The rat S-adenosylhomocysteine hydrolase promoter.

The 1.2-kb DNA sequence flanking the transcription start of the AdoHcy hydrolase gene was cloned into the luciferase reporter plasmid pGL3-basic, and promoter activity was measured in transiently transfected CHO cells. Deletion analysis showed that most promoter activity was located within a 153 bp fragment immediately upstream from the predominant transcription start. The 153 bp fragment includes sites for AP-2, glucocorticoid-responsive element, SP-1, and a TATA-like sequence TATTTAAA. Mutational analysis demonstrated that the SP-1 site nearest the start of transcription contributed significantly to promoter activity, whereas, the other elements, including the appropriately positioned TATTTAAA sequence, had little affect on promoter activity.

The gene and pseudogenes of rat S-adenosyl-L-homocysteine hydrolase.

Two rat liver genomic DNA libraries constructed in lambda DASH and lambda Charon 4A were screened for sequences with similarity to S-adenosyl-L-homocysteine (AdoHcy) hydrolase cDNA. Of 36 clones purified, two contained the AdoHcy hydrolase gene sequence and 34 contained pseudogene sequences. The AdoHcy hydrolase gene, which has been sequenced in its entirety, spans approximately 15 kb and consists of 10 exons. Primer extension and S1 experiments show that transcription is initiated from two major initiation sites located at positions -63 and -62 from the starting codon and from several minor sites. The promoter region is located in a CpG island, sequence TATTTAAA is present 23 bases upstream from the transcription start site, and an inverted CCAAT box is located 285 bp upstream from the transcription start site. Other potential transcription-factor binding sites including SP1, AP-2, GRE and Oct-1 sites were identified in the 5'-flanking region. Several different processed pseudogenes were found and analyzed.

The role of cysteine 78 in fluorosulfonylbenzoyladenosine inactivation of rat liver S-adenosylhomocysteine hydrolase.

Inactivation of rat liver S-adenosylhomocysteine hydrolase by the site-directed reagent 5'-p-fluorosulfonylbenzoyladenosine (FSBA) is associated with the formation of a disulfide bond between Cys-78 and Cys-112 (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362; Gomi, T., Ogawa, H., and Fujioka, M. (1986) J. Biol. Chem. 261, 13422-13425). To characterize the inactivation mechanism more precisely, the properties of four hydrolase proteins mutated at Cys-78 or Cys-112 were compared to those of the wild-type enzyme. When Cys-78 was mutated to either a serine or an alanine, proteins with greatly reduced enzymatic activity were obtained, large effects on kinetic constants were observed, and enzymatic activity was not affected by incubation with FSBA. When Cys-112 was mutated to either a serine or an alanine, the activity was similar to the wild-type protein, only small changes in the kinetic constants were observed, and the enzyme was inactivated more rapidly upon incubation with FSBA. FSBA inactivation of the C112A mutant protein was accompanied by the formation of a disulfide between Cys-78 and Cys-52. The data indicate that FSBA initially reacts with Cys-78 and that Cys-78 has an important role in the structure of the enzyme.

Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism.

Although the first observations of the inhibitory effect of methylation on gene activity were made almost a decade ago, the mechanism by which methyl groups affect transcription is still obscure. Here we use engineered promoters methylated in vitro in transient transfections to study the mechanism by which methylation mediates promoter repression. The results clearly show that the location of the methyl groups within the promoter region determines the extent of promoter repression. The most effective suppression was observed when methylation was in the preinitiation domain. The results also support a previous suggestion that a mediator protein is involved in the mechanism of promoter inhibition. The suppressor effect of methylation at sequences flanking the TATA box can be partially overcome in the presence of the simian virus 40 enhancer. In addition, results obtained by transient thymidine labeling of Ltk- cells that were transfected with a methylated thymidine kinase gene from herpes simplex virus, at the level of approximately one template per cell, further support the conclusion that methylation affects primarily transcription preinitiation.

Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus.

The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulatus, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medium; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis.

Inhibition of promoter activity by methylation: possible involvement of protein mediators.

