Publications by authors named "Kwang-Lae Hoe"

63 Publications

CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.

PLoS One 2021 16;16(4):e0246264. Epub 2021 Apr 16.

Department of Bioactive Material Sciences, Jeonbuk National University, Jeonju, Jeollabuk-do, Republic of Korea.

Tamoxifen (TAM) is a selective estrogen receptor modulator used for breast cancer patients. Prolonged use of tamoxifen is not recommended for some patients. In this study, we aimed to identify molecular targets sensitive to TAM using a genome-wide gene deletion library screening of fission yeast heterozygous mutants. From the screening, casein kinase 1 gamma 2 (CSNK1G2), a serine-/threonine protein kinase, was the most sensitive target to TAM with a significant cytotoxicity in estrogen receptor-positive (ER+) breast cancer cells but with only a slight toxicity in the case of ER- cells. In addition, tumor sphere formation and expression of breast stem cell marker genes such as CD44/CD2 were greatly inhibited by CSNK1G2 knockdown in ER+ breast cancer cells. Consistently, CSNK1G2 altered ERα activity via phosphorylation, specifically at serine (Ser)167, as well as the regulation of estrogen-responsive element (ERE) of estrogen-responsive genes such as CTSD and GREB1. However, ERα silencing almost completely blocked CSNK1G2-induced TAM sensitivity. In ER+ breast cancer cells, combined treatment with TAM and CSNK1G2 knockdown further enhanced the TAM-mediated decrease in phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) signaling but not extracellular signal-regulated kinase (ERK) signaling. Inversely, in ER- cells treated with TAM, only ERK and PI3K signaling was altered by CSNK1G2 knockdown. The CK1 inhibitor, D4476, partly mimicked the CSNK1G2 knockdown effect in ER+ breast cancer cells, but with a broader repression ranging from PI3K/AKT/mTOR/S6K to ERK signaling. Collectively, these results suggest that CSNK1G2 plays a key role in sensitizing TAM toxicity in ER+ and ER- breast cancer cells via differently regulating PI3K/AKT/mTOR/S6K and ERK signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246264PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051802PMC
April 2021

Systematic Target Screening Revealed That Tif302 Could Be an Off-Target of the Antifungal Terbinafine in Fission Yeast.

Biomol Ther (Seoul) 2021 03;29(2):234-247

Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea.

We used a heterozygous gene deletion library of fission yeasts comprising all essential and non-essential genes for a microarray screening of target genes of the antifungal terbinafine, which inhibits ergosterol synthesis via the Erg1 enzyme. We identified 14 heterozygous strains corresponding to 10 non-essential [7 ribosomal-protein (RP) coding genes, , , and ] and 4 essential genes (, , , and ). Expectedly, their mRNA and protein levels had decreased compared to the control strain SP286. When we studied the action mechanism of the non-essential target genes using cognate haploid deletion strains, knockout of SAGA-subunit genes caused a down-regulation in transcription compared to the control strain ED668. However, knockout of RP genes conferred no susceptibility to ergosterol-targeting antifungals. Surprisingly, the RP genes participated in the transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of mRNA. To understand the action mechanism of the interaction between the drug and the novel essential target genes, we performed isobologram assays with terbinafine and econazole (or cycloheximide). Terbinafine susceptibility of the heterozygous strain was attributed to both decreased mRNA levels and inhibition of translation. Moreover, Tif302 was required for efficacy of both terbinafine and cycloheximide. Based on a molecular modeling analysis, terbinafine could directly bind to Tif302 in yeasts, suggesting Tif302 as a potential off-target of terbinafine. In conclusion, this genome-wide screening system can be harnessed for the identification and characterization of target genes under any condition of interest.
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http://dx.doi.org/10.4062/biomolther.2020.166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921855PMC
March 2021

A randomized, open-label, crossover study to compare the safety and pharmacokinetics of two tablet formulations of tenofovir (tenofovir disoproxil and tenofovir disoproxil fumarate) in healthy subjects.

Int J Clin Pharmacol Ther 2020 Dec;58(12):749-756

Purpose: This study was performed to compare the pharmacokinetic properties and assess bioequivalence for the test formulation (HUG116 tablet; tenofovir disoproxil) and reference formulation (Viread tablet; tenofovir disoproxil fumarate).

