3-Methylhistidine in actin and other muscle proteins

1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7.6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation.     

Characterization and quantitation of myosin heavy-chain mRNA from embryonic cardiac tissue

The messenger RNA (mRNA) coding for myosin heavy chain from the 16-day-old chick embryonic cardiac tissue was purified by a rapid isolation procedure and characterized. The mRNA can be translated with fidelity under optimally chosen conditions. The protein synthesized in response to the RNA was a polypeptide of 200,000 molecular weight, identical to the authentic myosin heavy chain from the homologous chick heart tissue. The purity of the mRNA was assessed by electrophoresis in denaturing gels, by immunoprecipitation of the translation product, and by analysis of the kinetics of hybridization with the complementary DNA (cDNA). The cDNA reassociated with myosin heavy-chain mRNA with kinetics characteristic of a pure mRNA. The sequence complexity data indicated that in the 16-day-old chick embryonic heart cells there is a single mRNA sequence coding for myosin heavy chain in contrast to two different mRNA sequences reportedly present in the skeletal muscle cells (M. Patrinou-Georgoulas and H. A. John, 1977, Cell12, 491).     

Myofibrillar protein turnover. Synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states.

The turnover of 3-methylhistidine (N tau-methylhistidine) and in some cases actin, myosin heavy chain and aldolase in skeletal muscle was measured in a number of experiments in growing and adult rats in the fed and overnight-starved states. In growing fed rats in three separate experiments, measurements of the methylation rate of protein-bound 3-methylhistidine by either [14C]- or [3H]-methyl-labelled S-adenosylmethionine show that 3-methylhistidine synthesis is slower than the overall rate of protein synthesis indicated by [14C]tyrosine incorporation. Values ranged from 36 to 51%. However, in one experiment with rapidly growing young fed rats, acute measurements over 1 h showed that 3-methylhistidine synthesis could be increased to the same rate as the overall rate. After overnight starvation in these rats, the steady-state synthesis rate of 3-methylhistidine was 38.8% of the overall rate. This was a similar value to that in adult non-growing rats, in which measurements of the relative labelling of 3-methylhistidine and histidine after a single injection of [14C]histidine indicated that 3-methylhistidine synthesis was 37% of the overall rate in the fed or overnight-starved state. According to measurements of actin, myosin heavy-chain and aldolase synthesis in the over-night-starved state with young rats, with a variety of precursors, slow turnover of 3-methylhistidine results from the specific slow turnover of actin, since turnover rates of myosin heavy chain, mixed protein and aldolase were 2.5, 3 and 3.4 times faster respectively. However, in the fed state synthesis rates of actin were increased disproportionately to give similar rates for all proteins. These results show that (a) 3-methylhistidine turnover in muscle is less than half the overall rate in both young and adult rats, (b) slow 3-methylhistidine turnover reflects the specifically slow turnover of actin compared with myosin heavy chain and other muscle proteins, and (c) during growth the synthesis rate of actin is particularly sensitive to the nutritional state and can be increased to a similar rate to that of other proteins.     

A model for the active site of skeletal muscle myosin.

A model for a main element of the active site of skeletal muscle myosin is presented that relates directly to the 92 amino acid fragment (p₁₀) of myosin recently described by Elzinga &; Collins (1977). In this model, the substrate, an eight-membered cyclic complex of MgATP, fits tightly into a 16 amino acid segment of p₁₀ and interacts with seven of its amino acids. A main feature of the model is the important role played by the one molecule of N^τ-methylhistidine² that is present in each myosin heavy chain. At the site, it is postulated that this rare amino acid functions as a donor ligand to Mg²⁺. Once N^τ-methylhistidine is put in place next to the metal, the other amino acids that appear to form a pocket come easily into position around the MgATP. These amino acids with their postulated functions are: tyrosine 72, which through a Mg-bound water, or perhaps directly, is attached to the Mg; histidine 76, which donates a proton to the P_γ of ATP; lysine 78, which binds electrostatically to P_β of ATP; phenylalanines 80 and 81, which flank the purine ring of ATP; and aspartate 66, which forms a hydrogen bond to the 6-amino group of adenine. The Mg-coordination role ascribed to N^τ-methylhistidine 69 in skeletal muscle myosin could be taken by histidine 69 in cardiac myosin and in other muscle myosins that do not contain the methylated amino acid.The choice of p₁₀ to contain a main element of the active site is based on: (a) the presence in p₁₀ of the essential sulfhydryl groups, SH1 and SH2, whose modification affects the ATPase activity of myosin; (b) the presence in ρ₁₀ of N^τ-methylhistidine, an unusual amino acid whose methylation in skeletal muscle we take as an indicator for a special function at the active site; (c) the position of p₁₀ in the primary structure near the junction between subfragment 1 and subfragment 2 (the hinge region) where, we postulate, enzymatic events at the active site are coupled to movements of the hinge that occur during contraction; (d) indications that the DTNB light chain, probably involved in regulation, is also near the hinge; (e) the effects of MgATP at the active site on the chemical reactivity of three SH groups (SH1, SH2 and SH3) located near the hinge; and (f) the effect of hinge cleavage on the oxygen exchange reaction catalyzed at the active site. The correlation of all these observations forms the basis for our placement of part of the active site on p₁₀ near the subfragment 1-subfragment 2 hinge.     

