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FEBS Letters Apr 2016In silico analysis of the uncharacterized open reading frame YMR210w in Saccharomyces cerevisiae revealed that it possesses both an α/β hydrolase domain (ABHD) and a...
In silico analysis of the uncharacterized open reading frame YMR210w in Saccharomyces cerevisiae revealed that it possesses both an α/β hydrolase domain (ABHD) and a typical lipase (GXSXG) motif. The purified protein displayed monoacylglycerol (MAG) lipase activity and preferred palmitoyl-MAG. Overexpression of YMR210w in the known MAG lipase mutant yju3Δ clearly revealed that the protein had MAG lipase activity, hence we named the ORF MGL2. Overexpression of YMR210w decreased the cellular triacylglycerol levels. Analysis of the overexpressed strains showed reduction in the lipid droplets number and size. Phenotype studies revealed that the double deletion yju3Δmgl2Δ displayed a growth defect that was partially restored by MGL2 overexpression.
Topics: Amino Acid Motifs; Amino Acid Sequence; Carbon Radioisotopes; Genetic Complementation Test; Lipid Droplets; Lipoylation; Membrane Lipids; Microbial Viability; Monoacylglycerol Lipases; Mutagenesis, Site-Directed; Mutation; Phospholipids; Phylogeny; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spectrometry, Mass, Electrospray Ionization; Staining and Labeling
PubMed: 26991558
DOI: 10.1002/1873-3468.12136 -
PLoS Genetics Sep 2011The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential...
The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.
Topics: Centromere; Chromatin Immunoprecipitation; Chromosomal Instability; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA-Binding Proteins; Gene Expression; Histones; Humans; Kinetochores; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 21980305
DOI: 10.1371/journal.pgen.1002303 -
BMB Reports Feb 2022Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2...
Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of β-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator. [BMB Reports 2022;55(2): 98-103].
Topics: Carrier Proteins; Cell Line, Tumor; Chromosomal Proteins, Non-Histone; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Muscle Proteins; NIMA-Related Kinases; Neoplasms; Neoplastic Stem Cells; Phenotype; Pyruvate Kinase; Signal Transduction; Thyroid Hormones; Thyroid Hormone-Binding Proteins
PubMed: 35000669
DOI: 10.5483/BMBRep.2022.55.2.118 -
Proceedings of the National Academy of... Mar 1997Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in...
Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues. Calbindin-D28k is also present in pancreatic beta cells, but its function in these cells is not known. To determine a role for calbindin-D28k in the beta cell, rat calbindin-D28k was overexpressed in the pancreatic beta cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 microM calcium ionophore. In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (-345/+1) or five copies of the Far-FLAT minienhancer (-247/-198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. These studies provide the first direct evidence (to our knowledge) for a role for calbindin in beta cell function.
Topics: Animals; Calbindin 1; Calbindins; Calcimycin; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; Gene Expression Regulation; Genes, Reporter; Glucose Transporter Type 1; Insulin; Insulinoma; Ionophores; Islets of Langerhans; Monosaccharide Transport Proteins; Radioimmunoassay; Rats; S100 Calcium Binding Protein G; Transfection; Tumor Cells, Cultured
PubMed: 9050887
DOI: 10.1073/pnas.94.5.1961 -
Molecular Biology of the Cell May 2011Yeast Btn2 facilitates the retrieval of specific proteins from late endosomes (LEs) to the Golgi, a process that may be adversely affected in Batten disease patients. We...
Yeast Btn2 facilitates the retrieval of specific proteins from late endosomes (LEs) to the Golgi, a process that may be adversely affected in Batten disease patients. We isolated the putative yeast orthologue of a human complex I deficiency gene, designated here as BTN3, as encoding a Btn2-interacting protein and negative regulator. First, yeast overexpressing BTN3 phenocopy the deletion of BTN2 and mislocalize certain trans-Golgi proteins, like Kex2 and Yif1, to the LE and vacuole, respectively. In contrast, the deletion of BTN3 results in a tighter pattern of protein localization to the Golgi. Second, BTN3 overexpression alters Btn2 localization from the IPOD compartment, which correlates with a sharp reduction in Btn2-mediated [URE3] prion curing. Third, Btn3 and the Snc1 v-SNARE compete for the same binding domain on Btn2, and this competition controls Btn2 localization and function. The inhibitory effects upon protein retrieval and prion curing suggest that Btn3 sequesters Btn2 away from its substrates, thus down-regulating protein trafficking and aggregation. Therefore Btn3 is a novel negative regulator of intracellular protein sorting, which may be of importance in the onset of complex I deficiency and Batten disease in humans.
Topics: Adaptor Proteins, Vesicular Transport; Amino Acid Transport Systems; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Endosomes; Gene Expression Regulation, Fungal; Heat-Shock Proteins; Neuronal Ceroid-Lipofuscinoses; Prions; Proprotein Convertases; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport; Qb-SNARE Proteins; R-SNARE Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Two-Hybrid System Techniques; Vesicular Transport Proteins
PubMed: 21441304
DOI: 10.1091/mbc.E10-11-0878 -
The Journal of Clinical Investigation Jun 1995Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate...
Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate limiting enzyme in lipolysis; however, direct evidence to prove this concept has been lacking. The present study was designed to establish the function of HSL in adipocytes. A 2360-bp fragment containing the entire HSL coding region was cloned into the vector pCEP4 and was used to transfect the 3T3-F442A adipogenic cell line. Nondifferentiated, transfected cells were screened for HSL overexpression by indirect immunofluorescence microscopy and confirmed by immunoblotting cell extracts with anti-HSL/fusion protein antibodies and by Northern blots for HSL mRNA. Stable transfectants overexpressing HSL were obtained and cloned. Compared with undifferentiated 3T3-F442A cells transfected with pCEP4 not containing the insert (vector alone) where HSL expression was very low, undifferentiated HSL transfectants had up to a 100-fold increase in HSL activity. Likewise, immunoreactive HSL protein and HSL mRNA levels were increased up to 100-fold in HSL transfectants. When confluent cells were allowed to differentiate by exposure to insulin, HSL expression increased in vector alone transfected cells, but remained below that observed in HSL transfectants. A similar degree of differentiation was seen in both vector alone and HSL transfectants when based on the induction of lipoprotein lipase. Cellular triglyceride content increased dramatically in the vector alone transfected cells while triglyceride content was markedly reduced in the HSL transfectants. The expression of late markers of adipocyte differentiation, such as aP2 and GPDH, was diminished and appeared to vary with the degree to which HSL was overexpressed and the cellular triglyceride content was reduced. Thus, the overexpression of HSL in 3T3-F442A cells prevents differentiated adipocytes from taking on the appearance of fat cells, i.e., accumulating triglyceride. Furthermore, the overexpression of HSL directly or indirectly attenuates the expression of several genes that appear during late adipocyte differentiation.
Topics: 3T3 Cells; Adipocytes; Animals; Carrier Proteins; Cell Differentiation; Cell Line; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Gene Expression; Lipoprotein Lipase; Mice; Neoplasm Proteins; Nerve Tissue Proteins; Phosphorylation; RNA, Messenger; Sterol Esterase; Transfection; Triglycerides
PubMed: 7769105
DOI: 10.1172/JCI117967 -
Cells Jul 2022The spindle position checkpoint (SPOC) of budding yeast delays mitotic exit in response to misaligned spindles to ensure cell survival and the maintenance of genomic...
The spindle position checkpoint (SPOC) of budding yeast delays mitotic exit in response to misaligned spindles to ensure cell survival and the maintenance of genomic stability. The GTPase-activating protein (GAP) complex Bfa1-Bub2, a key SPOC component, inhibits the GTPase Tem1 to induce mitotic arrest in response to DNA and spindle damage, as well as spindle misorientation. However, previous results strongly suggest that Bfa1 exerts a GAP-independent function in blocking mitotic exit in response to misaligned spindles. Thus, the molecular mechanism by which Bfa1 controls mitotic exit in response to misaligned spindles remains unclear. Here, we observed that overexpression of the N-terminal domain of Bfa1 (Bfa1-D16), which lacks GAP activity and cannot localize to the spindle pole body (SPB), induced cell cycle arrest along with hyper-elongation of astral microtubules (aMTs) as Bfa1 overexpression in Δ. We found that Δ cells overexpressing Bfa1 or Bfa1-D16 inhibited activation of Mob1, which is responsible for mitotic exit. In anaphase-arrested cells, Bfa1-D16 overexpression inhibited Tem1 binding to the SPB as well as Bfa1 overexpression. Additionally, endogenous levels of Bfa1-D16 showed minor SPOC activity that was not regulated by Kin4. These results suggested that Bfa1-D16 may block mitotic exit through inhibiting Tem1 activity outside of SPBs. Alternatively, Bfa1-D16 dispersed out of SPBs may block Tem1 binding to SPBs by physically interacting with Tem1 as previously reported. Moreover, we observed hyper-elongated aMTs in , and mutants that induce anaphase arrest and cannot undergo mitotic exit at restrictive temperatures, suggesting that aMT dynamics are closely related to the regulation of mitotic exit. Altogether, these observations suggest that Bfa1 can control the SPOC independent of its GAP activity and SPB localization.
Topics: Cell Cycle Proteins; Cytoskeletal Proteins; GTPase-Activating Proteins; Mitosis; Monomeric GTP-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 35883622
DOI: 10.3390/cells11142179 -
Genes & Development Jul 2002A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during...
