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Genetics Jun 2018Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a...
Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a variety of cancers showed that increased expression of MMR proteins often correlated with tumor aggressiveness, metastasis, and early recurrence. To better understand these observations, we used The Cancer Genome Atlas and Gene Expression across Normal and Tumor tissue databases to analyze MMR protein expression in cancers. We found that the MMR genes and are overexpressed more frequently than , and that and are often cooverexpressed as a result of copy number amplifications of these genes. These observations encouraged us to test the effects of upregulating MMR protein levels in baker's yeast, where we can sensitively monitor genome instability phenotypes associated with cancer initiation and progression. Msh6 overexpression (two- to fourfold) almost completely disrupted mechanisms that prevent recombination between divergent DNA sequences by interacting with the DNA polymerase processivity clamp PCNA and by sequestering the Sgs1 helicase. Importantly, cooverexpression of Msh2 and Msh6 (∼eightfold) conferred, in a PCNA interaction-dependent manner, several genome instability phenotypes including increased mutation rate, increased sensitivity to the DNA replication inhibitor HU and the DNA-damaging agents MMS and 4-nitroquinoline N-oxide, and elevated loss-of-heterozygosity. Msh2 and Msh6 cooverexpression also altered the cell cycle distribution of exponentially growing cells, resulting in an increased fraction of unbudded cells, consistent with a larger percentage of cells in G1. These novel observations suggested that overexpression of MSH factors affected the integrity of the DNA replication fork, causing genome instability phenotypes that could be important for promoting cancer progression.
Topics: Cell Cycle; DNA Mismatch Repair; DNA Replication; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; MutS Homolog 2 Protein; MutS Homolog 3 Protein; Proliferating Cell Nuclear Antigen; Protein Binding; RecQ Helicases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Up-Regulation
PubMed: 29654124
DOI: 10.1534/genetics.118.300923 -
BMC Cancer Oct 2023Cell division cycle 6 (CDC6) is a key licensing factor in the assembly of pre-replicative complexes at origins of replication. The role of CDC6 in the pathogenesis of in...
BACKGROUND
Cell division cycle 6 (CDC6) is a key licensing factor in the assembly of pre-replicative complexes at origins of replication. The role of CDC6 in the pathogenesis of in diffuse larger B-cell lymphoma (DLBCL) remains unknown. We aim to investigate the effects of CDC6 on the proliferation, apoptosis and cell cycle regulation in DLBCL cells, delineate its underlying mechanism, and to correlate CDC6 expression with clinical characteristics and prognosis of patients with DLBCL.
METHODS
Initial bioinformatic analysis was performed to screen the potential role of CDC6 in DLBCL. Lentiviral constructs harboring CDC6 or shCDC6 was transfected to overexpress or knockdown CDC6 in SUDHL4 and OCI-LY7 cells. The cell proliferation was evaluated by CCK-8 assay, cell apoptosis was detected by Annexin-V APC/7-AAD double staining, and cell cycle was measured by flow cytometry. Real time quantitative PCR and western blot was used to characterize CDC6 expression and its downstream signaling pathways. The clinical data of DLBCL patients were retrospectively reviewed, the CDC6 expression in DLBCL or lymph node reactive hyperplasia tissues was evaluated by immunohistochemistry.
RESULTS
In silico data suggest that CDC6 overexpression is associated with inferior prognosis of DLBCL. We found that CDC6 overexpression increased SUDHL4 or OCI-LY7 cell proliferation, while knockdown of CDC6 inhibited cell proliferation in a time-dependent manner. Upon overexpression, CDC6 reduced cells in G1 phase and did not affect cell apoptosis; CDC6 knockdown led to significant cell cycle arrest in G1 phase and increase in cell apoptosis. Western blot showed that CDC6 inhibited the expression of INK4, E-Cadherin and ATR, accompanied by increased Bcl-2 and deceased Bax expression. The CDC6 protein was overexpressed DLBCL compared with lymph node reactive hyperplasia, and CDC6 overexpression was associated with non-GCB subtype, and conferred poor PFS and OS in patients with DLBCL.
CONCLUSION
CDC6 promotes cell proliferation and survival of DLBCL cells through regulation of G1/S cell cycle checkpoint and apoptosis. CDC6 is overexpressed and serves as a novel prognostic marker in DLBCL.
Topics: Humans; Hyperplasia; Retrospective Studies; Cell Line, Tumor; Lymphoma, Large B-Cell, Diffuse; Prognosis; Apoptosis; Cell Proliferation; Gene Expression Regulation, Neoplastic; Nuclear Proteins; Cell Cycle Proteins
PubMed: 37833632
DOI: 10.1186/s12885-023-11186-6 -
Cancer Science Mar 2013Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here,...
Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here, we identified novel mechanisms of ErbB2 downregulation. ErbB2 constitutively binds to an adaptor protein FRS2β. We found that FRS2β bound to CD2AP and CIN85, which induces endosomal trafficking that targets lysosomes. FRS2β colocalized with CIN85 in the cytoplasm. Expression of wild type FRS2β but not its CIN85 non-binding mutant, downregulated the ErbB2 protein and inhibited anchorage-independent cell growth. Moreover, the E3 ubiquitin-protein ligase Cbl was contained within a complex of FRS2β and CIN85. Knockdown of both CIN85 and CD2AP or of Cbl, or treatment with lysosomal degradation inhibitors diminished FRS2β downregulation of ErbB2. In addition, knockdown of endogenous FRS2β caused upregulation of ErbB2 in primary neural cells. Finally, immunohistochemical analysis showed that human breast cancer tissues that overexpress ErbB2 expressed low levels of FRS2β. Thus, an FRS2β-CIN85/CD2AP-Cbl axis for downregulation of ErbB2 may regulate ErbB2 protein levels in physiological and pathological settings. Molecular targeting drugs that can increase or stabilize the ErbB2-FRS2β-CIN85/CD2AP-Cbl axis may have promise for the control of ErbB2-overexpressing tumors.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Motifs; Breast Neoplasms; Cells, Cultured; Down-Regulation; Female; Gene Knockdown Techniques; Humans; Neurons; Phosphoproteins; Proto-Oncogene Proteins c-cbl; Receptor, ErbB-2; Signal Transduction
PubMed: 23279575
DOI: 10.1111/cas.12086 -
Microbial Cell Factories Aug 2022Glutathione is a valuable tri-peptide that is industrially produced by fermentation using the yeast Saccharomyces cerevisiae, and is widely used in the pharmaceutical,...
BACKGROUND
Glutathione is a valuable tri-peptide that is industrially produced by fermentation using the yeast Saccharomyces cerevisiae, and is widely used in the pharmaceutical, food, and cosmetic industries. It has been reported that addition of L-serine (L-Ser) is effective at increasing the intracellular glutathione content because L-Ser is the common precursor of L-cysteine (L-Cys) and glycine (Gly) which are substrates for glutathione biosynthesis. Therefore, we tried to enhance the L-Ser biosynthetic pathway in S. cerevisiae for improved glutathione production.
RESULTS
The volumetric glutathione production of recombinant strains individually overexpressing SER2, SER1, SER3, and SER33 involved in L-Ser biosynthesis at 48 h cultivation was increased 1.3, 1.4, 1.9, and 1.9-fold, respectively, compared with that of the host GCI strain, which overexpresses genes involved in glutathione biosynthesis. We further examined simultaneous overexpression of SHM2 and/or CYS4 genes involved in Gly and L-Cys biosynthesis, respectively, using recombinant GCI strain overexpressing SER3 and SER33 as hosts. As a result, GCI overexpressing SER3, SHM2, and CYS4 showed the highest volumetric glutathione production (64.0 ± 4.9 mg/L) at 48 h cultivation, and this value is about 2.5-fold higher than that of the control strain.
CONCLUSIONS
This study first revealed that engineering of L-Ser and Gly biosynthetic pathway are useful strategies for fermentative glutathione production by S. cerevisiase.
Topics: Biosynthetic Pathways; Cysteine; Fermentation; Glutathione; Metabolic Engineering; Phosphoglycerate Dehydrogenase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine
PubMed: 35933377
DOI: 10.1186/s12934-022-01880-8 -
Frontiers in Immunology 2021Tenascin-C (TNC) is an extracellular matrix glycoprotein that is expressed during embryogenesis. It is not expressed in normal adults, but is up-regulated under...
Tenascin-C (TNC) is an extracellular matrix glycoprotein that is expressed during embryogenesis. It is not expressed in normal adults, but is up-regulated under pathological conditions. Although TNC knockout mice do not show a distinct phenotype, analyses of disease models using TNC knockout mice combined with experiments revealed the diverse functions of TNC. Since high TNC levels often predict a poor prognosis in various clinical settings, we developed a transgenic mouse that overexpresses TNC through Cre recombinase-mediated activation. Genomic walking showed that the transgene was integrated into and truncated the gene. While homozygous transgenic mice showed a severe neurological phenotype, heterozygous mice were viable, fertile, and did not exhibit any distinct abnormalities. Breeding hemizygous mice with Nkx2.5 promoter-Cre or α-myosin heavy chain promoter Cre mice induced the heart-specific overexpression of TNC in embryos and adults. TNC-overexpressing mouse hearts did not have distinct histological or functional abnormalities. However, the expression of proinflammatory cytokines/chemokines was significantly up-regulated and mortality rates during the acute stage after myocardial infarction were significantly higher than those of the controls. Our novel transgenic mouse may be applied to investigations on the role of TNC overexpression in various tissue/organ pathologies using different Cre donors.
