Posttranslational Modification of Calmodulin in Rat Brain and Pituitary

The posttranslational modification of calmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation, calmodulin converting enzyme, and calmodulin (lysine) N-methyltransferase activities were determined. Incubation of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl-3H]-S-adenosyl-L-methionine and identification of labeled proteins by gel electrophoresis and fluorography indicated that calmodulin is a substrate for protein carboxylmethyltransferase in all tissues tested. In hippocampus, caudate nucleus, cerebral cortex, and anterior pituitary, but not in cerebellum, superior colliculus, brainstem, or diencephalon, a second methylated protein was found when calmodulin was added to incubation mixtures. This protein was shown to be identical to the previously described product of calmodulin converting enzyme. Converted calmodulin was isolated by fast protein liquid chromatography and shown to be des(Lys)calmodulin, lacking the carboxy terminal lysine residue of calmodulin. The anterior pituitary had by far the highest levels of calmodulin converting enzyme; this enzyme, in turn, was identified as a cobalt-stimulated carboxylpeptidase B. In contrast to the regional differences in these parameters, the levels of calmodulin (lysine) N-methyltransferase did not differ greatly among brain regions, although regional differences in the activity of this enzyme were statistically significant.     

Purification and Properties of Calmodulin-Lysine N-Methyltransferase from Rat Brain Cytosol

A S-adenosylmethionine:protein-lysine N-methyltransferase (EC 2.1.1.43) has been purified from rat brain cytosol 7,080-fold with a yield of 8%, using octopus calmodulin as a substrate. It contains a lysine residue that is not fully methylated. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and phosphocellulose and octopus calmodulin-Sepharose affinity chromatographies. Among protein substrates, it was highly specific toward octupus calmodulin. The Km values for octopus calmodulin and S-adenosyl-L-methionine were found to be 2.2 X 10(-8) M and 0.8 X 10(-6) M, respectively. The molecular weight was estimated to be 57,000 by gel filtration and the pH optimum was between 7.5 and 8.5. The enzyme was stimulated in the presence of 10(-7) M Mn2+ and 10(-4) M Ca2+. HPLC of the acid hydrolysate of methyl-3H-labeled calmodulin showed the formation of epsilon-N-mono, epsilon-N-di, and epsilon-N-trimethyllysine. Reverse-phase HPLC of tryptic peptides of the methyl-3H-labeled calmodulin demonstrated that the labeled N-methyllysine lies in the 107-126 peptide. These findings suggest that this enzyme methylated a specific lysine residue of octopus calmodulin.     

Novel eye-specific calmodulin methylation characterized by protein mapping in Drosophila melanogaster

Post-translational methylation of the epsilon-amino group of lysine residues regulates a number of protein functions. Calmodulin, a key modulator of intracellular calcium signaling, is methylated on lysine 115 in many species. Although the amino acid sequence of calmodulin is highly conserved in eukaryotes, it has been shown that lysine 115 is not methylated in Drosophila calmodulin and no other methylation site has been reported. In this study, we characterized in vivo modification states of Drosophila calmodulin using proteomic methodology involving the protein mapping of microdissected Drosophila tissues on 2-D gels. We found that Drosophila calmodulin was highly expressed in methylated forms in the compound eye, whereas its methylation was hardly detected in other tissues. We identified that lysine 94 located in an EF-hand III is the methylation site in Drosophila calmodulin. The predominance of methylated calmodulin in the compound eye may imply the involvement of calmodulin in photoreceptor-specific functions through methylation.     

Identification of glutathione S-transferase as a substrate and glutathione as an inhibitor of in vitro calmodulin-stimulated protein methylation in rat liver cytosol

This report describes the isolation of the major calmodulin-stimulated methyl acceptor protein of adult rat liver cytosol. This Mr 29,000 methyl acceptor protein (MeAP29) has been purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and Sephadex G-75. Affinity chromatography on glutathione-Sepharose and assays of enzyme activity indicate that MeAP29 is a member of the glutathione S-transferase family. We further show that glutathione can act as an inhibitor of calmodulin-stimulated in vitro methylation of MeAP29 and that MeAP29 methylation is enhanced in non-dialyzed liver cytosol from rats with lowered glutathione levels.     

Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination

Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.     

Analysis of the state of posttranslational calmodulin methylation in developing pea plants

A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [³H]methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase (DM Roberts et al. [1986] J Biol Chem 261: 1491-1494) raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.     

