Protein methylases in Trypanosoma brucei brucei: activities and response to DL-alpha-difluoromethylornithine

Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-L-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent Km for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent Km for S-AdoMet of 8.4 microM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-L-homocysteine and its analogue sinefungin with apparent Ki values of 12.9 and 1.6 microM, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.     

Inhibition of tRNA methylation in vitro and in whole cells by an oncostatic S-adenosyl-homocysteine (SAH) analogue: 5''-deoxy 5''-S-isobutyl adenosine (SIBA).

A high increase in the amount of methylated tRNA bases was found in vivo in Rous sarcoma virus infected and transformed chick embryo fibroblasts in comparison with normal cells, tRNA methylases extracted from transformed cells showed also higher activity in vitro with a heterologous substrate. 5'-deoxy-5'-S-isobutyl adenosine, (a structural analogue of S-adenosyl-L homocysteine), which inhibits virus-induced cell transformation, inhibits also the increase of incorporation of labelled methyl groups into tRNA in infected and transformed cells. When normal cells are grown in the presence of this inhibitor, undermethylated tRNAs are obtained. The effect of the drug is different in normal, infected and transformed cells. The methylation of the different bases is inhibited in vitro and in vivo to various extent. The effect of this substance on tRNA methylation may be the cause of its inhibitory effect on cell transformation.     

Inhibition of sterol transmethylation by S-adenosylhomocysteine analogs.

Structural analogs of S-adenosylhomocysteine were tested in vitro for inhibition of the yeast S-adenosylmethionine:delta 24-sterol-C-methyltransferase enzyme. A wide inhibitory range by these compounds was observed, suggesting which structural features of the parent compound are important for binding to the enzyme. No analog tested had inhibitory activity specific only for this enzyme. The most active compound was sinefungin, a metabolite of Streptomyces griseolus, which was also able to inhibit growth of yeast cultures. Sterol extracts of cells grown in the presence of sinefungin revealed a dramatic increase in the levels of zymosterol, the sterol substrate in the transmethylation under study, and a concomitant decrease in the levels of ergosterol. Evidence is presented that sinefungin is transported inside the cell by the same permease as S-adenosylmethionine. We conclude that sinefungin is blocking the in vivo methylation of sterols in yeast. The implications of this finding are discussed.     

Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

Radical S-adenosylmethionine (SAM) enzymes use the oxidizing power of a 5′-deoxyadenosyl 5′-radical to initiate an amazing array of transformations, usually through the abstraction of a target substrate hydrogen atom. A common reaction of radical SAM (RS) enzymes is the methylation of unactivated carbon or phosphorous atoms found in numerous primary and secondary metabolites, as well as in proteins, sugars, lipids, and RNA. However, neither the chemical mechanisms by which these unactivated atoms obtain methyl groups nor the actual methyl donors are conserved. In fact, RS methylases have been grouped into three classes based on protein architecture, cofactor requirement, and predicted mechanism of catalysis. Class A methylases use two cysteine residues to methylate sp²-hybridized carbon centers. Class B methylases require a cobalamin cofactor to methylate both sp²-hybridized and sp³-hybridized carbon centers as well as phosphinate phosphorous atoms. Class C methylases share significant sequence homology with the RS enzyme, HemN, and may bind two SAM molecules simultaneously to methylate sp²-hybridized carbon centers. Lastly, we describe a new class of recently discovered RS methylases. These Class D methylases, unlike Class A, B, and C enzymes, which use SAM as the source of the donated methyl carbon, are proposed to methylate sp²-hybridized carbon centers using methylenetetrahydrofolate as the source of the appended methyl carbon.     

Biological methylations in the crab Carcinus maenas; in vitro inhibition by a fraction purified from androgen gland extracts]

Extracts from muscles, testis, seminal vesicles and ovaries of the Crab, Carcinus maenas, have been studied in vitro, in presence of [14C]-methyl S-adenosylmethionine, with an E. coli tRNA as methyl acceptor. The highest level of methylases is found in the testis. It has been reported previously that a purified fraction extracted from the androgenic glands of Carcinus maenas inhibits the vitellogenesis in ovaries. We now show that the same fraction inhibits tRNA methylation in an extract of testis as methylase; a 50% inhibition is obtained with about 10 mug of a purified fraction corresponding to 15 glands. With an enzymatic preparation from the ovaries, a 50% inhibition of the tRNA methylase is observed with the purified extract from 4 glands.     