Materials And Methods: A randomized, open-label, single-dosing, two-treatment, two-period, two-sequence cross-over study was conducted in 50 healthy subjects. All subjects were randomly assigned to one of the two sequences, and they received a single dose of test or reference formulation in the first period and the alternative formulation during the next period under fasting conditions. Serial blood samples for pharmacokinetic evaluation were collected up to 72 hours post dose, and the pharmacokinetic parameters were estimated by noncompartmental methods. Throughout the study, tolerability was assessed based on adverse events, vital signs, and clinical laboratory tests.

Results: The test formulation showed similar pharmacokinetic profiles to those of the reference formulation. The geometric mean ratio and 90% confidence interval (CI) of the test formulation to the reference formulation for maximum plasma concentration (C) was 0.93 (0.87 - 0.99), and the corresponding value for the area under the concentration-time curve from time zero to time of last quantifiable concentration (AUC) was 0.94 (0.89 - 0.99). Both CIs were within the conventional bioequivalence range of 0.8 - 1.25. The tolerability profile was not significantly different between the test and reference formulations.

Conclusion: This study found that the PKs of the test formulation (HUG116 tablet; tenofovir disoproxil) and reference formulation (Viread tablet; tenofovir disoproxil fumarate) were similar, and the test formulation met the regulatory criteria for assuming bioequivalence with the reference formulation.
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http://dx.doi.org/10.5414/CP203651DOI Listing
December 2020

Co-treatment of birinapant with TRAIL synergistically induces apoptosis by downregulating cFLIP(L) in MDA-MB-453 cell lines.

Biochem Biophys Res Commun 2020 12 18;533(3):289-295. Epub 2020 Sep 18.

Rare Disease Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea. Electronic address:

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received much attention owing to its ability to specifically induce cell death in cancer. However, several types of cancer, including some forms of breast cancer, are resistant to TRAIL. Various chemotherapeutic agents, phytochemicals, and TRAIL combination therapies have been proposed to resolve TRAIL resistance. Here, we explored the sensitization effect of birinapant on TRAIL-induced apoptosis in the MDA-MB-453 cell line. Although neither birinapant nor TRAIL showed any cytotoxic effect when used alone, apoptosis was induced when birinapant and TRAIL were used together. Our data suggest that the combination of birinapant and TRAIL induces downregulation of FLICE-like inhibitory protein (cFLIP) (L) protein expression. Interestingly, cFLIP(L) overexpression reversed apoptosis caused by co-treatment with TRAIL. Taken together, our results indicate that a combination of birinapant and TRAIL may be a promising treatment for TRAIL-resistant breast cancer.
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http://dx.doi.org/10.1016/j.bbrc.2020.09.031DOI Listing
December 2020

Bacteria-derived metabolite, methylglyoxal, modulates the longevity of through TORC2/SGK-1/DAF-16 signaling.

Proc Natl Acad Sci U S A 2020 07 7;117(29):17142-17150. Epub 2020 Jul 7.

Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Korea;

Gut microbes play diverse roles in modulating host fitness, including longevity; however, the molecular mechanisms underlying their mediation of longevity remain poorly understood. We performed genome-wide screens using 3,792 mutants and identified 44 mutants that modulated longevity. Three of these mutants modulated longevity via the bacterial metabolite methylglyoxal (MG). Importantly, we found that low MG-producing mutants, , extended the lifespan of through activation of the DAF-16/FOXO family transcription factor and the mitochondrial unfolded protein response (UPR). Interestingly, the lifespan modulation by did not require insulin/insulin-like growth factor 1 signaling (IIS) but did require TORC2/SGK-1 signaling. Transcriptome analysis revealed that activated novel class 3 DAF-16 target genes that were distinct from those regulated by IIS. Taken together, our data suggest that bacteria-derived MG modulates host longevity through regulation of the host signaling pathways rather than through nonspecific damage on biomolecules known as advanced glycation end products. Finally, we demonstrate that MG enhances the phosphorylation of hSGK1 and accelerates cellular senescence in human dermal fibroblasts, suggesting the conserved role of MG in controlling longevity across species. Together, our studies demonstrate that bacteria-derived MG is a novel therapeutic target for aging and aging-associated pathophysiology.
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http://dx.doi.org/10.1073/pnas.1915719117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382248PMC
July 2020

Econazole Induces p53-Dependent Apoptosis and Decreases Metastasis Ability in Gastric Cancer Cells.

Biomol Ther (Seoul) 2020 Jul;28(4):370-379

Rare Disease Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.