To study the relationship between DNA methylation and promoter activity we have methylated in vitro the promoters of the mouse metallothionein I gene and the herpes simplex virus thymidine kinase gene. We have transiently transfected these promoters fused to the human growth hormone in their methylated or unmethylated state into mouse L or F9 cells. Promoters methylated by methylase (M.) Hpa II and M.Hha I caused inhibition of reporter gene expression in L cells but not in F9 cells, while methylation of all CpGs by M.Sss I caused inhibition in both cell lines. Repression of promoter activity by M.Hpa II and M.Hha I methylation, but not by M.Sss I methylation, could be alleviated by cotransfection with an excess of untranscribable DNA methylated with M.Sss I. The methylated sites in nuclei isolated from the transfected L cells, but not F9 cells, were found to be protected from Msp I digestion. Taken together these results suggest that a factor present in L cells and missing in F9 cells mediates the methylation-directed inhibition of promoter activity. The ability of methylated DNA to overcome the inhibition seems to reflect competition for the mediator factor. Interestingly, treatment with Zn2+ ions brought about activation of the methylated promoter of the metallothionein gene. Similarly, butyrate could override the repression of the thymidine kinase methylated promoter. These activations were not accompanied by demethylation of the promoter or displacement of the mediator factor.

Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding.

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme. In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit. The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+. From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme. In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme. Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively. These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site. This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 719-723).

Affective disorders and S-adenosylmethionine: a new hypothesis.

S-Adenosyl-L-methionine (AdoMet) is a safe and probably effective antidepressant agent in certain forms of clinical depression. This article presents a new hypothesis to account for the mechanism of action of S-adenosylmethionine in such illnesses, based upon the known biochemistry of this compound, and upon current knowledge of clinical and genetic aspects of affective disorders. Giulio Cantoni, S. Harvey Mudd and V. Andreoli postulate that at least some major mood disorders are due to abnormalities affecting the AdoMet-dependent methylation of a substance in the CNS. For convenience and without prejudging the chemical structure of this substance, they call it 'barinine'. The model requires that barinine be subject to AdoMet-dependent methylation and that methylbarinine be subject to metabolic demethylation to regenerate the original barinine. Methylbarinine should be mood elevating, whereas barinine itself should not be. Depression is a result of abnormalities lowering the normal steady-state concentration of methylbarinine, whereas mania results from an abnormal elevation of methylbarinine.

Expression of rat liver S-adenosylhomocysteinase cDNA in Escherichia coli and mutagenesis at the putative NAD binding site.

The cDNA for rat liver S-adenosylhomocysteinase has been cloned, and the nucleic acid sequence has been determined. By comparison of the deduced amino acid sequence for S-adenosylhomocysteinase with that of the dinucleotide binding region for other proteins, the sequence from amino acids 213 to 244 in rat liver S-adenosylhomocysteinase was proposed to be part of the NAD binding site (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). A vector has been constructed that expresses S-adenosylhomocysteinase in Escherichia coli in the presence of isopropyl beta-D-thiogalactopyranoside by inserting the coding sequence of rat liver S-adenosylhomocysteinase cDNA downstream from the lac promoter of plasmid pUC118. The enzyme that is produced comprises as much as 10% of the soluble cellular proteins. The purified enzyme is a tetramer, contains 4 mol of tightly bound NAD, and has kinetic properties indistinguishable from those of the liver enzyme. Tryptic peptide mapping and NH2-terminal sequence analysis indicate that the recombinant enzyme is structurally identical to the liver enzyme except for the absence of the NH2-terminal blocking group. The rat liver enzyme has a blocked NH2-terminal alanine residue (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). By oligonucleotide-directed mutagenesis mutant vectors have been generated that express proteins in which each of the glycines in the Gly-Xaa-Gly-Xaa-Xaa-Gly sequence of the putative NAD binding site (residues 219-224) was changed to valine. Immunoblot analysis of extracts of the cells transformed with these vectors reveals the presence of immunoreactive proteins with the subunit molecular weight of S-adenosylhomocysteinase. The mutant proteins have no catalytic activity, contain no bound NAD, and do not form the same quaternary structure as the recombinant S-adenosylhomocysteinase.

Relationship between transient DNA hypomethylation and erythroid differentiation of murine erythroleukemia cells.