Consequences of binding an S-adenosylmethionine analogue on the structure and dynamics of the thiopurine methyltransferase protein backbone

In humans, the enzyme thiopurine methyltransferase (TPMT) metabolizes 6-thiopurine (6-TP) medications, commonly used for immune suppression and for the treatment of hematopoietic malignancies. Genetic polymorphisms in the TPMT protein sequence accelerate intracellular degradation of the enzyme through an ubiquitylation and proteasomal-dependent pathway. Research has led to the hypothesis that these polymorphisms destabilize the native structure of TPMT, resulting in the formation of misfolded or partially unfolded states, which are subsequently recognized for intracellular degradation. Addition of the cosubstrate, S-adenosylmethionine (SAM), prevents degradation of the TPMT polymorphs in experimental assays, presumably by stabilizing the native structure. Using a bacterial orthologue of TPMT from Pseudomonas syringae, we have used NMR spectroscopy to describe the consequences of binding sinefungin, a SAM analogue, on the structure and dynamics of the TPMT protein backbone. NMR chemical shift mapping experiments localize sinefungin to a highly conserved site in classical methyltransferases. Distal chemical shift changes involving the presumed active site cover imply indirect conformational changes induced by sinefungin, which may play a role in substrate recognition or the catalytic mechanism. Analysis of protein backbone dynamics based on NMR relaxation reveals a combination of complementary effects. Whereas the peripheral, inserted structural elements of the TPMT topology are conformationally stabilized by the presence of sinefungin, a consistent increase in backbone mobility is observed for the central, conserved structural elements. The potential implications for the structural and dynamic effects of binding sinefungin for the catalytic mechanism of the enzyme and the stabilization of the degradation-susceptible TPMT polymorphs are discussed.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.     

Relationship Between Chemical Structure and Antileishmanial Effect of Sinefungine Analogues

Abstract

The synthesis of various analogues of sinefungin (l), having structures 2-5, has been developed by means of an original approach which uses radical chemistry. The study of their biological activities revealed that for the antileishmanial effect of sinefungin, the presence of the amino group at C-6′ in the (S)-configuration and the presence of the carboxyl group at C-9′ are necessary.     

A sensitive assay for histone methyltransferase

A method is described for the assay of histone methyltransferase using soluble histones as substrate. The precipitation of the methylated protein on chromatography paper allows for greater sensitivity and more rapid sample processing than have previously been reported. After incubation of the enzyme in the presence of radioisotopically labeled S-adenosyl-l-methionine and soluble rat brain histone, the residual S-adenosyl-l-methionine is removed by extensive washing in 1.1 m trichloroacetic acid. The amount of methyl groups incorporated into histones is measured by liquid seintillation counting. This procedure can probably be used to assay other protein methylases. A comparison is made between this assay and one using chromosomal bound histones as substrates.     

Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells.

The activity of rRNA methylases was stimulated by high-energy precursors of RNA (ribonucleoside triphosphates) and inhibited by degradation products of RNA (ribonucleotides and oligoribonucleotides). The response of methylases from rat Novikoff ascites tumor and liver to these metabolites was strikingly different. The highly active tumor enzymes responded preferentially to inhibition by catabolic metabolites, whereas the less active liver enzymes responded exclusively to stimulation by anabolic metabolites. When the activity of rRNA methylases was assayed in response to increasing concentration of S-adenosylmethionine, the tumor enzymes responded with a hyperbolic substrate dependence curve and the liver enzymes with a sigmoidal curve. In the presence of an inhibitory dinucleotide, ApA, the tumor enzymes responded with a sigmoidal curve; in the presence of a stimulator, adenosine 5'-triphosphate, the liver enzymes responded with a hyperbolic substrate concentration curve. When normal rats were subject to a series of treatments by thioacetamide, a hepatocarcinogen, the liver nucleolar rRNA methylases became responsive to inhibition by ApA and relatively unresponsive to stimulation by adenosine 5'-triphosphate. When tumor-bearing rats were treated with polyinosinate:polycytidylate, an antitumor agent, the tumor nucleolar rRNA methylases became unresponsive to inhibition by ApA and more responsive to stimulation by adenosine 5'-triphosphate. A correlation was noted between increased methylation efficiency in vivo and increased stability of nucleolar RNA during incubation in vitro, or vice versa. These results are interpreted to indicate that rRNAmethylases are regulated by cellular metabolites during the nucleolar biosynthesis of ribosomes and that rRNA methylases may provide a favorable site for selective action by cancer chemotherapeutic agents.     

Transmethylation and control of pattern formation in hydrozoa

Abstract. The control of pattern formation, cell differentiation and cell proliferation in hydroids involves inhibitory signals. In an attempt to identify their chemical nature, compounds from coelenterates which interfere with metamorphosis and pattern-forming processes in Hydractinia and Eirene were isolated. The most strongly metamorphosis-inhibiting compounds were determined to be N -methylpicolinic acid (homarine), N -methylnicotinic acid (trigonelline) and N -trimethylglycine (betaine). The overal concentration of these compounds within tissues is in the range of several millimoles, but micromolar quantities were found to affect development. Thus, the substances must be mainly present in a stored or an inactivated form. The compounds appear to exert their influence by transfering methyl groups to as yet unknown targets. Chemically related compounds that are not able to function as methyl donors have no or only a much lower inhibitory influence, while potential methyl or ethyl donors such as methionine and ethionine have a strong inhibitory influence. Cycloleucine, a competitor with methionine in the production of S-adenosyl-methionine (SAM), and sinefungin, a competitor with SAM in transmethylation, interfere with the intrinsic morphogenetically active compounds identified. One of the spatial patterns controlled by inhibitory signals is the distance between polyps in colonies. In Eirene , the addition of N -methylpicolinic acid led to an increase in the interpolyp distance, while sinefungin produced a decrease in this distance. The addition of sinefungin also stimulated stolon branching. Thus, control of methylation appears to play a key role in the control of metamorphosis and pattern formation in hydrozoa.     

