Possible regulation of trehalose metabolism by methylation in Saccharomyces cerevisiae

The current study was undertaken to correlate post‐translational protein modification by methylation with the functionality of enzymes involved in trehalose metabolism in Saccharomyces cerevisiae. Trehalose is an economically important disaccharide providing protection against various kinds of stresses. It also acts as a source of cellular energy by storing glucose. Methyl group donor S‐adenosyl L ‐methionine (AdoMet) and methylation inhibitor‐oxidized adenosine (AdOx) were used for the methylation study. AdoMet delayed initial growth of the cells but the overall growth rate remained same suggesting its interference in G1 phase of the cell cycle. Metabolic‐altered enzyme activities of acid trehalase (AT), neutral trehalase (NT), and trehalose‐6‐phosphate synthase (TPS) were observed when treated with AdOx and AdoMet separately. A positive effect of methylation was observed in TPS, hence, it was purified in three different conditions, using AdoMet, AdOx, and control. Differences in mobility of methylated, methylation‐inhibited, and control TPS during acidic native gel electrophoresis confirmed the occurrence of induced methylation. Hydrolysis under alkaline pH conditions revealed that methylation of TPS was different than O‐methylation. MALDI‐TOF analysis of trypsin‐digested samples of purified methylated, methylation‐inhibited, and control TPS revealed that an increase of 18 Da mass in methylated peptides suggesting the introduction of methyl ester in TPS. Results of amino acid analysis corroborated the presence of methyl cysteine. The data presented here strongly suggests that trehalose production was enhanced due to methylation of TPS arising from carboxymethylation of cysteine residues. J. Cell. Physiol. 226: 158–164, 2010. © 2010 Wiley‐Liss, Inc.     

The Saccharomyces cerevisiae poly(A)-binding protein is subject to multiple post-translational modifications,including the methylation of glutamic acid

Poly(A)-binding protein in mouse and man was recently found to be highly post-translationally modified. Here we analysed an ortholog of this protein, Pab1 from Saccharomyces cerevisiae, to assess the conservation and thus likely importance of these modifications. Pab1 showed the presence of six sites of methylated glutamate, five sites of lysine acetylation, and one phosphorylation of serine. Many modifications on Pab1 showed either complete conservation with those on human or mouse PABPC1, were present on nearby residues and/or were present in the same domain(s). The conservation of methylated glutamate, an unusual modification, was of particular note and suggests a conserved function. Comparison of methylated glutamate sites in human, mouse and yeast poly(A)-binding protein, along with methylation sites catalysed by CheR l-glutamyl protein methyltransferase from Salmonella typhimurium, revealed that the methylation of glutamate preferentially occurs in EE and DE motifs or other small regions of acidic amino acids. The conservation of methylated glutamate in the same protein between mouse, man and yeast suggests the presence of a eukaryotic l-glutamyl protein methyltransferase and that the modification is of functional significance.     

PLMLA: prediction of lysine methylation and lysine acetylation by combining multiple features

Post-translational lysine methylation and acetylation are two major modifications of lysine residues. They play critical roles in various biological processes, especially in gene regulation. Identification of protein methylation and acetylation sites would be a foundation for understanding their modification dynamics and molecular mechanism. This work presents a method called PLMLA that incorporates protein sequence information, secondary structure and amino acid properties to predict methylation and acetylation of lysine residues in whole protein sequences. We apply an encoding scheme based on grouped weight and position weight amino acid composition to extract sequence information and physicochemical properties around lysine sites. The prediction accuracy for methyllysine and acetyllysine are 83.02% and 83.08%, respectively. Feature analysis reveals that methyllysine is likely to occur at the coil region and acetyllysine prefers to occur at the helix region of protein. The upstream residues away from the central site may be close to methylated lysine in three-dimensional structure and have a significant influence on methyllysine, while the positively charged residues may have a significant influence on acetyllysine. The online service is available at http://bioinfo.ncu.edu.cn/inquiries_PLMLA.aspx.     

Cytotoxicity of the Vibrio vulnificus MARTX toxin Effector DUF5 is linked to the C2A Subdomain

The multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxins are bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species' MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full‐length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate‐3721 and arginine‐3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5_Ah domain was also cytotoxic, while the weakly conserved C1–C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain. Proteins 2014; 82:2643–2656. © 2014 Wiley Periodicals, Inc.     

Inhibition of Orientia tsutsugamushi infection by a truncated recombinant 56‐kDa outer membrane protein

Aims: The objective of this study was to evaluate recombinant 56‐kDa outer membrane protein as a potential inhibitor to infection from Orientia tsutsugamushi. Methods and Results: The 56‐kDa protein was cloned and expressed in an Escherichia coli system, and the degree of target cell attachment to immobilized 56‐kDa protein was measured in a cell adhesion assay. The results showed that the 56‐kDa protein has an ability to attach HeLa cells. Furthermore, treatment of target cells with a truncated 56‐kDa 1–418 (amino acid residues) protein inhibited target cell infection by O. tsutsugamushi, demonstrated with an indirect immunofluorescence antibody assay. Conclusions: The truncated 56‐kDa protein (a.a. 1–418) plays an important role in O. tsutsugamushi infection, and the 56‐kDa protein could be useful and effective in the inhibition of O. tsutsugamushi attachment and infection. Significance and Impact of the Study: The attachment of the 56‐kDa protein to target cells was directly determined by in vitro adherence test, and the invasion of target cells by O. tsutsugamushi was inhibited by treating the target cells with a truncated 56‐kDa protein.     