The state of DNA methylation in mouse erythroleukemia (MEL) cells has been analyzed in relation to commitment to differentiation in response to treatment with hexamethylenebisacetamide (HMBA). Previous experiments have shown that induction by HMBA involves transient genome-wide hypomethylation of DNA that is achieved by replacement of 5-methylcytosine with cytosine residues. The experiments described in the present communication revealed that hypomethylation is a very early event in the process of differentiation. Exposure of the cells to 3-deazaadenosine, an adenosine analog, in combination with homocysteine, resulted in the intracellular accumulation of 3-deazaadenosylhomocysteine, which caused an inhibition of HMBA-induced hypomethylation that was correlated with a comparable inhibition of differentiation. While these experiments suggest that hypomethylation is a necessary step in the process of differentiation, other experiments reported here indicate that hypomethylation of DNA may be necessary but not sufficient to trigger the whole program of differentiation in MEL cells. We found, for example that exposure of the cells to cycloheximide during the first 24 hr of induction by HMBA resulted in complete inhibition of differentiation without significant effect on the HMBA-induced hypomethylation. This result also indicates that the enzymatic machinery required for the hypomethylation of DNA is present in uninduced cells.

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum as deduced from the cDNA sequence.

S-Adenosyl-L-homocysteine hydrolase has been cloned from a lambda gt11 cDNA library prepared from Dictyostelium discoideum that had been starved for 3 hours. The sequence of the cloned cDNA was determined and the deduced amino acid sequence was compared to the amino acid sequence of rat AdoHcy hydrolase. When the sequences from the two species were aligned, 74% of the amino acids were in identical positions. If conservative changes were taken into account the homology was 84%. Because differences have been reported in the binding characteristics of NAD+ to the D. discoideum and rat AdoHcy hydrolases, changes in the amino acids of the putative NAD+-binding site were of particular interest. Six changes were observed in this region but the changes appeared to be in regions that are not critical to the three dimensional folding of the NAD+-binding site.

Molecular cloning, sequence analysis, and expression in Escherichia coli of the cDNA for guanidinoacetate methyltransferase from rat liver.

Five cDNA clones encoding rat liver guanidinoacetate methyltransferase (S-adenosyl-L-methionine: guanidinoacetate N-methyltransferase, EC 2.1.1.2) were isolated from a lambda gt11 cDNA library by use of a polyclonal antibody to the purified enzyme. Sequence analysis of the longest cDNA indicated that it consisted of 711 base pairs (bp) of coding region, 51 bp of 5' noncoding region, and 162 bp of 3' noncoding region excluding the poly(A) tail. The amino acid sequence deduced from the cDNA contained the sequences of NH2-terminal and three tryptic peptides. The predicted amino acid composition and molecular weight were in excellent agreement with those obtained with the purified enzyme. Introduction of the cDNA into plasmid pUC118 having the lac promoter resulted in a production in Escherichia coli of a Mr 26,000 polypeptide in the presence of isopropyl beta-D-thiogalactopyranoside. This protein represented as much as 5% of the bacterial soluble protein and showed the guanidinoacetate methyltransferase activity. Sequence analysis and tryptic peptide mapping indicated that the enzyme obtained by the recombinant DNA procedures was structurally identical to the liver enzyme, except for the absence of the NH2-terminal blocking group. Also, the enzyme showed kinetic properties indistinguishable from those of the liver enzyme.

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from rat liver as derived from the cDNA sequence.

Rat liver cDNA libraries constructed in lambda gt11 were screened for reactivity with polyclonal antibodies to native S-adenosyl-L-homocysteine (AdoHcy) hydrolase (adenosylhomocysteinase; EC 3.3.1.1). Five clones were isolated and sequenced. The amino acid sequence, deduced from the cDNA sequence, contained the sequence of eight peptides obtained by tryptic and cyanogen bromide fragmentation of rat liver AdoHcy hydrolase. Identification of the amino- and carboxyl-terminal peptides in the amino acid sequence showed that the complete sequence was obtained. A "fingerprint" sequence was found that is characteristic of dinucleotide-binding domains of many proteins. For AdoHcy hydrolase, this region from the lysine at position 213 to the aspartate at position 244, containing the sequence Gly-Xaa-Gly-Xaa-Xaa-Gly at positions 219-224, is presumably the site of binding for NAD+, which is required for the activity of the enzyme.

S-adenosylhomocysteinase: mechanism of reversible and irreversible inactivation by ATP, cAMP, and 2'-deoxyadenosine.

