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The Journal of Biological Chemistry Oct 2021Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is...
Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein.
Topics: Cell Line; Gene Expression; Humans; Protein Processing, Post-Translational; Recombinant Proteins; Tropomyosin
PubMed: 34478714
DOI: 10.1016/j.jbc.2021.101154 -
Theoretical Biology & Medical Modelling Apr 2006Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA) regulates the...
BACKGROUND
Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA) regulates the probability with which a gene template is switched from the inactive to the active state without affecting the rate at which RNA molecules are produced from the template. With graded, also called rheostat-like, induction the gene template has continuously varying levels of transcriptional activity, and the TA regulates the rate of RNA production. Support for each of these two mechanisms arises primarily from experimental studies measuring reporter proteins in individual cells, rather than from direct measurement of induction events at the gene template.
METHODS AND RESULTS
In this paper, using a computational model of stochastic gene expression, we have studied the biological and experimental conditions under which a binary induction mode operating at the gene template can give rise to differentially expressed "phenotypes" (i.e., binary, hybrid or graded) at the protein level. We have also investigated whether the choice of reporter genes plays a significant role in determining the observed protein expression patterns in individual cells, given the diverse properties of commonly-used reporter genes. Our simulation confirmed early findings that the lifetimes of active/inactive promoters and half-lives of downstream mRNA/protein products are important determinants of various protein expression patterns, but showed that the induction time and the sensitivity with which the expressed genes are detected are also important experimental variables. Using parameter conditions representative of reporter genes including green fluorescence protein (GFP) and beta-galactosidase, we also demonstrated that graded gene expression is more likely to be observed with GFP, a longer-lived protein with low detection sensitivity.
CONCLUSION
The choice of reporter genes may determine whether protein expression is binary, graded or hybrid, even though gene induction itself operates in an all-or-none fashion.
Topics: Computer Simulation; Gene Expression; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Half-Life; Luciferases; Models, Genetic; Phenotype; Promoter Regions, Genetic; Proteins; Stochastic Processes; Templates, Genetic; Time Factors; Trans-Activators; Transcription, Genetic; Transcriptional Activation; beta-Galactosidase
PubMed: 16597340
DOI: 10.1186/1742-4682-3-18 -
Journal of Dairy Science Mar 2021During industrial handling, Lactococcus lactis needs to adapt to different culture conditions by regulating its metabolic pathways. Modifying culture conditions may be...
During industrial handling, Lactococcus lactis needs to adapt to different culture conditions by regulating its metabolic pathways. Modifying culture conditions may be an important way to control the biomass and functional metabolites of lactic acid bacteria. In this study, we identified the differentially expressed genes and proteins of L. lactis under different culture conditions by integrating transcriptomics and proteomics. We also analyzed the data using a bioinformatic approach to reveal the regulatory mechanisms affected by culture conditions. The transcriptome and proteome studies indicated that different culture conditions (fructose, calcium ion, palmitic acid, low pH) affected gene and protein expressions. The levels of differentially expressed proteins did not significantly correlate with the expression levels of their corresponding genes. Our results highlight the importance of comparative transcriptomics and proteomics analyses. In this study, fructose and pH significantly affected sugar metabolism of L. lactis. When lactose was replaced by fructose, fructokinase expression was promoted, and fructose metabolism was accelerated, whereas starch and sucrose metabolism and galactose metabolism system were inhibited. Low pH may be beneficial to homofermentation of L. lactis, which may also metabolize galactose through the tagatose pathway and the Leloir pathway. Fatty acid metabolism and fatty acid biosynthesis were significantly downregulated under calcium ion and palmitic acid. The purine metabolism was upregulated under fructose treatment and downregulated under palmitic acid treatment.
Topics: Animals; Gene Expression Profiling; Lactococcus lactis; Proteome; Proteomics; Transcriptome
PubMed: 33455780
DOI: 10.3168/jds.2020-18895 -
Journal of Nippon Medical School =... Mar 2020Basophils were reported to be associated with allergy pathogenesis and the efficacy of allergen immunotherapy. Using a purified cedar allergen, we recently studied the...