A rapid method for the analysis of N tau-methylhistidine in human urine

A novel, automated method is described for the determination of N^τ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for N^τ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of N^τ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment.     

Molecular cloning and expression of cardiac-specific myosin heavy chain gene sequences in chick embryo

A recombinant DNA plasmid, pMHC8, that contains gene sequences for embryonic chick cardiac myosin heavy chain was constructed, identified and characterized. The identity of the clone was established by hybridization with labeled probes that afford screening of MHC²² with high specificity, by inhibition of MHC synthesis in the in vitro hybrid-arrested translation assay, and by tissue-specific hybridization of labeled pMHC8 DNA to MHC messenger RNA.The pMHC8 DNA probe is highly specific for chick heart muscle tissue, since it hybridized poorly to chick skeletal muscle RNA and did not detectably hybridize to adult rat heart RNA. Upon screening the embryonic chick heart cells in culture, no detectable level of MHC mRNA was observed in dividing myoblasts, but the mRNA appeared in differentiated cardiac myocytes paralleling morphogenetic changes in the embryonic cells.     

Immunochemical analysis of C-protein isoform transitions during the development of chicken skeletal muscle

Isoforms of C-protein in adult chickens which differ in fast (pectoralis major, PM) and slow (anterior latissimus dorsi, ALD) skeletal muscles can be distinguished immunochemically with monoclonal antibodies (McAbs) specific for the respective fast (MF-1) and slow (ALD-66) protein variants (Reinach et al., 1982 and 1983). The expression of these C-proteins during chick muscle development in vivo has been analyzed by immunoblot and immunofluorescence procedures. Neither MF-1 nor ALD-66 reacted with whole-cell lysates or myofibrils from PM of 12-day-old embryos. However, both McAbs bound to peptides of 145 kDa in PM from late embryonic and young posthatched chickens. All of the myofibers in these muscles reacted with both antibodies, but the binding of the anti-slow McAb (ALD-66) diminished progressively with age and was completely negative with PM by 2 weeks after hatching. In contrast, the ALD muscle from 17 days in ovo thru adulthood only reacted with ALD-66; no binding of MF-1 could be detected at these stages. Since both fast and slow myosin light chains (LC) coexist within embryonic pectoralis and ALD muscles (e.g., G. F. Gauthier, S. Lowey, P. A. Benfield, and A. W. Hobbs, 1982, J. Cell Biol.92, 471–484) yet segregate to specific fast and slow muscle fibers at different stages of development, the temporal transitions of C-protein and myosin LC were compared during myogenesis. “Slow-type” C-protein appeared after the disappearance of slow myosin light chains, whereas the accumulation of the “fast-type” light chains occurred before the expression of “fast-type” C-protein. The pattern of isoform transitions appears to be far more complex than previously suspected.     

