyl- l-homocysteine . It has an apparent molecular weight on gel filtration of approx. 5 · 10 5. A K m value for S- adenosyl- l-methionine of 2.5 · 10 −6 M was determined, while the dissociation constant K i for S- adenosyl- l-homocysteine , which acts as a competitor, was 1.4 · 10 −6 M.

The Periodic Synthesis of S-Adenosylmethionine:Protein Methyltransferases during the HeLa S-3 Cell Cycle

Abstract The activities of three S-adenosylmethionine:protein methyltransferases (protein methylase I, II, and III) were measured throughout the mitotic cycle of synchronized HeLa S-3 cells in an attempt to determine the biological function of these enzymes. 1. The activities of protein methylase I and II, which catalyze the methylation of protein-arginine residues and carboxyl groups, increase more or less continuously until they have doubled by the end of G2 phase. These increases were not obviously correlated with major cell cycle events such as DNA replication or histone synthesis and modification. 2. The activity of protein methylase III (S-adenosylmethionine:protein-lysine methyltransferase) remains relatively constant through G1 and early S phase of the cell cycle, then begins increasing until it reaches over three to four times its initial level by the end of G2. The increase in protein methylase III activity is temporally correlated with the increased mono- and dimethylation of the e-amino groups of histone lysine residues. 3. By inhibiting protein and RNA synthesis it was determined that this increase in protein methylase III activity in late S and G2 of the HeLa cell cycle was probably due to the de novo synthesis of enzyme molecules of those times. 4. The same 3- to 4-fold increase in the activity of protein methylase III occurs at the usual time after mitosis even if DNA and histone synthesis were prevented by treatment of the cells with cytosine arabinoside. In such treated cells, the enzyme apparently overmethylated the already mono- and dimethylated lysine residues of previously synthesized histone molecules to the di- and trimethyllysine state. These data suggest that protein methylase III synthesis is not triggered by the appearance of its normal substrate but rather is coupled with some other unknown cell cycle event.

Protein methylases during the development of rat brain

Various protein methylases ( S-adenosylmethionine : protein methyltransferases) are evenly distributed in the cerebrum of rat. Protein methylase I and II are cytosolic and protein methylase III is nuclear bound. A substantial amount of protein methylase I was also found in the synaptosome. While the amount and the rate of precursor incorporation of protein in rat brain increased during the early phase of life, the amount and the rate of radioactive precursor incorporation of DNA and RNA gradually decreased. The amount of protein methylase I ( S-adenosylmethionine : protein arginine methyltransferase) per cell or in the total brain increases after birth until 10 days of life, and thereafter remains unchanged. Protein methylase III activity ( S-adenosylmethionine : protein-lysine methyltransferase) also increases until 10 days after birth, however, it decreases significantly thereafter. Contrary to the above two enzymes, protein methylase II activity ( S-adenosylmethionine : protein carboxyl methyltransferase) increases continually until puberty. Since myelination begins at about 10 days after birth in rat, and degenerative mutant jimpy mice contain normal amounts of the protein methylases, the above result suggests that methylation of protein in the brain might not be related to the formation of myelin.