Basophils were reported to be associated with allergy pathogenesis and the efficacy of allergen immunotherapy. Using a purified cedar allergen, we recently studied the effectiveness of sublingual immunotherapy for patients with Japanese cedar pollinosis. Patients were classified as high responders (HR) and nonresponders (NR), and comprehensive microarray analysis was used to examine peripheral basophils in both groups. A total of 153 genes were differentially expressed in HR and NR patients. Most of these differentially expressed genes encoded intracellular molecules, and expression levels were higher in HR patients than in NR patients. mRNA expression of the gene encoding D4, zinc, and double plant homeodomain (PHD) fingers family 2 (DPF2) was significantly correlated with copy number variation (CNV). Genetic variation in the DPF2 gene and its expression in basophils might be associated with the efficacy of sublingual immunotherapy.
Topics: Basophils; DNA Copy Number Variations; DNA-Binding Proteins; Gene Expression; Humans; RNA, Messenger; Rhinitis, Allergic, Seasonal; Sublingual Immunotherapy; Transcription Factors
PubMed: 31564689
DOI: 10.1272/jnms.JNMS.2020_87-104 -
Genome Biology and Evolution 2010There is great variation in the rates of sequence evolution among proteins encoded by the same genome. The strongest correlate of evolutionary rate is expression level:...
There is great variation in the rates of sequence evolution among proteins encoded by the same genome. The strongest correlate of evolutionary rate is expression level: highly expressed proteins tend to evolve slowly. This observation has led to the proposal that a major determinant of protein evolutionary rate involves the toxic effects of protein that misfolds due to transcriptional and translational errors (the mistranslation-induced misfolding [MIM] hypothesis). Here, I present a model that explains the correlation of evolutionary rate and expression level by selection for function. The basis of this model is that selection keeps expression levels near optima that reflect a trade-off between beneficial effects of the protein's function and some nonspecific cost of expression (e.g., the biochemical cost of synthesizing protein). Simulations confirm the predictions of the model. Like the MIM hypothesis, this model predicts several other relationships that are observed empirically. Although the model is based on selection for protein function, it is consistent with findings that a protein's rate of evolution is at most weakly correlated with its importance for fitness as measured by gene knockout experiments.
Topics: Computer Simulation; Evolution, Molecular; Gene Expression; Genome; Models, Biological; Mutation; Proteins; Selection, Genetic; Statistics as Topic; Time Factors
PubMed: 20884723
DOI: 10.1093/gbe/evq059 -
Technology and Health Care : Official... 2019The human hematopoietic stem/progenitor cell 117 (HSPC117) protein is involved in many important biological processes.
BACKGROUND
The human hematopoietic stem/progenitor cell 117 (HSPC117) protein is involved in many important biological processes.
OBJECTIVE
This study was designed to identify the level of HSPC117 mRNA expression in 10 min pig tissue samples and HSPC117 subcellular localization in the PK15 cell line.
METHODS
In this study, 10 tissue samples of min pigs were collected, and EGFP-HSPC117 vectors were constructed to express EGFP-HSPC117 fusion proteins in PK15 cells.
RESULTS
HSPC117 mRNA was expressed in all of the tissue samples, although the levels of expression in fat and lung tissues were significantly lower than in other tissues (P< 0.01). After generating and detecting the EGFP-HSPC117 fusion protein, fluorescence was found to be distributed throughout the cytoplasm and nucleus during interphase; however, the fluorescence was concentrated in the nuclear area in mitotic cells.
CONCLUSIONS
These results indicate that the HSPC117 gene is expressed in many min pig tissues. The HSPC117 protein was distributed throughout the cells during interphase, but was concentrated in the nuclear area in mitotic cells.
Topics: Animals; Biological Phenomena; Cell Line; Epithelial Cells; Gene Expression; Proteins; RNA; Reverse Transcriptase Polymerase Chain Reaction; Swine
PubMed: 31045548
DOI: 10.3233/THC-199028 -
The American Journal of Pathology Mar 2002Autosomal-dominant polycystic kidney disease, one of the most frequent human genetic disorders, is genetically heterogeneous. Most cases result from mutations of PKD1 or...
