Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.
Animals ; Cell Line ; Chloride Channels ; physiology ; Cricetinae ; Cystic Fibrosis Transmembrane Conductance Regulator ; biosynthesis ; genetics ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Membrane Potentials ; genetics ; Protein Processing, Post-Translational ; Protein Splicing

The RNA-directed DNA Methylation (RdDM) is one type of epigenetic modification which was firstly discovered in plant. RdDM can directly cause DNA modifications of the genome through RNA-DNA interactions. In plant, both of RdDM and mRNA degradation induced by siRNA can silence sequence specific genes through RNA. They play very significant roles in chromosome rearrangement, defence of virus invasion, regulation of gene expression and many processes of plant development. However, the mechanisms of RdDM are still unclear. In this paper the basic characteristics of RdDM were briefly summarized and advances in studies on mechanisms of RdDM were reviewed. These include the kinds of DNA methyltransferases and their functional mechanisms in RdDM, the relationships between DNA methylation and chromatin modification, and important proteins involved in the RdDM process. In plants, RdDM may occur at both the transcriptional and post-transcriptionnal levels, both of which induce gene silencing. Methylation of the target gene promoter correlates with transcriptional gene silencing (TGS) whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS). RdDM and RNAi all depend on the similar siRNA and enzymes, such as DCL3, RdR2, SDE4 and AGO4. There are at least three kinds of DNA methyltransferases, DRM1/2, MET1 and CMT3, in pants. They can interact with and modifies all cytidines within the DNA regions homologous to RNA sequence. Furthermore, methylation of lysine 9 in Histone H3 can affect the methylation of cytidines.
DNA Methylation ; DNA Modification Methylases ; metabolism ; Gene Silencing ; Plants ; genetics ; metabolism ; Protein Processing, Post-Translational ; RNA ; metabolism

OBJECTIVETo investigate the influence of fluorosis on nicotinic acetylcholine receptors (nAChRs) in protein and gene levels in SH-SY5Y cells and the mechanism of the receptor modification.

METHODSSH-SY5Y cells, a human neuroblastoma cell line, were incubated with different concentrations of fluoride or with antioxidant for 48 hours. The functions of cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and protein oxidation detected by carbonyl content; the alpha3 and alpha7 nAChR subunits in protein level were measured by Western blotting and in mRNA level by RT-polymerase chain reaction (RT-PCR).

RESULTSIn high-dose group as compared to the control, the decreased MTT (49%), increased protein oxidation (72%), and lower expression of alpha3 (51%) and alpha7 (47%) nAChR subunit proteins were obviously observed in SH-SY5Y cells. There were no changes in expression of nAChR subunit mRNAs between the cells treated with fluoride and those un-treated in controls. Prior treatment with antioxidant resulted in preventing the decrease of nAChR protein in cells exposed to the high doses of fluoride.

CONCLUSIONFluorosis should result in damage of cells and the declined expression of nAChRs in protein levels, but no influences on gene expression of the receptors in human neuroblastoma neurons. The decreased nAChR proteins might be involved in the mechanism of oxidative stress induced by fluorosis.

Cell Line, Tumor ; Fluoride Poisoning ; metabolism ; Fluorides ; toxicity ; Humans ; Neuroblastoma ; metabolism ; pathology ; Protein Processing, Post-Translational ; drug effects ; Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Nicotinic ; biosynthesis ; genetics

Filamentous fungi can secrete large amounts of proteins, glycosylate proteins and grow faster than plant, insect or mammalian cells. With the advances in fungal molecular genetics, strain improvement, and especially fungal genomics, filamentous fungi are developed as microbial cell factories for the production of heterologous proteins. This review focuses on recent developments of filamentous fungi as production hosts, such as protein quality control mechanisms, the secretion pathways, protein modification, strain stability, and most importantly the application of functional genomics in protein expression. At the same time, the strategies for improving heterologous protein production were also discussed in details.
Biotechnology ; methods ; trends ; Cloning, Molecular ; Fermentation ; genetics ; physiology ; Fungi ; genetics ; metabolism ; Genetic Engineering ; Protein Processing, Post-Translational ; Recombinant Proteins ; biosynthesis ; genetics