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Differentiation of chick embryo myoblasts is transiently sensitive to functional denervation

Clonal analysis of myoblast differentiation has been used to assess effects of denervation on developing skeletal muscle: chick embryo legs denervated by spinal cord cautery yield reduced proportions of clonable myoblasts (P. H. Bonner, 1978, Develop. Biol., 66, 207–219). The present work examines the effects on clonable myoblasts of functional denervation by d-tubocurarine. Curare treatment during the third or fourth days of embryonic development had no effect on clonable myoblasts later in development, treatment during the fifth or sixth days resulted in reduced proportions of clonable myoblasts, and treatment during the eighth or ninth days again had no effect. Clonal analysis of treated and control embryo leg muscle cells was performed between Days 10 and 18. Embryos were also permanently denervated by spinal cord cautery late in the sixth day. These embryos showed no effect of denervation on clonable myoblast proportion. It is concluded that the differentiation of skeletal muscle myoblasts is affected by interference with normal nerve-muscle relationships only during a “window” of sensitivity and that this “window” extends approximately from Hamburger and Hamilton stage 27 to stage 30.     

Quantitative aspect of the myofibrillar protein turnover in transient state on dietary protein depletion and repletion revealed by urinary excretion of Nτ-Methylhistidine

The fractional rates of synthesis and breakdown of myosin and actin in skeletal muscle of younn adult male rats were measured during 2 weeks of ad libitum feeding of a protein-free diet, and 8 days of refeeding with an adequate protein diet. Daily urinary excretion of N^τ-Methylhistidine (3-methylhistidine) by the N^τ-methylhistidine pool of the body gave the fractional breakdown rate of the myosin-actin pool. The fractional synthesis rate of the myosin-actin pool was calculated from the fractional breakdown rate and the size of N^τ-methylhistidine pool in the body. The feeding of the protein-free diet resulted in a decreased in body weight and a decrease in daily urinary excretion of N^τ-methylhistidine. Refeeding caused an increase in body weight and a progressive increase in daily urinary excretion of N^τ-methylhistidine. At the start of the experiment, the fractional breakdown rate of the myosin-actin pool was 4% per day and with prolonged protein depletion, the rate decreased to 1.25% per day. The fractional synthesis rate also decreased more rapidly than the breakdown rate. On refeeding for one day with an adequate protein diet, the fractional synthesis rate increased from 0.75 to 5.75% per day. Accumulation of skeletal muscle protein by refeeding was accompanied by a difference between the faster rate of synthesis and slower rate of breakdown even though the fractional breakdown rate increased during the rehabilitation period.     

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC–tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.     

Different patterns of protein turnover in skeletal and gastrointestinal smooth muscle and the production of Nτ-methylhistidine during fasting in the rat

During four days of fasting in rats skeletal muscle protein synthesis fell pro-gressively, whereas skeletal muscle protein breakdown was unchanged until the third and fourth days when it rose dramatically. In contrast, the synthetic rate of smooth muscle protein was unchanged during three days of fasting despite a loss of protein content, indicating an abrupt rise in protein breakdown in this tissue on the first day of fasting which was sustained thereafter. Urinary excretion of N-methylhistidine was significantly increased throughout fasting. The concentration of free N-methylhistidine in plasma and in muscle tissue was elevated throughout the period of fasting. This elevation was not caused by reduced renal clearance, but appears to have been mainly the result of increased breakdown of N-methylhistidine-containing proteins in tissues other than skeletal muscle.     

Biological activity and the 3-methylhistidine content of actin and myosin

1. The 3-methylhistidine content of myosin varies according to muscle type. It is highest in myosin from white skeletal muscle and lower values are obtained from myosin of red skeletal and smooth muscle. 2. The 3-methylhistidine content of actin was similar in all of the types of muscle from which it was isolated. 3. The 3-methylhistidine of rabbit actin is localized in a single tryptic peptide that was readily modified during fractionation procedures. 4. Photo-oxidation studies indicated that the 3-methylhistidine residues are not essential for adeonsine triphosphatase and actin-combining activities of myosin. 5. During photooxidation G-actin lost completely the ability to polymerize to the F form before all the 3-methylhistidine was destroyed.     