Topics: Animals; Chromosome Walking; Cytokines; Gene Expression Regulation, Developmental; Genome; Homozygote; Inflammation Mediators; Integrases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardial Infarction; Neurodegenerative Diseases; Phenotype; Promoter Regions, Genetic; Tenascin; Ventricular Myosins
PubMed: 33763067
DOI: 10.3389/fimmu.2021.620541 -
FEBS Letters Jan 2017A screen to identify lysosomal-expressed ion channels led to the discovery of the human Sidt2 protein. Sidt2 is expressed within lysosomal organelles but as a result of...
A screen to identify lysosomal-expressed ion channels led to the discovery of the human Sidt2 protein. Sidt2 is expressed within lysosomal organelles but as a result of heterologous overexpression the protein is also detectable within the plasma membrane of human embryonic kidney cells. The overexpressed protein leads to cell depolarization upon sodium addition. Accordingly in whole-cell patch clamp experiments a spontaneous noninactivating monovalent cation current can be detected in Sidt2-overexpressing cells. Strong overexpression of Sidt2 in HEK293 cells is attended by a significant reduction/loss of detectable lysosomes, indicating that the overexpressed protein leads to lysosomal dysfunction, a hallmark of Alzheimer's disease. Sidt2 is located on chromosome 11q23, a locus repeatedly found by chromosomal mapping of Alzheimer's disease-related genes.
Topics: Alzheimer Disease; Amines; Animals; Cations; Cell Membrane; Cell Shape; Cell Size; Electric Conductivity; Evolution, Molecular; HEK293 Cells; Humans; Lysosomal Membrane Proteins; Lysosomes; Membrane Potentials; Membrane Proteins; Mice; Nucleotide Transport Proteins; Sodium; Transfection
PubMed: 27987306
DOI: 10.1002/1873-3468.12528 -
The FEBS Journal May 2012Adjusting protein synthesis by phosphorylating eukaryotic translation initiation factor 2 (eIF2α) is a major mechanism by which eukaryotes adapt to and overcome stress....
Adjusting protein synthesis by phosphorylating eukaryotic translation initiation factor 2 (eIF2α) is a major mechanism by which eukaryotes adapt to and overcome stress. The eIF2α kinase Gcn2 is essential for overcoming amino acid starvation in all eukaryotes. We have shown that to sense starvation, the Gcn2 RWD domain must directly contact its effector protein, Gcn1, and both must bind to the ribosome, suggesting that starvation is sensed within a Gcn1-Gcn2-ribosome complex. The mammalian protein IMPACT, highly expressed in neurons, and its yeast orthologue yeast IMPACT homologue (Yih1) harbour an RWD domain with Gcn1-binding activity. We have shown that Yih1 downregulates Gcn2 by competing with Gcn2 for Gcn1-binding. Here, we provide evidence that Yih1 forms a complex with ribosomes. In velocity sedimentation assays, overexpressed glutathione S-transferase (GST)-tagged Yih1 cosedimented with polyribosomes independently of Gcn1. Reduction of polyribosomes to monosomes concomitantly decreased GST-Yih1 sedimentation in the heavy fractions where polyribosomes are normally found. Furthermore, GST-Yih1 coprecipitated large ribosomal protein Rpl39 independently of Gcn1. GST-Yih1 overexpression did not significantly affect Gcn1-ribosome or Gcn2-ribosome cosedimentation. myc-tagged Yih1 expressed from its own promoter cosedimented with polyribosomes independently of Gcn1, indicating that Yih1-ribosome interaction occurs under physiological conditions. GST-IMPACT cosedimented with yeast ribosomes and coprecipitated Rpl39 in a Gcn1-independent fashion, suggesting that Yih1/IMPACT-ribosome association is evolutionarily conserved. Moreover, GST-IMPACT coprecipitated actin as found for GST-Yih1. Taken together, our findings strongly suggest that IMPACT/Yih1 associates with ribosomes and that these ribosomes may simultaneously carry Gcn1 and Gcn2. Close physical proximity of Yih1 to the Gcn1-Gcn2-ribosome complex would allow cells to quickly inhibit Gcn2 whenever or wherever necessary.