Specificity of the histone lysine methyltransferases from rat brain chromatin

The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to specific epsilon-N-lysine residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or nonspecifically associated histones, while histones H3 and H4 within newly synthesized chromatin are methylated. These enzymes can be solubilized by limited digestion (10-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. Histone H3 lysine methyltransferase remained associated with a short DNA fragment throughout purification. Dissociation of the enzyme from the DNA fragment with DNAase digestion resulted in complete loss of enzyme activity; however, when this enzyme remained associated with DNA it was quite stable. Activity of the dissociated enzyme could not be restored upon the addition of sheared calf thymus or Escherichia coli DNA. Histone H3 lysine methyltransferase was found to methylate lysine residues in chromosomal bound or soluble histone H3, while H3 associated with mature nucleosomes was not methylated. The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest was extremely labile, losing all activity upon further purification. We isolated a methyltransferase by DEAE-cellulose chromatography, which would transfer methyl groups to arginine residues in soluble histone H4. However, this enzyme would not methylate nucleosomal or chromosomal bound histone H4, nor were methylated arginine nucleosomal or chromosomal bound histone H4, nor were methylated arginine residues detectable upon incubating intact nuclei or chromatin with S-adenosylmethionine.     

PROTEIN METHYLATION BY CEREBRAL TISSUE

Abstract— Transfer of the methyl group of S -adenosyl [Me-¹⁴C]methionine into cerebral proteins, an encephalitogenic protein and histones was studied using extracts of bovine and rat brains. The brain extract contains multiple substrate proteins and their lysine and arginine residues were methylated to form N^e-mono-, -di- and -trimethyl-lysine and N ^G-mono-, N ^G, N ^G- and N^G,N^G-dimethylarginine residues respectively, at different rates. The enzyme which catalyses the methylation of arginine residues was differentiated by ammonium sulphate fractionation from that methylating lysine residues. Methylation of arginine and lysine residues of proteins was stepwise in general, from mono- to dimethyl-arginine and from mono- to di- and trimethyl-lysines. Two different enzymes which methylate histone and the encephalitogenic basic protein respectively were obtained from the cytoplasmic fraction of rat brain and their enzymic properties were examined.     

A kinetic study of human protein arginine N-methyltransferase 6 reveals a distributive mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.     

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins

SET domain lysine methyltransferases are known to catalyze site and state-specific methylation of lysine residues in histones that is fundamental in epigenetic regulation of gene activation and silencing in eukaryotic organisms. Here we report the three-dimensional solution structure of the SET domain histone lysine methyltransferase (vSET) from Paramecium bursaria chlorella virus 1 bound to cofactor S-adenosyl-L-homocysteine and a histone H3 peptide containing mono-methylated lysine 27. The dimeric structure, mimicking an enzyme/cofactor/substrate complex, yields the structural basis of the substrate specificity and methylation multiplicity of the enzyme. Our results from mutagenesis and enzyme kinetics analyses argue that a general base mechanism is less likely for lysine methylation by SET domains; and that the only invariant active site residue tyrosine 105 in vSET facilitates methyl transfer from cofactor to the substrate lysine by aligning intermolecular interactions in the lysine access channel of the enzyme.     

Purification and kinetic properties of ox brain histamine N-methyltransferase.

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.     

In vitro methylation of yeast tRNAAsp by rat brain cortical tRNA-(adenine-1) methyltransferase.

Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s). The enzyme(s) can methylate E. coli tRNA and to a lower degree yeast tRNA. Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme. The digestions of in vitro methylated [Me-3H]-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop. Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme. This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp.     

A cytochrome c methyltransferase from Crithidia oncopelti.

The mitochondrial cytochrome c-557 of Crithidia oncopelti contains two lysine residues and an N-terminal proline residue that are methylated in vivo by the methyl group of methionine. The purified cytochrome can act as a methyl acceptor for a methyltransferase activity in the cell extract that uses S-adenosylmethionine as methyl donor. Crithidia cytochrome c-557 is by far the best substrate for this methyltransferase of those tested, in spite of the fact that methylation sites are already almost fully occupied. The radioactive uptake of [14C]methyl groups from S-adenosylmethionine occurred only at a lysine residue (-8) and the N-terminal proline residue. This methyltransferase appears to differ from that of Neurospora and yeast [Durban, Nochumson, Kim, Paik & Chan (1978) J. Biol. Chem. 253, 1427-1435; DiMaria, Polastro, DeLange, Kim & Paik (1979) J. Biol. Chem. 254, 4645-4652] in that lysine-72 of horse cytochrome c is a poor acceptor. Also, the Crithidia methyltransferase appears to be stable to carry lysine methylation much further to completion than do the enzymes from yeast and Neurospora, which produce very low degrees of methylation in native cytochromes c.     