Targeted Identification of Protein Interactions in Eukaryotic mRNA Translation

To identify protein–protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP‐MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP‐tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross‐validation approach is employed to identify the most statistically significant protein–protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae’s mRNA translation proteins and complexes are identified.     

Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients

Background

Antibodies against spliceosome Sm proteins (anti-Sm autoantibodies) are specific to the autoimmune disease systemic lupus erythematosus (SLE). Anti-Sm autosera have been reported to specifically recognize Sm D1 and D3 with symmetric di-methylarginines (sDMA). We investigated if anti-Sm sera from local SLE patients can differentially recognize Sm proteins or any other proteins due to their methylation states.

Results

We prepared HeLa cell proteins at normal or hypomethylation states (treated with an indirect methyltransferase inhibitor adenosine dialdehyde, AdOx). A few signals detected by the anti-Sm positive sera from typical SLE patients decreased consistently in the immunoblots of hypomethylated cell extracts. The differentially detected signals by one serum (Sm1) were pinpointed by two-dimensional electrophoresis and identified by mass spectrometry. Three identified proteins: splicing factor, proline- and glutamine-rich (SFPQ), heterogeneous nuclear ribonucleoprotein D-like (hnRNP DL) and cellular nucleic acid binding protein (CNBP) are known to contain methylarginines in their glycine and arginine rich (GAR) sequences. We showed that recombinant hnRNP DL and CNBP expressed in Escherichia coli can be detected by all anti-Sm positive sera we tested. As CNBP appeared to be differentially detected by the SLE sera in the pilot study, differential recognition of arginine methylated CNBP protein by the anti-Sm positive sera were further examined. Hypomethylated FLAG-CNBP protein immunopurified from AdOx-treated HeLa cells was less recognized by Sm1 compared to the CNBP protein expressed in untreated cells. Two of 20 other anti-Sm positive sera specifically differentiated the FLAG-CNBP protein expressed in HeLa cells due to the methylation. We also observed deferential recognition of methylated recombinant CNBP proteins expressed from E. coli by some of the autosera.

Conclusion

Our study showed that hnRNP DL and CNBP are novel antigens for SLE patients and the recognition of CNBP might be differentiated dependent on the level of arginine methylation.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.     

Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

Chemical synthesis and in vivo hyperexpression of a modular gene coding for Escherichia coli translational initiation factor IF1

Summary An artificial gene encoding the Escherichia coli translational initiation factor IF1 was synthesized based on the primary structure (71 amino acid residues) of the protein. Codons for individual amino acids were selected on the basis of the preferred codon usage found in the structural genes for the initiation factor IF2 of E. coli and Bacillus stearothermophilus, both of which can be expressed at high levels in E. coli cells. We gave the IF1 gene a modular structure by introducing specific restriction enzyme sites into the sequence, resulting in units of three to ten codons. This was conceived to facilitate site-directed mutagenesis of the gene and thus to obtain IF1 with specific amino acid alterations at desired positions. The IF1 gene was assembled by shot-gun ligation of 9 synthetic oligodeoxyri-bonucleotides ranging in size from 31 to 65 nucleotides and cloned into an expression vector to place the gene under the control of an inducible promoter. Upon induction, E. coli cells harbouring the artificial gene were found to produce large amounts (60 mg/100 g cells) of a protein indistinguishable from natural IF1 in both chemecal and biological properties.     

Lewis acid catalysed methylation of N‐(9H‐fluoren‐9‐yl)methanesulfonyl (Fms) protected lipophilic α‐amino acid methyl esters

This work reports an efficient Lewis acid catalysed N‐methylation procedure of lipophilic α‐amino acid methyl esters in solution phase. The developed methodology involves the use of the reagent system AlCl₃/diazomethane as methylating agent and α‐amino acid methyl esters protected on the amino function with the (9H‐fluoren‐9‐yl)methanesulfonyl (Fms) group. The removal of Fms protecting group is achieved under the same conditions to those used for Fmoc removal. Thus the Fms group can be interchangeable with the Fmoc group in the synthesis of N‐methylated peptides using standard Fmoc‐based strategies. Finally, the absence of racemization during the methylation reaction and the removal of Fms group were demonstrated by synthesising a pair of diastereomeric dipeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Methylation of Drosophila histones at proline, lysine, and arginine residues during heat shock

