The Further Research Of Mouse N5-glutamine Methyltransferase Gene MHemk

Introduction.Methylation is a common and important epigenetic modification mechanism in cells,which modulate important cellular and metabolic processes by regulating gene expression or protein translation. S-Adenosyl-L-methionine(SAM) is a ubiquitous methyl donor in vivo. SAM-dependent methyltransferases belong to a large family of ubiquitous enzymes that transfer methyl groups from SAM to a wide variety of substrates,such as nucleic acids(DNA or RNA),proteins,and many small molecules.The DNA and protein methyltranferases are the most important and prevalent methylases and have been extensively studied.The post-transcriptional methylation of DNA and post-translational methylation of proteins mediated by methyltranferases provide the mechanisms by which epigenetic information is imparted to these molecules.Such information can alter the targeting and timing of gene expression and activity of certain proteins.DNA methyltransferases play important roles not only in the maintenance of DNA methylation and genome stability but also in the early mammalian embryo development. In bacterial,protist or plant DNA,three methylated bases,namely 5-methylcytosine(m5C),N6-methyladenine(m6A) and N4-methylcytosine(m4C) were found,whereas in mammalian DNA only 5-methylcytosine(m5C) was found.Protein methylation is a post-translational modification of proteins,which could serve to modulate intra- or inter-molecular interactions of the target proteins.There is more current understanding about arginine and lysine methyltransferases, which are involved in methylation of histones and play an important regulatory role in gene transcription.In addition,N5-glutamine methyltransferases were identified in bacteria and yeast,which are involved in methylation of peptide chain release factors(RFs) and play an important regulatory role in protein translation.Beginning with the ESTs which were highly expressed in undifferentiated human embryonic stem(ES) cells and using homology search in mouse dbEST database,we previously cloned two splice variants of a novel putative mouse N5-glutamine methyltransferase (Hemk),termed mHemk1 and mHemk2(GenBank accession number AY456393 and AY583759),respectively.The longer variant mHemk1 is 1792 bp in length,encoding a putative mouse protein of 23 kDa, consisting of 214 amino acid residues.The shorter variant mHemk2 is 1696 bp in length encoding a putative mouse protein of 14.7 kDa, consisting of 138 amino acid residues.In protein data bank annotations, Hemk has previously been classed among methyltransferases methylating N6-adenine or N4-cytidine in DNA because of the presence of S-adenosyl-L-methionine(SAM) binding motif and of an NPPY motif in the protein.These motifs were thought to be the hallmarks of this class of DNA methyltransferases,.However,no DNA methylase activity has been detected for the two variants of mHemk so far,although their subcellular localization was in both nucleus and cytoplasm with the predominant nuclear localization,indicating that mHemk is not a DNA methyltransferase.In contrast,the mHemk-homologous proteins Hemk in bacteria and YDR140w in yeast have been proved to be the members of Hemk methyltransferase family,which act as a protein methyltransferase catalyzing the methylation of polypeptide chain release factors 1(eRF1) in yeast or RF1 and RF2 in bacteria with SAM as the methyl donor, indicating they are N5-glutamine methyltransferases.The NPPY motif-characteristic of N6-DNA methyltransferase was recently found to be shared by Hemk proteins,although all of these Hemk homologues have no DNA methyltransferase activity.Our previous studies showed that the mHemk mRNAs were abundantly expressed in undifferentiated ES cells,testis and brain,weakly expressed in differentiated ES cells and kidney,and not expressed in muscle,heart,placenta,pancreas,lung and stomach,and showed that the knock-down of mHemk by siRNA leads to a decrease in protein translation and cell proliferation.However,whether mHemk is a N5-glutamine methyltransferase remains to be further confirmed.Abstract 1.A novel endoplasmic reticulum retention s ignal RFSK at the C-terminusAim:Studying the subcellular localization of mHemk to find and validate the novel endoplasmic reticulum retention signal.Method:We previously cloned and identified two splice variants of a novel mouse N5-glutamine methyltransferase gene mHemk,termed mHemk1 and mHemk2(GenBank accession number AY456393 and AY583759,respectively).In this study,we further analyzed the subcellular localization of the proteins they encode.Bioinformatics analysis predicted a potential endoplasmic reticulum membrane retention signal RFSK or KSLK at the C-terminus of mHemk1 or mHemk2 protein, and predicted a preferential cytoplasmic localization of the two splice variants.The plasmids were constructed to express the fusion proteins of the enhanced green fluorescent protein(EGFP) with each of the two splice variants and their mutants lacking the predicted endoplasmic reticulum-retention signals,and were transfected into the MCF-7 and Hela cells.Then,Use an immunofluorescence staining was used to observe the distribution of the proteins in these two kinds of cells.Result:The fusion protein EGFP-mHemk1 localized mainly in the cytoplasm and endoplasmic reticulum,in contrast,the fusion protein EGFP-mHemk1-M localized mainly in the nucleus.Differently,both EGFP-mHemK2 and EGFP-mHemK2-M localized in both the cytoplasm and the nucleus.Conclusion:RFSK motif in mHemk1 protein is a novel endoplasmic reticulum retention signal.Nevertheless,there was no strong evidence to support that KSLK motif is an endoplasmic reticulum retention signal in mHemk2 protein.AbstractT 2.The expression of a novel mouse N5-glutamine methyItransferase gene mHemk and its human homologous gene hHemk1 in the baculovirus expression systemAim:To construct the baculovirus expression system of gene hHernk1 which was the human homologous gene of mouse gene mHemk, and to express proteins encoded by genes mHemk and hHemk1 in Sf9 insect cells for the researches on their functions and structures.Method:Gene hHemk1,which was the homologous gene of mouse genes mHemk,was inserted into pFastBac-HT-A vector to construct a recombinant bacmid pFastBac-HT-A/hHemk1.Then the new recombinant bacmid pFastBac-HT-A/hHemk1 and the recombinant bacmids pFastBac-HT-A/mHemk1 and pFastBac-HT-A/mHemk2 were transfected into Sf9 insect cells.The baculovirus produced by transfected insect cells was harvested and then infected normal Sf9 insect cells.Finally,the proteins extracted from the normal or transfected Sf9 insect cells were detected by Western Blot.Results:The positive recombinant bacmid pFastBac-HT-A/hHemk1 which contains hHemk1 gene was detected by PCR.The baculovirus is produced after transfecting the recombinant bacmids pFastBac-HT-A /mHemk1,pFastBac-HT-A/mHemk2 or pFastBac-HT- A/hHemk1 into Sf9 insect cells and was harvested.Western Blot performed for the total proteins from the infected Sf9 insect cells by baculovirus showed that the protein mHemk1 and the protein hHemk1 were expressed in Sf9 insect cells,but the protein mHemk2 expression was not detected in Sf9 insect cells.Conclusion:We have established the baculovirus expression systems of gene mHemk1 and hHemk1.mHemk and hHemk1 proteins were expressed successfully in Sf9 insect cells.