Dimeric bisbenzimidazoles: Cytotoxicity and effects on DNA methylation in normal and cancer human cells

Cancer cells are characterized by hypermethylation of the promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors reactivate the genes, pointing to DNA methyltransferases as potential targets for anticancer therapy. Dimeric bisbenzimidazoles varying in the length of an oligomeric linker between two bisbenzimidazole residues (DB(n), where n is the number of methylene groups in the linker) were earlier shown to efficiently inhibit methylation of DNA duplexes by murine DNA methyltransferase Dnmt3a. Here, some of the compounds were tested for cytotoxicity, cell penetration, and effect on genomic DNA methylation in F-977 fetal lung fibroblasts and HeLa cervical cancer cells. Within the 0–60 μM concentration range, only DB(11) exerted a significant toxic effect on normal cells, whereas the effects of DB(n) on cancer cells were not significant. DB(1) and DB(3) slightly stimulated proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) penetrated into the nuclei of HeLa and F-977 cells and accumulated predominantly in or near the nucleolus, while DB(11) was incapable of nuclear penetration. HeLa cells incubated with 26 μM DB(1) or DB(3) displayed a decrease in methylation of the 18S rRNA gene, which was in the regions of predominant accumulation of DB(1) and DB(3). The same DB(3) concentration exerted a similar effect on F-977 cells. However, the overall genomic DNA methylation level remained unchanged in both of the cell lines. The results indicated that DB(n)-type compounds can be used to demethylate certain genes and are thereby promising as potential anticancer agents.     

Distinction between smooth muscle,fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

Summary FITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological dedifferentiation to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.This work was supported by the Life Insurance Medical Research Fund of Australia and New Zealand, the National Heart Foundation of Australia, the Deutsche Forschungsgemeinschaft and the Wellcome Trust (London). We thank Janet D. McConnell for excellent technical assistance     

Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms

A procedure was developed to determine the percentage of skeletal muscle actin and cardiac actin present in different striated muscle tissues. The method was applied to 2 mg of actin mixtures isolated from various origins. All samples show simultaneous expression of both striated muscle isoactins, with the cardiac actin being the major form (congruent to 80%) in 11-day-old chick embryonic leg muscle, decreasing to approximately 50% values in the late fetal stage of chicken, mouse, and in fused mouse muscle cell cultures and becoming the minor species (less than 5%) in adult skeletal muscle tissues. We also find a significant amount (up to 20%) of the skeletal muscle isoform in adult heart (ventricle) of porcine, bovine, and human origin and no differences in muscle actin ratios in human atrium and ventriculum cells. Similarly, no significant variation in the actin ratios was observed between a normal heart and a heart from a patient with hereditary obstructive myopathy. For those cells and tissues where comparison with levels of mRNA was possible we mostly find a good correlation between the relative ratios of expression of cardiac and skeletal actin proteins and mRNAs.     

Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs

Background

Age-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach.

Results

We observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age.

Conclusions

This study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-653) contains supplementary material, which is available to authorized users.     

Accelerated protein turnover in the skeletal muscle of dystrophic mice

The excretion of 3-methylhistidine increased in the urine of dystrophic mice C57BL/6J. The content of 3-methylhistidine residue decreased in the muscle proteins of dystrophic mice, but not in other organs. Methylated proteins in the skeletal muscle, actin and myosin, were partially purified from the dystrophic and control muscles. The amount of 3-methylhistidine residue in unit weight of the actin and myosin preparations was normal in dystrophic muscle. These three facts indicate that the turnover rates of actin and myosin are increased in the muscle of the dystrophic mice.     

Nerve-dependent changes in clonable myoblast populations

Between the 3rd and 12th days of development at least four distinct classes of clonable myoblast are present in chick embryo leg skeletal muscle (N. K. White, P. H. Bonner, D. R. Nelson, and S. D. Hauschka, 1975, Develop. Biol.44, 346–361). In the present study, the behavior of each class has been examined quantitatively by clonal growth and differentiation of cells derived from denervated and normal embryos. Embryos functionally denervated by cauterization of the posterior spinal cord or by injection of the neuromuscular blocking agent d-tubocurarine exhibit changes in the two broad categories of clonable myoblast—fresh medium-sufficient (FMS) and conditioned medium-requiring (CMR) muscle cells. Clonal analysis of cells derived from leg muscle tissue of 10- to 12-day-old embryos denervated early in development (Days 3 to 6) has shown that the proportion of FMS clonable myoblasts is reduced to about 60% of the level found in normally innervated leg muscle. The CMR class is composed of three subclasses: CMR-I, CMR-II, and CMR-III (White et al., 1975). Clonal analysis of cells from denervated muscle has shown that, while the total CMR clone proportion is unchanged, the three subclasses are rearranged. The proportions of CMR-I and CMR-II muscle clones are greatly increased and the CMR-III subclass is severely reduced or absent when compared to clones derived from normally innervated leg muscle tissue.