Econazole, a potent broad-spectrum antifungal agent and a Ca channel antagonist, induces cytotoxicity in leukemia cells and is used for the treatment of skin infections. However, little is known about its cytotoxic effects on solid tumor cells. Here, we investigated the molecular mechanism underlying econazole-induced toxicity and evaluated its regulatory effect on the metastasis of gastric cancer cells. Using the gastric cancer cell lines AGS and SNU1 expressing wild-type p53 we demonstrated that econazole could significantly reduce cell viability and colony-forming (tumorigenesis) ability. Econazole induced G0/G1 phase arrest, promoted apoptosis, and effectively blocked proliferation- and survival-related signal transduction pathways in gastric cancer cells. In addition, econazole inhibited the secretion of matrix metalloproteinase- 2 (MMP-2) and MMP-9, which degrade the extracellular matrix and basement membrane. Econazole also effectively inhibited the metastasis of gastric cancer cells, as confirmed from cell invasion and wound healing assays. The protein level of p53 was significantly elevated after econazole treatment of AGS and SNU1 cells. However, apoptosis was blocked in econazole-treated cells exposed to a p53-specific small-interfering RNA to eliminate p53 expression. These results provide evidence that econazole could be repurposed to induce gastric cancer cell death and inhibit cancer invasion.
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http://dx.doi.org/10.4062/biomolther.2019.201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327138PMC
July 2020

Optimization of a microarray for fission yeast.

Genomics Inform 2019 Sep 18;17(3):e28. Epub 2019 Sep 18.

Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea.

Bar-code (tag) microarrays of yeast gene-deletion collections facilitate the systematic identification of genes required for growth in any condition of interest. Anti-sense strands of amplified bar-codes hybridize with ~10,000 (5,000 each for up- and down-tags) different kinds of sense-strand probes on an array. In this study, we optimized the hybridization processes of an array for fission yeast. Compared to the first version of the array (11 µm, 100K) consisting of three sectors with probe pairs (perfect match and mismatch), the second version (11 µm, 48K) could represent ~10,000 up-/down-tags in quadruplicate along with 1,508 negative controls in quadruplicate and a single set of 1,000 unique negative controls at random dispersed positions without mismatch pairs. For PCR, the optimal annealing temperature (maximizing yield and minimizing extra bands) was 58°C for both tags. Intriguingly, up-tags required 3 higher amounts of blocking oligonucleotides than down-tags. A 1:1 mix ratio between up- and down-tags was satisfactory. A lower temperature (25°C) was optimal for cultivation instead of a normal temperature (30°C) because of extra temperature-sensitive mutants in a subset of the deletion library. Activation of frozen pooled cells for >1 day showed better resolution of intensity than no activation. A tag intensity analysis showed that tag(s) of 4,316 of the 4,526 strains tested were represented at least once; 3,706 strains were represented by both tags, 4,072 strains by up-tags only, and 3,950 strains by down-tags only. The results indicate that this microarray will be a powerful analytical platform for elucidating currently unknown gene functions.
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http://dx.doi.org/10.5808/GI.2019.17.3.e28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6808646PMC
September 2019

Arginase II activity regulates cytosolic Ca level in a p32-dependent manner that contributes to Ca-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells.

Exp Mol Med 2019 06 3;51(6):1-12. Epub 2019 Jun 3.

Department of Biology, School of Medicine, Kangwon National University, Chuncheon, 24341, South Korea.

Although arginase II (ArgII) is abundant in mitochondria, Ca-accumulating organelles, the relationship between ArgII activity and Ca translocation into mitochondria and the regulation of cytosolic Ca signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca]m as measured by CoCl-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII mice. Conversely, [Ca]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.
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http://dx.doi.org/10.1038/s12276-019-0262-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545325PMC
June 2019

Resveratrol is an arginase inhibitor contributing to vascular smooth muscle cell vasoconstriction via increasing cytosolic calcium.

Mol Med Rep 2019 May 14;19(5):3767-3774. Epub 2019 Mar 14.

Department of Biology, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea.