Origin and formation of 1,7-dimethylguanosine in tRNA chemical and enzymatic methylation

tRNA chemical methylation: 1. 1,7-Dimethylguanosine was found in in vivo methylated tRNA from liver and kidney of rat after exposure to a low dose of dimethylnitrosamine (4 mg/kg body weight). 2. At 4 h after dimethylnitrosamine administration, the 1,7-dimethylguanosine:7-methylguanine ratio (product ratio) for liver and kidney tRNA was 0.017 and 0.091, respectively. At 24 h after dimethylnitrosamine administration, the product ratio was lower in both hepatic and renal tRNA. 3. When dimethylnitrosamine was given in four separate daily injections, the product ratio in hepatic tRNA 4 h after the last dose was the same as for the same total dose given by a single injection, but in renal tRNA it was lower. No dialkyl compound was found in liver and kidney tRNA 24 h after the last multiple injection. tRNA enzymatic methylation: 1. Base analyses of Escherichia coli B tRNA methylated in vitro, by using S-adenosylmethionine as physiological methyl donor and enzyme preparations from liver and kidney of normal rat, indicated that 1,7-dimethylguanosine was also a product of enzymatic methylation. 2. The amount of 1,7-dimethylguanosine formed by kidney enzyme preparation was 3-times that produced by the liver extract. 3. A second type of enzymatic methylation assay where chemically methylated tRNA was used as substrate indicated that the 7-methylguanosine residues in the nucleic acid are not the substrate of the methylase activity forming the 1,7-dimethylguanosine moieties. Analogous data were obtained for the origin of 1,7-dimethylguanosine residues in tRNA chemical methylation by dimethyl sulphate.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

Tissue specificities of protease A-like esteroprotease in male mice

An esteroprotease hydrolyzing p-tosyl-L-arginine methyl ester (TAME) has been purified to homogeneity from male mice submandibular glands by the ammonium sulphate precipitation, Sephadex gel chromatography and DEAE-cellulose chromatography. The enzyme was shown as a single chain acidic protein (pI = 5.7) with the molecular weight of 27.5 K and evidence was obtained to reveal that it was similar to protease A. Using this enzyme as antigen we prepared anti-TAMEase antibody. The immunoblotting studies on tissue specificity using 20 different tissues from male mice revealed that cross-reactivities with anti-TAMEase antibody were observed in the crude extract from the sublingual gland, parotid gland and pancreas. The species specificity studies with the submandibular glands of 7 different species indicated that only the crude extract from rat submandibular glands reacted against anti-TAMEase antibody but it exerted a low TAMEase activity.     

Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9-AdoMet

The methylation of lysine residues of histones plays a pivotal role in the regulation of chromatin structure and gene expression. Here, we report two crystal structures of SET7/9, a histone methyltransferase (HMTase) that transfers methyl groups to Lys4 of histone H3, in complex with S-adenosyl-L-methionine (AdoMet) determined at 1.7 and 2.3 A resolution. The structures reveal an active site consisting of: (i) a binding pocket between the SET domain and a c-SET helix where an AdoMet molecule in an unusual conformation binds; (ii) a narrow substrate-specific channel that only unmethylated lysine residues can access; and (iii) a catalytic tyrosine residue. The methyl group of AdoMet is directed to the narrow channel where a substrate lysine enters from the opposite side. We demonstrate that SET7/9 can transfer two but not three methyl groups to unmodified Lys4 of H3 without substrate dissociation. The unusual features of the SET domain-containing HMTase discriminate between the un- and methylated lysine substrate, and the methylation sites for the histone H3 tail.     

Specific recognition of calmodulin from Dictyostelium discoideum by the ATP, ubiquitin-dependent degradative pathway

Calmodulin purified from Dictyostelium discoideum is selectively degraded by rabbit reticulocyte extracts in the presence of ubiquitin and ATP. This protein forms a 1:1 covalent conjugate with ubiquitin. Analyses of the cyanogen bromide fragments of the protein conjugate indicate that lysine 115 on calmodulin is the ubiquitin conjugation site. Bovine brain calmodulin which contains a trimethyllysine residue at this position is not a substrate for conjugation with ubiquitin, and its degradation rate is not affected by ATP and ubiquitin. These results suggest that the trimethyllysine residue in mammalian calmodulin may function in protecting the protein from degradation by the ATP, ubiquitin-dependent pathway. Since there are eight lysine residues in Dictyostelium calmodulin, the specific conjugation of ubiquitin to lysine 115 may provide a good model system to delineate the structural features required for the conjugation and to follow the degradative steps in the pathway.