Publications by authors named "Prakash Arumugam"

29 Publications

  • Page 1 of 1

Identification of pathways modulating vemurafenib resistance in melanoma cells via a genome-wide CRISPR/Cas9 screen.

G3 (Bethesda) 2021 Feb;11(2)

Bioinformatics Institute (BII), A*STAR, Singapore 138671, Singapore.

Vemurafenib is a BRAF kinase inhibitor (BRAFi) that is used to treat melanoma patients harboring the constitutively active BRAF-V600E mutation. However, after a few months of treatment patients often develop resistance to vemurafenib leading to disease progression. Sequence analysis of drug-resistant tumor cells and functional genomic screens has identified several genes that regulate vemurafenib resistance. Reactivation of mitogen-activated protein kinase (MAPK) pathway is a recurrent feature of cells that develop resistance to vemurafenib. We performed a genome-scale CRISPR-based knockout screen to identify modulators of vemurafenib resistance in melanoma cells with a highly improved CRISPR sgRNA library called Brunello. We identified 33 genes that regulate resistance to vemurafenib out of which 14 genes have not been reported before. Gene ontology enrichment analysis showed that the hit genes regulate histone modification, transcription and cell cycle. We discuss how inactivation of hit genes might confer resistance to vemurafenib and provide a framework for follow-up investigations.
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http://dx.doi.org/10.1093/g3journal/jkaa069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022920PMC
February 2021

Metagenomic analysis of microbial community and its role in bioelectrokinetic remediation of tannery contaminated soil.

J Hazard Mater 2021 Jan 21;412:125133. Epub 2021 Jan 21.

Electron Microscope Unit, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.

Tanneries create a serious threat to the environment by generating a significant amount of toxic metal-containing solid waste. This study deals with the application of bio-electrokinetic remediation (Bio-EK) of tannery effluent contaminated soil (TECS). Metagenomes representing the TECS sample were sequenced using the Illumina HiSeq platform. The bioreduction of hexavalent chromium Cr(VI)to trivalent chromium Cr (III) was achieved by BIO-EK techniques. NGS-data (Next Generation Sequencing) analysis was revealed that Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Planctomycetes were identified in the bio-electrokinetic system. Proteobacteria are responsible for the bioreduction of chromium hexavalent by the formation of FeS particles. The bio-generated FeS particles can be reduced the toxic chromium (VI) to non-toxic chromium (III) in soil. Simultaneously total chromium and organic content were significantly removed in BIO-EK (40 and 290 mg kg) when compared to control soil (182 and 240 mg kg). The presence of pollutant degrading microbes such as Desulfovibrio, Pseudomonas, Bacillus, Clostridium, Halanaerobium enhanced the bioreduction of the chromium during the electrokinetic remediation. This study can be claimed that the microbial cultures assisted electrokinetic remediation of total chromium, organic and iron in the tannery effluent contaminated soil was one of the suitable efficient techniques. In addition, the viability of the new combination technology developed (Electrokinetic + Bio) to treat low-permeability polluted soils was demonstrated.
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http://dx.doi.org/10.1016/j.jhazmat.2021.125133DOI Listing
January 2021

Bio-electrokinetic remediation of crude oil contaminated soil enhanced by bacterial biosurfactant.

J Hazard Mater 2021 Mar 13;405:124061. Epub 2020 Oct 13.

Research Chair in Laser Diagnosis of Cancers, Department of Physics and Astronomy, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Kingdom of Saudi Arabia; Department of Environmental Engineering, Kyungpook National University, 80 Daehak‑ro, Buk‑gu, Daegu 41566, South Korea. Electronic address:

The present study evaluating the coupling between bioremediation (BIO) and electrokinetic (EK) remediation of crude oil hydrocarbon by using bio-electrokinetic (BIO-EK) technique. The application of bacterial biosurfactant (BS) may increase the remediation efficiency by increasing the solubility of organic materials. In this work, the potential biosurfactant producing marine bacteria were isolated and identified by 16S rDNA analysis namely Bacillus subtilis AS2, Bacillus licheniformis AS3 and Bacillus velezensis AS4. Biodegradation efficiency of crude oil was found as 88%, 92% and 97% for strain AS2, AS3 and AS4 respectively, with the optimum temperature of 37 °C and pH 7. FTIR confirm the BS belongs to lipopeptide in nature. GCMS reveals that three isolates degraded the lower to higher molecular weight of the crude oil (C to C) effectively. Results showed that use of BS in electokinetic remediation enhance the biodegradation rate of crude oil contaminated soil about 92% than EK (60%) in 2 days operation. BS enhances the solubilization of hydrocarbon and it leads to the faster electromigration of hydrocarbon to the anodic compartment, which was confirmed by the presence of higher total organic content than the EK. This study proven that the BIO-EK combined with BS can be used to enhance in situ bioremediation of petroleum contaminated soils.
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http://dx.doi.org/10.1016/j.jhazmat.2020.124061DOI Listing
March 2021

Macrocyclization of an all-d linear α-helical peptide imparts cellular permeability.

Chem Sci 2020 Jun 11;11(21):5577-5591. Epub 2020 May 11.