The contractility of vascular smooth muscle cells (VSMCs) controls the lumen diameter of vessels, thus serving a role in regulating blood pressure and organ blood flow. Although arginases are known to have numerous effects in the biological activities of VSMCs, the effects of arginase II on the constriction of VSMCs has not yet been investigated. When conducting a natural products screen for an inhibitor against arginase, the present study identified that a relatively high concentration of resveratrol (RSV) exhibited arginase inhibitory activity. Therefore, the present study investigated whether RSV could regulate VSMCs contractions and the underlying mechanism. Arginase inhibition by RSV led to an increase in the concentration of the substrate L‑Arg and an accompanying increase in the cytosol Ca2+ concentration [(Ca2+)c] in VSMCs. The increased [Ca2+]c induced by RSV and L‑Arg treatments resulted in CaMKII‑dependent MLC20 phosphorylation. The effects of RSV on VSMCs were maintained even when VSMCs were pre‑treated with sirtinol, an inhibitor of Sirt proteins. In a vascular tension assay with de‑endothelialized aortic vessels, vasoconstrictor responses, which were measured using phenylephrine (PE), were significantly enhanced in the RSV‑ and L‑Arg‑treated vessels. Therefore, although arginase inhibition has exhibited beneficial effects in various diseases, care is required when considering administration of an arginase inhibitor to patients with vessels endothelial dysfunction as RSV can induce vessel contraction.
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http://dx.doi.org/10.3892/mmr.2019.10035DOI Listing
May 2019

Arginase II Contributes to the Ca/CaMKII/eNOS Axis by Regulating Ca Concentration Between the Cytosol and Mitochondria in a p32-Dependent Manner.

J Am Heart Assoc 2018 09;7(18):e009579

1 Department of Biology School of medicine Kangwon National University Chuncheon Korea.

Background Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase ( eNOS ). We tested the hypotheses that arginase II activity participates in the regulation of Ca/Ca/calmodulin-dependent kinase II / eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca ([Ca]c) and decreased mitochondrial Ca ([Ca]m) in microscopic and fluorescence-activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca/Ca/calmodulin-dependent kinase II . These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II -null mice resulted in reduced p32 protein level. In the aortas of wild-type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO -dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca]m were increased in the aortas from apolipoprotein E (ApoE) mice fed a high-cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca/Ca/calmodulin-dependent kinase II -dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca-dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.
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http://dx.doi.org/10.1161/JAHA.118.009579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222941PMC
September 2018

Mutation Analysis of Synthetic DNA Barcodes in a Fission Yeast Gene Deletion Library by Sanger Sequencing.

Genomics Inform 2018 Jun 30;16(2):22-29. Epub 2018 Jun 30.

Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea.

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (∼28%) and well above the predicted error of Sanger sequencing (∼2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.
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http://dx.doi.org/10.5808/GI.2018.16.2.22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187811PMC
June 2018

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.

Yonsei Med J 2018 May;59(3):366-375

Department of Biological Sciences, Kangwon National University, Chuncheon, Korea.

Purpose: Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms.

Materials And Methods: Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin.

Results: Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation.

Conclusion: Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.
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http://dx.doi.org/10.3349/ymj.2018.59.3.366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889988PMC
May 2018

Arginase II inhibition prevents interleukin-8 production through regulation of p38 MAPK phosphorylation activated by loss of mitochondrial membrane potential in nLDL-stimulated hAoSMCs.

Exp Mol Med 2018 02 2;50(2):e438. Epub 2018 Feb 2.

Department of Biological Sciences, Kangwon National University, Chuncheon, Republic of Korea.

Arginase inhibition exhibits beneficial effects in vascular endothelial and smooth muscle cells. In human aortic smooth muscle cells (hAoSMCs), native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. Therefore, we examined the effect of arginase inhibition on IL-8 production and the underlying mechanism. In hAoSMCs, reverse transcription-PCR, western blotting and immunocytochemistry with MitoTracker confirmed that arginase II was confined predominantly to mitochondria. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine ethyl ester. The MMP decreased upon nLDL stimulation but was restored upon arginase inhibition. MMP loss caused by nLDL was prevented by treatment with the intracellular Ca chelator BAPTA-AM. In mitochondrial Ca measurements using Rhod-2 AM, increased mitochondrial Ca levels by nLDL were inhibited upon preincubation with an arginase inhibitor. Among the polyamines, spermine, an arginase activity-dependent product, caused mitochondrial Ca movement. The nLDL-induced MMP change resulted in p38 mitogen-activated protein kinase (MAPK) phosphorylation and IL-8 production and was prevented by the arginase inhibitors BAPTA and ruthenium 360. In isolated AoSMCs from ApoE mice fed a high-cholesterol diet, arginase activity, p38 MAPK phosphorylation, spermine and mitochondrial Ca levels and keratinocyte-derived chemokine (KC) production were increased compared with wild-type (WT) mice. However, in AoSMCs isolated from arginase II-null mice, increases in MMP and decreases in mitochondrial Ca levels were noted compared with WT and were associated with p38 MAPK activation and IL-8 production. These data suggest that arginase activity regulates the change in MMP through Ca uptake that is essential for p38 MAPK phosphorylation and IL-8 production.
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http://dx.doi.org/10.1038/emm.2017.254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903817PMC
February 2018

Editor's Highlight: A Genome-wide Screening of Target Genes Against Silver Nanoparticles in Fission Yeast.