Autosomal-dominant polycystic kidney disease, one of the most frequent human genetic disorders, is genetically heterogeneous. Most cases result from mutations of PKD1 or PKD2 encoding polycystin-1 or polycystin-2, respectively. Polycystin-1 is a large transmembrane protein containing several domains involved in cell-cell and/or cell-matrix interactions. Polycystin-2 is transmembrane glycoprotein sharing homology with some families of cation channels. Despite a large number of reports, the tissue distribution of these two proteins, especially of polycystin-1, is still debated. We investigated the expression pattern of PKD1 and PKD2 transcripts and proteins during human embryogenesis and kidney development, using Northern blot analysis, in situ hybridization, and immunohistochemical methods. For each gene, the expression pattern of transcripts and protein was concordant. In human 5- to 6-week-old embryos, both genes are widely expressed, mainly in neural tissue, cardiomyocytes, endodermal derivatives, and mesonephros. At this age, PKD2 but not PKD1 expression is observed in the ureteric bud and the uninduced metanephros. Thereafter, PKD2 is diffusely expressed at all stages of nephron development, whereas high PKD1 expression first appears in differentiated proximal tubules. Proximal tubule expression of both genes decreases from weeks 20 to 24 onwards. PKD1 transcripts, later restricted to distal tubules in fetal nephrogenesis, are no longer detected in adult kidneys, which nevertheless maintain a faint expression of polycystin-1, whereas persistent expression of PKD2 transcripts and protein is observed throughout nephrogenesis. Overall, contrary to previous observations, we found profound differences in the spatiotemporal expression of PKD1 and PKD2 during nephrogenesis, PKD2 being expressed earlier and more diffusely than PKD1. These data suggest that polycystins could interact with different partners, at least during kidney development.
Topics: Adult; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Humans; Kidney; Membrane Proteins; Pregnancy; Protein Biosynthesis; Proteins; TRPP Cation Channels; Transcription, Genetic
PubMed: 11891195
DOI: 10.1016/S0002-9440(10)64919-X -
Virology Journal Sep 2010Some UL45 gene function of Herpesvirus was reported. While there was no any report of the duck enteritis virus (DEV) UL45 protein as yet.
BACKGROUND
Some UL45 gene function of Herpesvirus was reported. While there was no any report of the duck enteritis virus (DEV) UL45 protein as yet.
RESULTS
The UL45 gene and des-transmembrane domain of UL45 (named UL45Δ gene, 295-675bp of UL45) of DEV were amplified by PCR and subcloned into the prokaryotic expression vector pET-32a(+). The constructed recombinant plasmids were transformed into the host strain BL21(DE3) PLysS and induced by IPTG. SDS-PAGE analysis showed the UL45 gene couldn't express while UL45Δ gene was highly expressed. His Purify Kit or salting-out could purify the protein effectively. Using the purified protein to immunize New-Zealand rabbits and produce polyclonal antibody. The agar diffusion reaction showed the titer of antibody was 1:32. Western blot analysis indicated the purified rabbit anti-UL45Δ IgG had a high level of specificity and the UL45 gene was a part of DEV genome. The transcription phase study of UL45 gene showed that expression of UL45 mRNA was at a low level from 0 to 18 h post-infection (pi), then accumulated quickly at 24 h pi and peaked at 42 h pi. It can be detected till 72 h pi. Besides, western blot analysis of purified virion and different viral ingredients showed that the UL45 protein resided in the purified virion and the viral envelope.
CONCLUSIONS
The rabbit anti-UL45Δ IgG was produced successfully and it can serve as a good tool for penetrating studies of the function of DEV UL45 protein. The transcription phase and protein characteristics analysis indicated that DEV UL45 gene was a late gene and UL45 protein may be a viral envelope protein.