A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during meiosis I and from centromeric regions during meiosis II. The budding yeast protein Spo13 plays a key role in preventing centromeric cohesin from being lost during meiosis I. We have determined the molecular basis for the metaphase arrest obtained when SPO13 is overexpressed during the mitotic cell cycle. Overexpression of SPO13 inhibits anaphase onset by at least two mechanisms. First, Spo13 causes a transient delay in degradation of the anaphase inhibitor Pds1. Second, Spo13 inhibits cleavage of the cohesin subunit Scc1/Mcd1 or its meiosis-specific homolog, Rec8, by the separase Esp1. The finding that Spo13 did not prevent cleavage of another Esp1 substrate, Slk19, suggests that overexpression of SPO13 is sufficient to prevent cohesin cleavage by protecting specific substrates from separase activity.
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Fungal Proteins; Hydrolysis; Nuclear Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cohesins
PubMed: 12101125
DOI: 10.1101/gad.989302 -
Journal of Neuroinflammation Jul 2021Colony-stimulating factor 1 (CSF1) expression in the central nervous system (CNS) increases in response to a variety of stimuli, and CSF1 is overexpressed in many CNS...
BACKGROUND
Colony-stimulating factor 1 (CSF1) expression in the central nervous system (CNS) increases in response to a variety of stimuli, and CSF1 is overexpressed in many CNS diseases. In young adult mice, we previously showed that CSF1 overexpression in the CNS caused the proliferation of IBA1 microglia without promoting the expression of M2 polarization markers.
METHODS
Immunohistochemical and molecular analyses were performed to further examine the impact of CSF1 overexpression on glia in both young and aged mice.
RESULTS
As CSF1 overexpressing mice age, IBA1 cell numbers are constrained by a decline in proliferation rate. Compared to controls, there were no differences in expression of the M2 markers ARG1 and MRC1 (CD206) in CSF1 overexpressing mice of any age, indicating that even prolonged exposure to increased CSF1 does not impact M2 polarization status in vivo. Moreover, RNA-sequencing confirmed the lack of increased expression of markers of M2 polarization in microglia exposed to CSF1 overexpression but did reveal changes in expression of other immune-related genes. Although treatment with inhibitors of the CSF1 receptor, CSF1R, has been shown to impact other glia, no increased expression of oligodendrocyte lineage or astrocyte markers was observed in CSF1 overexpressing mice.
CONCLUSIONS
Our study indicates that microglia are the primary glial lineage impacted by CSF1 overexpression in the CNS and that microglia ultimately adapt to the presence of the CSF1 mitogenic signal.
Topics: Animals; Arginase; Calcium-Binding Proteins; Cell Lineage; Gliosis; Immunohistochemistry; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Microfilament Proteins; Neuroglia; Receptors, Immunologic; Sequence Analysis, RNA; Signal Transduction
PubMed: 34281564
DOI: 10.1186/s12974-021-02212-0 -
Environmental Science and Pollution... May 2019Abiotic stress can influence the interactions between a pathogen and its host. In this paper, we analyzed the effects of salicylic acid (SA) and pH on the morphological...
Abiotic stress can influence the interactions between a pathogen and its host. In this paper, we analyzed the effects of salicylic acid (SA) and pH on the morphological development and pathogenicity of Magnaporthe oryzae, the pathogen that causes rice (Oryza sativa) blast. A strain of rice blast that overexpresses biotrophy-associated secreted protein 1 (BAS1) and a wild-type (WT) strain were pretreated with different levels of pH and different concentrations of SA to analyze M. oryzae colony growth, sporulation, spore germination, dry weight of hypha, and appressorium formation. Disease incidence and the expression of defense-related genes in infected rice were analyzed after pretreatment with pH 5.00 or pH 8.00 and 200 μM SA. The results showed that both SA and pH had some influence on morphological development, including sporulation and appressorium formation of the BAS1-overexpression strain. In the 200 μM SA pretreatment, there was a lower incidence of disease and higher expression levels of the rice defense-related genes PR1a, PAL, HSP90, and PR5 on leaves inoculated with the BAS1-overexpession strain compared with the WT strain, whereas, LOX2 appeared to be downregulated in the BAS1-overexpession strain compared with the WT. In both pH treatments, disease incidence and expression of HSP90 were higher and the expression of PR1a and PR10a and LOX2 and PAL was lower in leaves inoculated with the BAS1-overexpression strain compared with leaves inoculated with the WT strain. We conclude that SA and pH affect morphological development of the BAS1-overexpression blast strain, but that these factors have little influence on the pathogenicity of the strain, indicating that BAS1-overexpression may have enhanced the tolerance of this rice blast strain to abiotic stressors. This work suggests new molecular mechanisms that exogenous SA and pH affect the interactions between M. oryzae and rice.
Topics: Fungal Proteins; Gene Expression Regulation, Plant; Host-Pathogen Interactions; Hydrogen-Ion Concentration; Magnaporthe; Oryza; Plant Diseases; Plant Leaves; Plant Proteins; Salicylic Acid; Spores, Fungal
PubMed: 29931642
DOI: 10.1007/s11356-018-2532-y