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Biochimica Et Biophysica Acta.... Aug 2020The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo...
The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.
Topics: Carboxy-Lyases; Galactokinase; Gene Expression Regulation, Fungal; Phosphoprotein Phosphatases; Phosphorylation; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; Transcriptome
PubMed: 32339526
DOI: 10.1016/j.bbamcr.2020.118727 -
Plant & Cell Physiology Feb 2019Orange carotenoid protein (OCP) plays a vital role in the thermal dissipation of excitation energy in the photosynthetic machinery of the cyanobacterium Synechocystis...
Orange carotenoid protein (OCP) plays a vital role in the thermal dissipation of excitation energy in the photosynthetic machinery of the cyanobacterium Synechocystis sp. PCC 6803. To clarify the role of OCP in the protection of PSII from strong light, we generated an OCP-overexpressing strain of Synechocystis and examined the effects of overexpression on the photoinhibition of PSII. In OCP-overexpressing cells, thermal dissipation of energy was enhanced and the extent of photoinhibition of PSII was reduced. However, photodamage to PSII, as monitored in the presence of lincomycin, was unaffected, suggesting that overexpressed OCP protects the repair of PSII. Furthermore, the synthesis de novo of proteins in thylakoid membranes, such as the D1 protein which is required for the repair of PSII, was enhanced in OCP-overexpressing cells under strong light, while the production of singlet oxygen was suppressed. Thus, the enhanced thermal dissipation of energy via overexpressed OCP might support the repair of PSII by protecting protein synthesis from oxidative damage by singlet oxygen under strong light, with the resultant mitigation of photoinhibition of PSII.
Topics: Bacterial Proteins; Light; Photosystem II Protein Complex; Synechocystis
PubMed: 30398652
DOI: 10.1093/pcp/pcy218 -
Biochimica Et Biophysica Acta. Reviews... Dec 2021Class III β-tubulin (βIII-tubulin) is frequently overexpressed in human tumors and is associated with resistance to microtubule-targeting agents, tumor aggressiveness,... (Review)
Review
Class III β-tubulin (βIII-tubulin) is frequently overexpressed in human tumors and is associated with resistance to microtubule-targeting agents, tumor aggressiveness, and poor patient outcome. Understanding the mechanisms regulating βIII-tubulin expression and the varied functions βIII-tubulin may have in different cancers is vital to assess the prognostic value of this protein and to develop strategies to enhance therapeutic benefits in βIII-tubulin overexpressing tumors. Here we gather all the available evidence regarding the clinical implications of βIII-tubulin overexpression in cancer, describe factors that regulate βIII-tubulin expression, and discuss current understanding of the mechanisms underlying βIII-tubulin-mediated resistance to microtubule-targeting agents and tumor aggressiveness. Finally, we provide an overview of emerging therapeutic strategies to target tumors that overexpress βIII-tubulin.
Topics: Humans; Neoplasms; Tubulin
PubMed: 34364992
DOI: 10.1016/j.bbcan.2021.188607 -
Journal of Experimental Botany Feb 2019The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively...
The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin-thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI-LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.
Topics: Chloroplast Thioredoxins; Chloroplasts; Gene Expression Regulation, Plant; Oxidation-Reduction; Photosynthesis; Photosystem II Protein Complex; Plant Proteins; Protein Serine-Threonine Kinases; Nicotiana
PubMed: 30476130
DOI: 10.1093/jxb/ery415 -
Cancer Science Jan 2022CD28, one of the costimulatory molecules, has a pivotal role in T-cell activation, and its expression is strictly regulated in normal T cells. Gain-of-function genetic...
CD28, one of the costimulatory molecules, has a pivotal role in T-cell activation, and its expression is strictly regulated in normal T cells. Gain-of-function genetic alterations involving CD28 have been frequently observed in adult T-cell leukemia/lymphoma (ATLL). These abnormalities, such as CD28 fusions and copy number variations, may not only confer continuous, prolonged, and enhanced CD28 signaling to downstream pathways but also induce overexpression of the CD28 protein. In this study, 120 ATLL cases were examined by immunohistochemistry for CD28 and its ligands CD80 and CD86, and their expression on tumor cells was semiquantitatively evaluated. CD28 was overexpressed in 55 (46%) cases, and CD80 or CD86 (CD80/CD86) was infrequently overexpressed in 12 (11%). Compared with non-overexpressers, CD28 overexpressers showed a higher frequency of CD28 genetic alterations and had an increased number of CD80/CD86-positive non-neoplastic cells infiltrating tumor microenvironment. In the entire ATLL patient cohort, CD28 overexpressers showed a significantly poorer overall survival (OS) compared with non-overexpressers (P = .001). The same was true for a subgroup who were treated with multidrug regimens with or without mogamulizumab. CD28 overexpression had no prognostic impact in the group who received allogeneic hematopoietic stem cell transplantation. In the multivariate analysis for OS, CD28 overexpression was selected as an independent risk factor. These results suggest ATLL patients with CD28 overexpression have more aggressive clinical course and are more refractory to treatment with multidrug chemotherapy. CD28 overexpression appears to be a novel unfavorable prognostic marker in ATLL patients, and further prospective studies are warranted to establish its prognostic significance.
Topics: Adult; Aged; Aged, 80 and over; B7-1 Antigen; B7-2 Antigen; CD28 Antigens; DNA Copy Number Variations; Female; Gene Expression Regulation, Neoplastic; Humans; Leukemia-Lymphoma, Adult T-Cell; Male; Middle Aged; Prognosis; Survival Analysis; Tumor Microenvironment; Up-Regulation
PubMed: 34738707
DOI: 10.1111/cas.15191 -
Systems Biology Dec 2005The overexpression of secreted proteins is of critical importance to the biotechnology and biomedical fields. A common roadblock to high yields of proteins is in the...