Topics: Binding Sites; Biological Evolution; Glutathione Transferase; Microfilament Proteins; Peptide Elongation Factors; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 22404850
DOI: 10.1111/j.1742-4658.2012.08553.x -
Biomedical Research (Tokyo, Japan) 2021Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is...
Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is identical to wild type TBC1D8B from amino acid positions 1 to 427 and possesses a modified sequence from position 428 to 435 (ECGGLFLL) because of the introduction of a premature stop codon at position 436 to narrow down the minimum requirement element. The N-terminal TBC1D8B contains two GRAM domains but not the TBC domain essential for Rab-GTPase activity. The N-terminal TBC1D8B overexpression significantly reduced the cultured HCT116 cell number. When we tested C-terminal TBC1D8B, containing the portion of TBC1D8B absent in the N-terminal TBC1D8B, the cell number reduction was not observed. The N-terminal TBC1D8B overexpression significantly increased the coronin 1B expression and reduced the phosphorylation of serine 51 in eIF2α, respective markers of apoptosis and cell death/survival. Also, caspase 3 and poly ADP-ribose polymerase increased cleavage in suspended cells overexpressing the N-terminal TBC1D8B. Taken together, it is not the TBC domain for Rab-GTPase activity, but amino acids 1 to 435, including the two GRAM domains, that is enough for TBC1D8B to cause spontaneous apoptosis. TBC1D8B could be a potential anticancer therapeutic molecule.
Topics: Antineoplastic Agents; Apoptosis; Calcium-Binding Proteins; Cell Death; Cloning, Molecular; Codon, Terminator; Eukaryotic Initiation Factor-2; GTPase-Activating Proteins; HCT116 Cells; Humans; Phosphorylation; Protein Domains; Transfection; Vesicular Transport Proteins
PubMed: 34092754
DOI: 10.2220/biomedres.42.95 -
Chemical Research in Toxicology Nov 2019Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride...
Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride accumulation and mediate fluoride toxicity. Consequently, deletion of fluoride exporter genes (FEX) in resulted in over 1000-fold increased fluoride sensitivity. We used this FEX knockout strain to identify genes, that when overexpressed, are able to partially relieve the toxicity of fluoride exposure. Overexpression of five genes, SSU1, YHB1, IPP1, PHO87, and PHO90, increase fluoride tolerance by 2- to 10-fold. Overexpression of these genes did not provide improved fluoride resistance in wild-type yeast, suggesting that the mechanism is specific to low fluoride toxicity in yeast. Ssu1p and Yhb1p both function in nitrosative stress response, which is induced upon fluoride exposure along with metal influx. Ipp1p, Pho87p, and Pho90p increase intracellular orthophosphate. Consistent with this observation, fluoride toxicity is also partially mitigated by the addition of high levels of phosphate to the growth media. Fluoride inhibits phosphate import upon stress induction and causes nutrient starvation and organelle disruption, as supported by gene induction monitored through RNA-Seq. The combination of observations suggests that transmembrane nutrient transporters are among the most sensitized proteins during fluoride-instigated stress.
Topics: Anion Transport Proteins; Calcium; Dioxygenases; Gene Expression Regulation, Fungal; Hemeproteins; Inorganic Pyrophosphatase; Mitochondria; Nitrate Transporters; Nitrates; Nitrosative Stress; Phosphates; RNA-Seq; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sodium Fluoride
PubMed: 31576749
DOI: 10.1021/acs.chemrestox.9b00315 -
Experimental Dermatology Oct 2017We previously observed that aquaporin-3 and aquaporin-10 are upregulated in the epidermis of hand dermatitis patients (Med. Hypotheses, 84, 2015, 498). To address the...
We previously observed that aquaporin-3 and aquaporin-10 are upregulated in the epidermis of hand dermatitis patients (Med. Hypotheses, 84, 2015, 498). To address the functional relevance of this upregulation, we overexpressed AQP3/AQP10 in mice using the human K1 promoter. Combining imiquimod with detergent-containing water challenge, a common trigger in hand and other dermatitis, resulted in an increase in acanthosis in mice overexpressing AQP3 or AQP3 and AQP10. Aquaporin overexpression also drove a trend towards greater weight loss in these animals. These data support a role for cutaneous aquaporins in the pathogenesis of dermatitis and as a potential target in their treatment.
Topics: Aminoquinolines; Animals; Aquaporin 3; Aquaporins; Cell Differentiation; Dermatitis; Detergents; Epidermis; Filaggrin Proteins; Imiquimod; Intermediate Filament Proteins; Keratin-10; Keratinocytes; Membrane Proteins; Mice; Up-Regulation; Weight Loss
PubMed: 28111811
DOI: 10.1111/exd.13307