Toxicol Sci 2018 01;161(1):171-185

Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea.

To identify target genes against silver nanoparticles (AgNPs), we screened a genome-wide gene deletion library of 4843 fission yeast heterozygous mutants covering 96% of all protein encoding genes. A total of 33 targets were identified by a microarray and subsequent individual confirmation. The target pattern of AgNPs was more similar to those of AgNO3 and H2O2, followed by Cd and As. The toxic effect of AgNPs on fission yeast was attributed to the intracellular uptake of AgNPs, followed by the subsequent release of Ag+, leading to the generation of reactive oxygen species (ROS). Next, we focused on the top 10 sensitive targets for further studies. As described previously, 7 nonessential targets were associated with detoxification of ROS, because their heterozygous mutants showed elevated ROS levels. Three novel essential targets were related to folate metabolism or cellular component organization, resulting in cell cycle arrest and no induction in the transcriptional level of antioxidant enzymes such as Sod1 and Gpx1 when 1 of the 2 copies was deleted. Intriguingly, met9 played a key role in combating AgNP-induced ROS generation via NADPH production and was also conserved in a human cell line.
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http://dx.doi.org/10.1093/toxsci/kfx208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837777PMC
January 2018

Genome-wide functional analysis using the barcode sequence alignment and statistical analysis (Barcas) tool.

BMC Bioinformatics 2016 Dec 23;17(Suppl 17):475. Epub 2016 Dec 23.

Korea Research Institute of Bioscience and Biotechnology (KRIBB), Personalized Genomic Medicine Research Center, Daejeon, Republic of Korea.

Background: Pooled library screen analysis using shRNAs or CRISPR-Cas9 hold great promise to genome-wide functional studies. While pooled library screens are effective tools, erroneous barcodes can potentially be generated during the production of many barcodes. However, no current tools can distinguish erroneous barcodes from PCR or sequencing errors in a data preprocessing step.

Results: We developed the Barcas program, a specialized program for the mapping and analysis of multiplexed barcode sequencing (barcode-seq) data. For fast and efficient mapping, Barcas uses a trie data structure based imperfect matching algorithm which generates precise mapping results containing mismatches, shifts, insertions and deletions (indel) in a flexible manner. Barcas provides three functions for quality control (QC) of a barcode library and distinguishes erroneous barcodes from PCR or sequencing errors. It also provides useful functions for data analysis and visualization.

Conclusions: Barcas is an all-in-one package providing useful functions including mapping, data QC, library QC, statistical analysis and visualization in genome-wide pooled screens.
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http://dx.doi.org/10.1186/s12859-016-1326-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5260075PMC
December 2016

Gene Deletion by Synthesis in Yeast.

Methods Mol Biol 2017 ;1472:169-85

Department of New Drug Discovery and Development, Chungnam National University, 125 Gwahak-ro, Daejeon, 34141, South Korea.

Targeted gene deletion is a useful tool for understanding the function of a gene and its protein product. We have developed an efficient and robust gene deletion approach in yeast that employs oligonucleotide-based gene synthesis. This approach requires a deletion cassette composed of three modules: a central 1397-bp KanMX4 selection marker module and two 366-bp gene-specific flanking modules. The invariable KanMX4 module can be used in combination with different pairs of flanking modules targeting different genes. The two flanking modules consist of both sequences unique to each cassette (chromosomal homologous regions and barcodes) and those common to all deletion constructs (artificial linkers and restriction enzyme sites). Oligonucleotides for each module and junction regions are designed using the BatchBlock2Oligo program and are synthesized on a 96-well basis. The oligonucleotides are ligated into a single deletion cassette by ligase chain reaction, which is then amplified through two rounds of nested PCR to obtain sufficient quantities for yeast transformation. After removal of the artificial linkers, the deletion cassettes are transformed into wild-type diploid fission yeast SP286 cells. Verification of correct clone and gene deletion is achieved by performing check PCR and tetrad analysis. This method with proven effectiveness, as evidenced by a high success rate of gene deletion, can be potentially applicable to create systematic gene deletion libraries in a variety of yeast species.
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http://dx.doi.org/10.1007/978-1-4939-6343-0_13DOI Listing
January 2018

Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation.