Binding of cytochrome b5 to membranes of isolated subcellular organelles from rat liver

The in vitro incorporation of a well-characterized integral protein cytochrome b5 into membranes of various subcellular organelles was investigated by biochemical and immunochemical methods. Microsomes, peroxisomes, and outer mitochondrial membranes, all containing endogenous cytochrome b5, incorporated large amounts of the hemoprotein in such a way that it was reducible by an inherent NADH cytochrome b5 reductase. Lysosomal membranes did not incorporate cytochrome b5. Inner mitochondrial and Golgi membranes, which do not naturally contain cytochrome b5, bound it in vitro but it was not reduced in the presence of NADH. These results show some discrepancies between the natural localization and the in vitro binding of cytochrome b5. They confirm one aspect of the fluid membrane theory and bring new elements to our understanding of the maintenance of the specific features of the membranes of subcellular organelles with respect to the cell dynamism.     

Genetic organization of the KpnI restriction--modification system.

The KpnI restriction-modification (KpnI RM) system was previously cloned and expressed in E. coli. The nucleotide sequences of the KpnI endonuclease (R.KpnI) and methylase (M. KpnI) genes have now been determined. The sequence of the amino acid residues predicted from the endonuclease gene DNA sequence and the sequence of the first 12 NH2-terminal amino acids determined from the purified endonuclease protein were identical. The kpnIR gene specifies a protein of 218 amino acids (MW: 25,115), while the kpnIM gene codes for a protein of 417 amino acids (MW: 47,582). The two genes transcribe divergently with a intergeneic region of 167 nucleotides containing the putative promoter regions for both genes. No protein sequence similarity was detected between R.KpnI and M.KpnI. Comparison of the amino acid sequence of M.KpnI with sequences of various methylases revealed a significant homology to N6-adenine methylases, a partial homology to N4-cytosine methylases, and no homology to C5-methylases.     

Stereochemical mechanisms of tRNA methyltransferases

Methylation of tRNA on the four canonical bases adds structural complexity to the molecule, and improves decoding specificity and efficiency. While many tRNA methylases are known, detailed insight into the catalytic mechanism is only available in a few cases. Of interest among all tRNA methylases is the structural basis for nucleotide selection, by which the specificity is limited to a single site, or broadened to multiple sites. General themes in catalysis include the basis for rate acceleration at highly diverse nucleophilic centers for methyl transfer, using S-adenosylmethionine as a cofactor. Studies of tRNA methylases have also yielded insights into molecular evolution, particularly in the case of enzymes that recognize distinct structures to perform identical reactions at the same target nucleotide.     

Exogenous AdoMet and its analogue sinefungin differentially influence DNA cleavage by R.EcoP15I--usefulness in SAGE

While it has been demonstrated that AdoMet is required for DNA cleavage by Type III restriction enzymes, here we show that in the presence of exogenous AdoMet, the head-to-head oriented recognition sites are cleaved only on a supercoiled DNA. On a linear DNA, exogenous AdoMet strongly drives methylation while inhibiting cleavage reaction. Strikingly, AdoMet analogue sinefungin results in cleavage at all recognition sites irrespective of the topology of DNA. The cleavage reaction in the presence of sinefungin is ATP dependent. The site of cleavage is comparable with that in the presence of AdoMet. The use of EcoP15I restriction in presence of sinefungin as an improved tool for serial analysis of gene expression is discussed.     

Genetic Adaptation Controlled by Methylations and Acetylations at the Nuclear and Cytosolic Levels: A Hypothetical Model

Metabolic sensors related to the maturation of metabolism seem to control a process of generic adaptation involving the silencing of genes and the expression of their copies more adapted to environmental changes. Nuclear methylases and histone deacetylases control the gene silencing process. Nuclear methylases compete with cytosolic methylases for the same methyl donnors, this will favor the expression of unmethylated more adapted gene copies, when cytosotic methylases take over. Methylated cytosolic compounds may then represent an index of this adaptation. If a more adapted gene copy is mutated, the regulatory ligand of the gene product that does not find its target may induce a reexpression of the silenced gene. The hypothetical model proposed considers that gene silencing and expression of a more adequate copy involves a nonspecific gene silencer switch that depends on the histone status; the silencer switch is counteracted by the ligand of the adapted gene copy product acting like an inducer.