The overexpression of secreted proteins is of critical importance to the biotechnology and biomedical fields. A common roadblock to high yields of proteins is in the endoplasmic reticulum (ER) where proofreading for properly folded proteins is often rate limiting. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. An obvious method for overcoming this block would be to increase the capacity of the folding process (overexpressing chaperones) or decreasing the proofreading process (blocking the down-regulation by the UPR). Unfortunately, these processes are tightly interlinked, whereby modification of one mechanism has unknown effects on the other. Although some success has been achieved in improving expression via co-overexpressing ER chaperones, the results have not lead to a global method for increasing all heterologously overexpressed proteins. Further, many diseases have been linked to extended periods of stress and are not treatable by these approaches. This work utilises both experimental analysis of the interactions within the ER and modelling in order to understand how these interactions affect early secretory pathway dynamics. This study shows that overexpression of the ER chaperone binding protein does not regulate Ire1p and the UPR as predicted by a model based on the published understanding of the molecular mechanism. A new model is proposed for Ire1p regulation and the UPR that better fits the experimental data and recent studies on Ire1p.
Topics: Computer Simulation; Fungal Proteins; Gene Expression Regulation, Fungal; HSP70 Heat-Shock Proteins; Membrane Glycoproteins; Models, Biological; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Up-Regulation
PubMed: 16986272
DOI: 10.1049/ip-syb:20050048 -
Journal of Bioscience and Bioengineering Mar 2024Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli....
Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli. Protein overexpression imposes a burden on metabolism because metabolic pathways must supply building blocks for protein and DNA synthesis. Different E. coli strains have distinct metabolic capacities. In this study, two proteins were overexpressed in four E. coli strains (MG1655(DE3), W3110(DE3), BL21star(DE3), and Rosetta(DE3)), and their effects on metabolic burden were investigated. Metabolomic analysis showed that E. coli strains overexpressing green fluorescent protein had decreased levels of several metabolites, with a positive correlation between the number of reduced metabolites and green fluorescent protein expression levels. Moreover, nucleic acid-related metabolites decreased, indicating a metabolic burden in the E. coli strains, and the growth rate and protein expression levels were improved by supplementation with the five nucleosides. In contrast, two strains overexpressing delta rhodopsin, a microbial membrane rhodopsin from Haloterrigena turkmenica, led to a metabolic burden and decrease in the amino acids Ala, Val, Leu, Ile, Thr, Phe, Asp, and Trp, which are the most frequent amino acids in the delta rhodopsin protein sequence. The metabolic burden caused by protein overexpression was influenced by the metabolic capacity of the host strains and the sequences of the overexpressed proteins. Detailed characterization of the effects of protein expression on the metabolic state of engineered cells using metabolomics will provide insights into improving the production of target compounds.
Topics: Green Fluorescent Proteins; Rhodopsin; Escherichia coli; Metabolome; Amino Acids; DNA
PubMed: 38281859
DOI: 10.1016/j.jbiosc.2023.12.003 -
Molecular and Biochemical Parasitology Sep 2020Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei,...
Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.
Topics: DNA-Directed RNA Polymerases; Dyneins; Gene Expression; Genes, Protozoan; Protozoan Proteins; Recombinant Proteins; Trypanosoma brucei brucei; Viral Proteins
PubMed: 32682799
DOI: 10.1016/j.molbiopara.2020.111300 -
Molecular and Cellular Biology Sep 1990By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the...
By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.
Topics: Animals; Cell Division; Cell Line; Colonic Neoplasms; Gene Expression; Genetic Vectors; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured
PubMed: 2388620
DOI: 10.1128/mcb.10.9.4650-4657.1990 -
The Journal of Biological Chemistry Mar 1993We have determined the patterns of mRNA and protein expression of 7 protein kinase C (PKC) isozymes in NIH 3T3 cells. Only PKC-alpha is expressed abundantly in NIH 3T3...
We have determined the patterns of mRNA and protein expression of 7 protein kinase C (PKC) isozymes in NIH 3T3 cells. Only PKC-alpha is expressed abundantly in NIH 3T3 cells; endogenous levels of the other 6 PKC isozymes are low or undetectable. We have overexpressed PKC-delta and -epsilon in these cells to observe activation/translocation of these two isozymes and the biological consequences of overexpression. Both PKC-delta and -epsilon, but not PKC-alpha, are partially associated with the insoluble fraction even in the absence of phorbol 12-myristate 13-acetate (PMA). Upon PMA stimulation, both PKC-delta and -epsilon translocate to the insoluble fraction of cell homogenates, as can be observed with the endogenous PKC-alpha. Overexpression of PKC-delta induces significant changes in morphology and causes the cells to grow more slowly and to a decreased cell density in confluent cultures. These changes are accentuated by treatment with PMA. Overexpression of PKC-epsilon does not lead to morphological changes, but causes increased growth rates and higher cell densities in monolayers. None of the PKC-delta overexpressers grow in soft agar with or without PMA, but all the cell lines that overexpress PKC-epsilon grow in soft agar in the absence of PMA, but not in its presence. NIH 3T3 cells that overexpress PKC-epsilon also form tumors in nude mice with 100% incidence. This indicates that high expression of PKC-epsilon contributes to neoplastic transformation.
Topics: 3T3 Cells; Animals; Biological Transport; Blotting, Northern; Blotting, Western; Brain; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cloning, Molecular; Enzyme Activation; Enzyme Stability; Indoles; Isoenzymes; Kinetics; Maleimides; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasms, Experimental; Protein Kinase C; Tetradecanoylphorbol Acetate
PubMed: 8454583
DOI: No ID Found