Yonsei Med J 2016 Nov;57(6):1329-38

Department of Biology, College of Natural Sciences, School of Medicine, Kangwon National University, Chuncheon, Korea.

Purpose: Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism.

Materials And Methods: Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay.

Results: SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. N(G)-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition.

Conclusion: These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.
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http://dx.doi.org/10.3349/ymj.2016.57.6.1329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011263PMC
November 2016

The effect of the cwf14 gene of fission yeast on cell wall integrity is associated with rho1.

J Microbiol 2016 Feb 2;54(2):98-105. Epub 2016 Feb 2.

Department of New Drug Discovery and Development, Chungnam National University, Daejeon, 305-764, Republic of Korea.

In all eukaryotic organisms, a wide range of morphologies are responsible for critical cellular function and development. In particular, the Rho GTPases, which are highly conserved from yeast to mammals, are key molecules in signaling pathways that control cell polarity processes and cell wall biosynthesis, which are fundamental aspects of morphogenesis. Therefore, using haploinsufficiency deletion mutants of the fission yeast Schizosaccharomyces pombe, we screened the slow-growing mutants and their morphogenesis, specifically focusing on regulation of their Rho GTPases. Based on this screening, we found that the cwf14 mutant of S. pombe exhibited the slow growth and abnormal phenotypes with an elongated cell shape and thicker cell wall when compared with wild-type cells. In particular, cells with the cwf14 deletion showed excessive Rho1 expression. However, the wildtype strain with ectopically expressed Rho1 did not exhibited any significant change in the level of cwf14, suggesting that cwf14 may act on the upstream of Rho1. Furthermore, the cells with a cwf14 deletion also have increased sensitivity to β-glucanase, a cell wall-digesting enzyme, which is also seen in Rho1-overexpressing cells. Overall, our results suggest that the cwf14 plays a key role in fission yeast morphogenesis and cell wall biosynthesis and/or degradation possibly via the regulation of Rho1 expression.
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http://dx.doi.org/10.1007/s12275-016-5569-yDOI Listing
February 2016

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast.

Biochem Biophys Res Commun 2015 Dec 3;468(4):606-10. Epub 2015 Nov 3.

Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. Electronic address:

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs.
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http://dx.doi.org/10.1016/j.bbrc.2015.10.165DOI Listing
December 2015

A Novel Arginase Inhibitor Derived from Scutellavia indica Restored Endothelial Function in ApoE-Null Mice Fed a High-Cholesterol Diet.

J Pharmacol Exp Ther 2015 Oct 11;355(1):57-65. Epub 2015 Aug 11.

Departments of Biological Sciences (H.M.H., S.R.) and Biochemistry (J.H.L.), College of Natural Sciences, and Departments of Molecular and Cellular Biochemistry (Y.M.K.), School of Medicine, Kangwon National University, Chuncheon, Gangwon-do; College of Pharmacy, Catholic University, Daegu (B.S.M.); Infectious Signaling Network Research Center, Department of Physiology, School of Medicine, (B.H.J.) and Department of New Drug Discovery and Development (K.L.H.), Chungnam National University, Daejeon, South Korea