Homogeneous S-adenosylhomocysteinase (AdoHcyase) from rat liver is a tetrameric enzyme that contains four molecules of tightly bound NAD per mole of enzyme. We report here that incubation of the rat liver enzyme with ATP, Mg2+, and KCl leads to conversion of the active enzyme to an inactive form with release of all enzyme-bound NAD which can be recovered quantitatively by gel filtration. At various concentrations of ATP, the release of NAD corresponds closely with the degree of inactivation, suggesting that the four subunits are equivalent. Hydrolysis of ATP is not required for the inactivation process since nonhydrolyzable ATP analogues can replace ATP in the inactivation process. The ATP-dependent inactivation is fully reversible upon incubation of the inactivated enzyme with NAD. The ATP-dependent inactivation of the enzyme appears to be analogues to the cAMP-dependent inactivation of the enzyme from Dictyostelium discoideum described earlier by Hohman et al. (1985) [Hohman, R. J., Guitton, M. C., & Veron, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4578-4581; Hohman, R. J., Veron, M., & Guitton, M. C. (1985) Curr. Top. Cell. Regul. 26, 233-245] but differs from the irreversible inactivation studied earlier by Abeles et al. (1982) [Abeles, R. H., Fish, S., & Lapinskas, B. (1982) Biochemistry 21, 5557-5562]. These authors have ascribed the time-dependent inactivation that results from incubation of the enzyme with 2'-deoxyadenosine at the C-3' and concluded that AdoHcyase "probably consists of two nonequivalent pairs of subunits".(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the S-adenosylhomocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on RNA methylation and synthesis.

The effects of 3-deazaaristeromycin and 3-deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S-Adenosylhomocysteine accumulated in cells incubated with 3-deazaaristeromycin while S-3-deazaadenosylhomocysteine was the major product in cells incubated with 3-deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S-adenosylhomocysteine or S-3-deazaadenosylhomocysteine, with S-adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6-methyladenosine and N6-methyl-2'-O-methyladenosine were inhibited to the greatest extent, while the synthesis of 7-methylguanosine and 2'-O-methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 microM 3-deazaaristeromycin or with 10 microM 3-deazaadenosine and 50 microM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 microM 3-deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.

Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation.

In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation. In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy[5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-light) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into light-light duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3H]adenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the 3H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.

Effects of S-adenosyl-methionine on plasma norepinephrine, blood pressure, and heart rate in healthy volunteers.

S-adenosyl-methionine (SAMe) is currently undergoing trials as a possible antidepressant. Because SAMe's mechanism of action is obscure and norepinephrine (NE) is often implicated in affective disorders, we studied the effects of SAMe on this neurotransmitter in volunteers. Plasma NE and 3-methoxy-4-hydroxyphenylglycol (MHPG) in the supine and standing position were studied before and after acute placebo or a single 400 mg dose of SAMe and following seven daily administrations; concomitant measures were heart rate (HR) and blood pressure. Subjects were unable to distinguish acute drug from placebo, and although chronic SAMe administration was open, they reported no behavioral effects. Standing HR and plasma NE were reduced following chronic SAMe. Qualitatively similar changes are obtained following chronic treatment with monoamine oxidase inhibitors (MAOIs). However, unlike MAOIs, chronic SAMe treatment was not associated with changes in plasma MHPG. Exaggerated standing NE is found in depressed patients; SAMe may reduce this abnormal response, providing a clue for its mechanism of action in depression.

Cholera toxin inhibits chemotaxis by a cAMP-independent mechanism.

Cholera toxin inhibits chemotaxis of the RAW264 mouse macrophage cell line. The degree of inhibition by cholera toxin increases upon incubation with the cells, suggesting that the entry of the toxin is required for inhibition of chemotaxis. In the absence of guanine nucleotides, cholera toxin catalyzes the [32P]ADP-ribosylation of RAW264 cell membrane proteins of Mr 41,000, Mr 45,000, and a doublet of Mr 48,000-50,000. GTP increases the labeling of the Mr 45,000 protein and the Mr 48,000-50,000 doublet, and it decreases the labeling of the Mr 41,000 protein. Experiments with cholera toxin treatment of intact cells indicate that the Mr 45,000 protein is the major membrane protein ADP-ribosylated by the toxin in vivo. Cholera toxin increases cAMP levels in RAW264 cells, but increased cAMP levels do not correlate with inhibition of chemotaxis, because isoproterenol and forskolin, which also increase cAMP levels, have no effect on chemotaxis.