Topics: Animals; Antibodies, Viral; Cloning, Molecular; Ducks; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Viral; Herpesviridae; Rabbits; Recombinant Proteins; Time Factors; Transcription, Genetic; Viral Structural Proteins; Virion
PubMed: 20843372
DOI: 10.1186/1743-422X-7-232 -
Annals of the New York Academy of... Jun 1998In summary, several important issues were discussed at the workshop. These included the importance of oligomer size for different functions of the sHsps, the highly... (Review)
Review
In summary, several important issues were discussed at the workshop. These included the importance of oligomer size for different functions of the sHsps, the highly significant observation that the sHsps can block apoptosis; and that some mechanisms of sHsps protection involve glutathione while others may be related to microfilament stability. Further evidence was presented for the chaperone functions of sHsps, and structural studies provided additional information relating sHsps' large oligomeric structure to chaperone function. Regulation of oligomer size by phosphorylation was also a prominent topic of discussion, as was the importance of the relationship of oligomer size to differing functions of Hsp27. An interesting contrast was noted between Hsp27, whose oligomer size is regulated by phosphorylation, and alpha beta-crystallin, whose large oligomer structure is unaffected by phosphorylation. This may prove to be of physiological significance, particularly in cell types that express both of these proteins. Observations of the translocation of MAPKAP kinase 2 from the nucleus to the cytoplasm in live cells suggest that the dynamics of phosphorylation of the sHsps may be more complex than previously thought. One topic that seems to have been settled is that the binding site of at least dimers of sHsps resides in the carboxyl-terminal region of the proteins. Ever-increasing numbers of studies are reporting interesting patterns of sHsp expression, phosphorylation, and subcellular localizations of the sHsps in various cells or tissues in normal and diseased states. Unfortunately, very little is understood about what these observations may mean. It can be expected, however, that as more is learned about the function and regulation of sHsps, the relationships between sHsps and cellular response to disease will become better understood. Lastly, the discovery that specific Hsp27-binding proteins may exist should open completely new avenues of investigation into the functions of sHsps. If the observations of unique subcellular localizations of sHsps in various cells types (e.g., striated muscle and Sertoli cells) can be related to expression of specific Hsp-binding proteins, considerable advances in our understanding of sHsps should result. It was abundantly clear from results presented and from ensuing discussions in the workshop that the study of sHsps is an exceedingly dynamic area of research (FIG. 2) with an ever-expanding significance for a number of other areas of current biological research.
Topics: Animals; Binding Sites; Gene Expression; Heat-Shock Proteins; Molecular Chaperones; Oxidative Stress; Protein Binding; RNA, Messenger; Rats
PubMed: 9668602
DOI: 10.1111/j.1749-6632.1998.tb08973.x -
The Biochemical Journal Apr 2002Many eukaryotic genes are expressed as multiple isoforms through the differential utilization of transcription/translation initiation sites or alternative splicing. The...
Many eukaryotic genes are expressed as multiple isoforms through the differential utilization of transcription/translation initiation sites or alternative splicing. The conventional approach for studying individual isoforms in a clean background (i.e. without the influence of other isoforms) has been to express them in cells or whole organisms in which the target gene has been deleted; this is time-consuming. Recently an efficient post-transcriptional gene-silencing method has been reported that employs a small interfering double-stranded RNA (siRNA). On the basis of this method we report a rapid alternative approach for isoform-specific gene expression. We show how the adaptor protein ShcA can be suppressed and expressed in an isoform-specific manner in a human cell line. ShcA exists in three isoforms, namely p66, p52 and p46, which differ only in their N-terminal regions and are derived from two different transcripts, namely p66 and p52/p46 mRNAs. An siRNA with a sequence shared by the two transcripts suppressed all of them. However, another siRNA whose sequence was present only in p66 mRNA suppressed only the p66 isoform, suggesting that the siRNA signal did not propagate to other regions of the target mRNA. The expression of individual isoforms was achieved by first down-regulating all isoforms by the common siRNA and then transfecting with an expression vector for each isoform that harboured silent mutations at the site corresponding to the siRNA. This allowed functional analysis of individual ShcA isoforms and may be more generally applicable for studying genes encoding multiple proteins.
Topics: Adaptor Proteins, Signal Transducing; Blotting, Western; Cloning, Molecular; Cytoplasm; DNA, Complementary; Down-Regulation; Gene Silencing; Genetic Techniques; HeLa Cells; Humans; Kinetics; Protein Biosynthesis; Protein Isoforms; Protein Structure, Tertiary; Proteins; RNA, Messenger; RNA, Small Interfering; RNA, Untranslated; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Transcription, Genetic; Transfection
PubMed: 11903040
DOI: 10.1042/0264-6021:3630001