Elevated endothelial arginase activity decreases nitric oxide (NO) production by competing with the substrate l-arginine, previously reported, and reciprocally regulating endothelial nitric oxide synthase (eNOS) activity. Thus, arginase inhibitors may help treat vascular diseases associated with endothelial dysfunction. A screening of metabolites from medicinal plants revealed that (2S)-5,2',5'-trihydroxy-7,8-dimethoxy flavanone (TDF) was a noncompetitive inhibitor of arginase. We investigated whether TDF reciprocally regulated endothelial NO production and its possible mechanism. TDF noncompetitively inhibited arginase I and II activity in a dose-dependent manner. TDF incubation decreased arginase activity and increased NO production in human umbilical vein endothelial cells and isolated mouse aortic vessels and reduced reactive oxygen species (ROS) generation in the endothelium of the latter. These TDF-mediated effects were associated with increased eNOS phosphorylation and dimerization but not with changes in protein content. Endothelium-dependent vasorelaxant responses to acetylcholine (Ach) were significantly increased in TDF-incubated aortic rings and attenuated by incubation with soluble guanylyl cyclase inhibitor. Phenylephrine-induced vasoconstrictor responses were markedly attenuated in TDF-treated vessels from wild-type mice. In atherogenic-prone ApoE(-/-) mice, TDF attenuated the high-cholesterol diet (HCD)-induced increase in arginase activity, which was accompanied by restoration of NO production and reduction of ROS generation. TDF incubation induced eNOS dimerization and phosphorylation at Ser1177. In addition, TDF improved Ach-dependent vasorelaxation responses and attenuated U46619-dependent contractile responses but did not change sodium nitroprusside-induced vasorelaxation or N-NAME-induced vasoconstriction. The findings suggest that TDF may help treat cardiovascular diseases by reducing pathophysiology derived from HCD-mediated endothelial dysfunction.
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http://dx.doi.org/10.1124/jpet.115.224592DOI Listing
October 2015

NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells.

Exp Mol Med 2015 Jun 12;47:e168. Epub 2015 Jun 12.

Department of Biology, College of Natural Sciences, Kangwon National University, Chuncheon, Korea.

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox(-/-) mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.
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http://dx.doi.org/10.1038/emm.2015.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491723PMC
June 2015

Systematic targeted gene deletion using the gene-synthesis method in fission yeast.

J Microbiol Methods 2014 Nov 20;106:72-77. Epub 2014 Aug 20.

Department of New Drug Discovery and Development, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea. Electronic address:

Genome-wide targeted gene deletion, a systematic method to study gene function by replacing target genes with deletion cassettes, using serial-PCR or block-PCR requires elaborate skill. We developed a novel gene-synthesis method to systematically prepare deletion cassettes on a 96-well basis in fission yeast. We designed the 2129-bp deletion cassette as three modules: a central 1397-bp KanMX4 selection marker module and two flanking 366-bp gene-specific artificial linker modules. The central KanMX4 module can be used in multiple deletion cassettes in combination with different sets of flanking modules. The deletion cassettes consisted of 147 oligonucleotides (93 for the central module+25 for each of the flanking modules+4 for the joints) and the oligonucleotides were designed as ~29mers using an in-house program. Oligonucleotides were synthesized on a 96-well basis and ligated into deletion cassettes without gaps by ligase chain reaction, which was followed by two rounds of nested PCR to amplify trace amounts of the ligated cassettes. After the artificial linkers were removed from the deletion cassettes, the cassettes were transformed into wild-type diploid fission yeast strain SP286. We validated the transformed colonies via check PCR and subjected them to tetrad analysis to confirm functional integrity. Using this method, we systematically deleted 563 genes in the fission yeast Schizosaccharomyces pombe with a >90% success rate and a point-mutation rate of ~0.4 mutations per kb. Our method can be used to create systematic gene deletions in a variety of yeasts especially when it included a bar-code system for parallel analyses.
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http://dx.doi.org/10.1016/j.mimet.2014.08.005DOI Listing
November 2014

Transactivation of bad by vorinostat-induced acetylated p53 enhances doxorubicin-induced cytotoxicity in cervical cancer cells.

Exp Mol Med 2014 Feb 14;46:e76. Epub 2014 Feb 14.

Department of New Drug Discovery and Development, Chungnam National University, Daejeon, Republic of Korea.

Vorinostat (VOR) has been reported to enhance the cytotoxic effects of doxorubicin (DOX) with fewer side effects because of the lower DOX dosage in breast cancer cells. In this study, we investigated the novel mechanism underlying the synergistic cytotoxic effects of VOR and DOX co-treatment in cervical cancer cells HeLa, CaSki and SiHa cells. Co-treatment with VOR and DOX at marginal doses led to the induction of apoptosis through caspase-3 activation, poly (ADP-ribose) polymerase cleavage and DNA micronuclei. Notably, the synergistic growth inhibition induced by the co-treatment was attributed to the upregulation of the pro-apoptotic protein Bad, as the silencing of Bad expression using small interfering RNA (siRNA) abolished the phenomenon. As siRNA against p53 did not result in an increase in acetylated p53 and the consequent upregulation of Bad, the observed Bad upregulation was mediated by acetylated p53. Moreover, a chromatin immunoprecipitation analysis showed that the co-treatment of HeLa cells with VOR and DOX increased the recruitment of acetylated p53 to the bad promoter, with consequent bad transactivation. Conversely, C33A cervical cancer cells containing mutant p53 co-treated with VOR and DOX did not exhibit Bad upregulation, acetylated p53 induction or consequent synergistic growth inhibition. Together, the synergistic growth inhibition of cervical cancer cell lines induced by co-treatment with VOR and DOX can be attributed to the upregulation of Bad, which is induced by acetylated p53. These results show for the first time that the acetylation of p53, rather than histones, is a mechanism for the synergistic growth inhibition induced by VOR and DOX co-treatments.
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http://dx.doi.org/10.1038/emm.2013.149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944441PMC
February 2014