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.
Animals ; Cell Line ; Endoplasmic Reticulum/metabolism ; Golgi Apparatus/metabolism ; Kinetics ; Peptides/genetics/*metabolism ; Pituitary Gland, Anterior/*cytology ; Protein Precursors/biosynthesis/genetics/*metabolism ; *Protein Processing, Post-Translational ; Protein Sorting Signals/genetics ; Protein Transport ; Rats ; Recombinant Proteins/biosynthesis/metabolism ; Retroviridae/genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism ; Secretory Vesicles/metabolism ; Somatostatin/biosynthesis/genetics/metabolism/secretion

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.
Animals ; Cell Line ; Endoplasmic Reticulum/metabolism ; Golgi Apparatus/metabolism ; Kinetics ; Peptides/genetics/*metabolism ; Pituitary Gland, Anterior/*cytology ; Protein Precursors/biosynthesis/genetics/*metabolism ; *Protein Processing, Post-Translational ; Protein Sorting Signals/genetics ; Protein Transport ; Rats ; Recombinant Proteins/biosynthesis/metabolism ; Retroviridae/genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism ; Secretory Vesicles/metabolism ; Somatostatin/biosynthesis/genetics/metabolism/secretion

To investigate the effect of Kozak sequence (+4A or +4G) on expression of green fluorescent protein (GFP) gene in HEK293 cells. The eukaryotic expression vectors containing GFP gene with different Kozak sequence (+4A or +4G) were constructed by classic DNA recombination methods, including PCR, enzyme digestion, ligation, transformation, identification, et al. Two different Kozak sequences (+4A or +4G) were obtained through PCR with different mutagenic primers. The right recombinant plasmids pHGFP-A and pHGFP-G were transfected into HEK293 cells by liposome-mediated gene transfer method. The expression level of GFP was observed by fluorescent microscope, flow cytometry and Western blot. The flow cytometry revealed that the expression levels of GFP fluorescence in pHGFP-A and pHGFP-G transfected cells were about 15% and 45%, respectively. Western blot showed the specific bands of about 27 kD (GFP) both in pHGFP-G and pHGFP-A sample lanes; and the GFP expression density of pHGFP-G was about 3.87-fold as that of pHGFP-A by ImageJ software analysis. These results indicated that the +4G in Kozak sequence (when -3 site is purine base pair) plays an important role in GFP protein translation, which enhances the GFP expression up to 4-fold in HEK293 cells.
Actins ; biosynthesis ; genetics ; Cell Line ; Cloning, Molecular ; Gene Expression Regulation ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Protein Processing, Post-Translational ; RNA, Messenger ; metabolism ; Recombination, Genetic ; Transfection

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P< 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.
Tumor Markers, Biological/isolation & purification ; Tissue Distribution ; Stomach Ulcer/metabolism ; Stomach Neoplasms/*metabolism ; Proteome/analysis ; *Protein Processing, Post-Translational ; Phosphorylation ; Nicotinamide N-Methyltransferase/immunology/*metabolism ; Mice, Inbred BALB C ; Mice ; Humans ; Carcinoma/*metabolism ; Blotting, Western/methods ; Antibodies, Monoclonal/biosynthesis ; Animals

Eukaryotic DNA replication is tightly restricted to only once per cell cycle in order to maintain genome stability. Cells use multiple mechanisms to control the assembly of the prereplication complex (pre-RC), a process known as replication licensing. This review focuses on the regulation of replication licensing by posttranslational modifications of the licensing factors, including phosphorylation, ubiquitylation and acetylation. These modifications are critical in establishing the pre-RC complexes as well as preventing rereplication in each cell cycle. The relationship between rereplication and diseases, including cancer and virus infection, is discussed as well.
Acetylation ; Animals ; Cell Cycle ; DNA Replication ; genetics ; physiology ; DNA Replication Timing ; DNA, Neoplasm ; biosynthesis ; genetics ; Genomic Instability ; Host-Pathogen Interactions ; Humans ; Models, Biological ; Neoplasms ; drug therapy ; genetics ; metabolism ; Phosphorylation ; Protein Processing, Post-Translational ; Ubiquitination ; Virus Diseases ; genetics ; metabolism

Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase-histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2.
Bacteria ; enzymology ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Host-Pathogen Interactions ; Phosphorylation ; Polysaccharides, Bacterial ; biosynthesis ; Porphyromonas gingivalis ; enzymology ; Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Protein Tyrosine Phosphatases ; chemistry ; genetics ; metabolism ; Protein-Tyrosine Kinases ; chemistry ; genetics ; metabolism ; Quorum Sensing ; Signal Transduction ; Streptococcus gordonii ; enzymology ; Virulence Factors ; metabolism