Heat shock or arsenite treatment alter the pattern of histone methylation in Drosophila cells. Both types of stress induce a rapid increase in the methylation level of histone H2B. The methylated amino acid residue of H2B has been identified by thin layer chromatography and electrophoresis as methylproline and is located at the N-terminal end of H2B. Heat shock also induces a decrease in the level of methylation of histone H3. Under normal growth temperature conditions, histone H3 is shown to be methylated on lysine residues. However under heat shock conditions, there is a decrease in the extent of methylation of lysine residues and the appearance of new methylation on arginine residues in H3. These new heat shock-induced methylated residues have been identified as the symmetrical and asymmetrical forms of dimethylarginine. The methylated amino acid residue of histone H4 is lysine with mono-, di-, and trimethyl forms found in both control and heat or chemically stressed cells. These stress-induced changes in the methylation level of the N-terminal proline residue of histone H2B and shift in the methylation sites of histone H3 may be involved in the restructuration of chromatin accompanying the inactivation of normal genes in response to stress. Moreover, we suggest that the hypermethylation of H2B may also be involved in its protection from increased ubiquitin-mediated proteolytic activity under these conditions of cellular stress.     

Genetics of ribosomal protein methylation in Escherichia coli. I. A mutant deficient in methylation of protein L11.

Summary Several thousand mutagenized clones of Escherichia coli were screened for methyl group incorporation into protein in crude extracts, in order to isolate mutants lacking the full complement of methyl groups in ribosomal proteins. One mutant isolated by this method and designated prm-1 incorporated 6–7 methyl groups per ribosome upon incubation of its ribosomes with a partially purified enzyme preparation from E. coli wild-type. The methyl groups were located exclusively in the 50S particle and for the most part (85%) in protein L11. Three methylated amino acids were detected: -N-trimethyllysine, -N-monomethyllysine, and an uncharacterized amino acid. These accounted respectively for 4.6, 1.3 and 0.9 methyl groups per ribosome. These results indicate that protein L11 in wild-type contains a stoichiometric amount of these methylated amino acids which are absent in mutant prm-1. Since this mutant is fully viable, its methylation deficiency does not result in a major defect in ribosome assembly or functioning.     

Enhancing survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> by increasing the periplasmic expression of Cu,Zn superoxide dismutase from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The gene for the Cu,Zn superoxide dismutase (Cu,ZnSOD) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli LMG194. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the E. coli periplasmic expression vector pBAD/gIIIA, yielding pBAD-1. E. coli was transformed using the constructed plasmid pBAD-1 and induced by adding 0.02% l-arabinose to express Cu,ZnSOD protein. The results indicated that Cu,ZnSOD enzyme activity in the periplasmic space was about fivefold to sixfold higher in the recombinant E. coli strains bearing the sod gene than in the control strains. The yields of Cu,ZnSOD were about threefold higher at 48 h than at 24 h in the recombinant E. coli cells. Significantly higher survival of strains was obtained in cells bearing the sod gene than in the control cells when the cells were treated by heat shock and superoxide-generating agents, such as paraquat and menadione.     

Identification of the mutations in the T-protein gene causing typical and atypical nonketotic hyperglycinemia

We have investigated the molecular lesions of T-protein deficiency causing typical or atypical nonketotic hyperglycinemia (NKH) in two unrelated pedigrees. A patient with typical NKH was identified as being homozygous for a missense mutation in the T-protein gene, a G-to-A transition leading to a Gly-to-Asp substitution at amino acid 269 (G269D). Sibling patients of a second family with atypical NKH had two different missense mutations in the T-protein gene (compound heterozygote), a G-to-A transition leading to a Gly-to-Arg substitution at amino acid 47 (G47R) in one allele, and a G-to-A transition leading to an Arg-to-His substitution at amino acid 320 (R320H) in the other allele. Gly 269 is conserved in T-proteins of various species, even in E. coli, whereas Gly 47 and Arg 320 are replaced by Ala and Leu, respectively, in E. coli. The mutation occurring in more conservative amino acid residues thus results in more deleterious damage to the T-protein, and gives the severe clinical phenotype, viz., typical NKH.     

The SET8 H4K20 protein lysine methyltransferase has a long recognition sequence covering seven amino acid residues

The SET8 histone lysine methyltransferase, which monomethylates the histone 4 lysine 20 residue plays important roles in cell cycle control and genomic stability. By employing peptide arrays we have shown that it has a long recognition sequence motif covering seven amino acid residues, viz. R¹⁷–H¹⁸–(R¹⁹KY)–K²⁰–(V²¹ILFY)–(L²²FY)–R²³. Celluspots peptide array methylation studies confirmed specific monomethylation of H4K20 and revealed that the symmetric and asymmetric methylation on R¹⁷ of the H4 tail inhibits methylation on H4K20. Similarly, dimethylation of the R located at the −3 position also reduced methylation of p53 K382 which had been shown previously to be methylated by SET8. Based on the derived specificity profile, we identified 4 potential non-histone substrate proteins. After relaxing the specificity profile, we identified several more candidate substrates and showed efficient methylation of 20 novel non-histone peptides by SET8. However, apart from H4 and p53 none of the identified novel peptide targets was methylated at the protein level. Since H4 and p53 both contain the target lysine in an unstructured part of the protein, we conclude that the long recognition sequence of SET8 makes it difficult to methylate a lysine in a folded region of a protein, because amino acid side chains essential for recognition will be buried.