Integration profiling of gene function with dense maps of transposon integration.

Genetics 2013 Oct 26;195(2):599-609. Epub 2013 Jul 26.

Section on Eukaryotic Transposable Elements, Program in Cellular Regulation and Metabolism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.

Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average, we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.
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http://dx.doi.org/10.1534/genetics.113.152744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781984PMC
October 2013

Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach.

Biochem Biophys Res Commun 2013 Jul 10;436(4):613-8. Epub 2013 Jun 10.

Bioinformatics Lab, Healthcare Group, SK Telecom, 9-1, Sunae-dong, Pundang-gu, Sungnam-si, Kyunggi-do 463-784, Republic of Korea.

Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at http://pombe.kaist.ac.kr/compendium.
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http://dx.doi.org/10.1016/j.bbrc.2013.05.138DOI Listing
July 2013

A genome-wide resource of cell cycle and cell shape genes of fission yeast.

Open Biol 2013 May 22;3(5):130053. Epub 2013 May 22.

Cell Cycle Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.
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http://dx.doi.org/10.1098/rsob.130053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866870PMC
May 2013

The fission yeast GATA factor, Gaf1, modulates sexual development via direct down-regulation of ste11+ expression in response to nitrogen starvation.

PLoS One 2012 10;7(8):e42409. Epub 2012 Aug 10.

Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon, Korea.

Gaf1 is the first GATA family zinc-finger transcription factor identified in Schizosaccharomyces pombe. Here, we report that Gaf1 functions as a negatively acting transcription factor of ste11(+), delaying the entrance of cells exposed to transient nitrogen starvation into the meiotic cycle. gaf1Δ strains exhibited accelerated G(1)-arrest upon nitrogen starvation. Moreover, gaf1Δ mutation caused increased mating and sporulation frequency under both nitrogen-starved and unstarved conditions, while overexpression of gaf1(+) led to a significant impairment of sporulation. By microarray analysis, we found that approximately 63% (116 genes) of the 183 genes up-regulated in unstarved gaf1Δ cells were nitrogen starvation-responsive genes, and furthermore that 25 genes among the genes up-regulated by gaf1Δ mutation are Ste11 targets (e.g., gpa1(+), ste4(+), spk1(+), ste11(+), and mei2(+)). The phenotype caused by gaf1Δ mutation was masked by ste11Δ mutation, indicating that ste11(+) is epistatic to gaf1(+) with respect to sporulation efficiency, and accordingly that gaf1(+) functions upstream of ste11(+) in the signaling pathway governing sexual development. gaf1Δ strains showed accelerated ste11(+) expression under nitrogen starvation and increased ste11(+) expression even under normal conditions. Electrophoretic mobility shift assay analysis demonstrated that Gaf1 specifically binds to the canonical GATA motif (5'-HGATAR-3') spanning from -371 to -366 in ste11(+) promoter. Consequently, Gaf1 provides the prime example for negative regulation of ste11(+) transcription through direct binding to a cis-acting motif of its promoter.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042409PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416868PMC
March 2013

The CCR4-NOT complex is implicated in the viability of aneuploid yeasts.

PLoS Genet 2012 21;8(6):e1002776. Epub 2012 Jun 21.

Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability.
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http://dx.doi.org/10.1371/journal.pgen.1002776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380822PMC
September 2012

Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B.

Mol Biol Cell 2011 Dec 30;22(23):4486-502. Epub 2011 Sep 30.

Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.

Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.
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http://dx.doi.org/10.1091/mbc.E11-07-0608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226469PMC
December 2011

Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

Mol Cell 2011 Apr;42(2):160-71

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.
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http://dx.doi.org/10.1016/j.molcel.2011.02.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083633PMC
April 2011