MSD International , Translation Medicine Research Centre , 8 Biomedical Grove, #04-01/05 Neuros Building , Singapore , 138665 , Singapore . Email:

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein-protein interactions (PPIs). Indeed, the vast diversity of chemical space conferred through their primary, secondary and tertiary structures allows these molecules to be applied to targets that are typically deemed intractable small molecules. However, the development of peptide therapeutics has been hindered by their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies are aimed at addressing these issues. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may improve peptide properties by (i) restricting conformational freedom to improve target affinities, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. As a second strategy, molecules constructed entirely from d-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-d α-helical stapled and stitched peptides. As a template, we used a recently reported all d-linear peptide that is a potent inhibitor of the p53-Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-d-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all d-peptide was determined to exhibit increased α-helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.
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http://dx.doi.org/10.1039/c9sc06383hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441689PMC
June 2020

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry.

J Vis Exp 2019 10 30(152). Epub 2019 Oct 30.

Department of Neuroscience, Cell Biology, and Physiology, Wright State University; Department of Surgery, Boonshoft School of Medicine, Wright State University;

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2' deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.
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http://dx.doi.org/10.3791/59930DOI Listing
October 2019

Chemogenomic profiling in yeast reveals antifungal mode-of-action of polyene macrolactam auroramycin.

PLoS One 2019 10;14(6):e0218189. Epub 2019 Jun 10.

Bioinformatics Institute, Singapore.

In this study, we report antifungal activity of auroramycin against Candida albicans, Candida tropicalis, and Cryptococcus neoformans. Auroramycin, a potent antimicrobial doubly glycosylated 24-membered polyene macrolactam, was previously isolated and characterized, following CRISPR-Cas9 mediated activation of a silent polyketide synthase biosynthetic gene cluster in Streptomyces rosesporous NRRL 15998. Chemogenomic profiling of auroramycin in yeast has linked its antifungal bioactivity to vacuolar transport and membrane organization. This was verified by disruption of vacuolar structure and membrane integrity of yeast cells with auroramycin treatment. Addition of salt but not sorbitol to the medium rescued the growth of auroramycin-treated yeast cells suggesting that auroramycin causes ionic stress. Furthermore, auroramycin caused hyperpolarization of the yeast plasma membrane and displayed a synergistic interaction with cationic hygromycin. Our data strongly suggest that auroramycin inhibits yeast cells by causing leakage of cations from the cytoplasm. Thus, auroramycin's mode-of-action is distinct from known antifungal polyenes, reinforcing the importance of natural products in the discovery of new anti-infectives.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218189PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557514PMC
February 2020

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

BMC Genomics 2019 May 14;20(1):374. Epub 2019 May 14.

Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore, 138671, Republic of Singapore.

Background: Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.

Results: Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.

Conclusions: We describe the NRPS-t1PKS cluster 'BIIRfg' compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.
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http://dx.doi.org/10.1186/s12864-019-5762-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518819PMC
May 2019

Chemical-genetic interaction landscape of mono-(2-ethylhexyl)-phthalate using chemogenomic profiling in yeast.

Chemosphere 2019 Aug 15;228:219-231. Epub 2019 Apr 15.

Bioinformatics Institute, 30 Biopolis Street, #07-01, Matrix, Singapore 138671, Singapore. Electronic address:

Integration of chemical-genetic interaction data with biological functions provides a mechanistic understanding of how toxic compounds affect cells. Mono-(2-ethylhexyl)-phthalate (MEHP) is an active metabolite of di-(2-ethylhexyl)-phthalate (DEHP), a commonly used plasticizer. MEHP adversely affects human health causing hepatotoxicity and reproductive toxicity. How MEHP affects cellular physiology is not fully understood. We utilized a genome-wide competitive fitness-based assay called 'chemogenomic profiling' to determine the genetic interaction map of MEHP in Saccharomyces cerevisiae. Gene Ontology enrichment analysis of 218 genes that provide resistance to MEHP indicated that MEHP affects seven cellular processes namely: (1) cellular amino acid biosynthetic process, (2) sterol biosynthetic process, (3) cellular transport, (4) transcriptional and translational regulation, (5) protein glycosylation, (6) cytokinesis and cell morphogenesis and (7) ionic homeostasis. We show that MEHP protects yeast cells from membrane perturbing agents such as amphotericin B, dihydrosphingosine and phytosphingosine. Moreover, we also demonstrate that MEHP compromises the integrity of the yeast plasma membrane and cell wall. Our work provides a basis for further investigation of MEHP toxicity in humans.
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http://dx.doi.org/10.1016/j.chemosphere.2019.04.100DOI Listing
August 2019

Hypoculoside, a sphingoid base-like compound from Acremonium disrupts the membrane integrity of yeast cells.

Sci Rep 2019 01 24;9(1):710. Epub 2019 Jan 24.

Bioinformatics Institute, 30 Biopolis Street, #07-01, Matrix, 138671, Singapore.

We have isolated Hypoculoside, a new glycosidic amino alcohol lipid from the fungus Acremonium sp. F2434 belonging to the order Hypocreales and determined its structure by 2D-NMR (Nuclear Magnetic Resonance) spectroscopy. Hypoculoside has antifungal, antibacterial and cytotoxic activities. Homozygous profiling (HOP) of hypoculoside in Saccharomyces cerevisiae (budding yeast) revealed that several mutants defective in vesicular trafficking and vacuolar protein transport are sensitive to hypoculoside. Staining of budding yeast cells with the styryl dye FM4-64 indicated that hypoculoside damaged the vacuolar structure. Furthermore, the propidium iodide (PI) uptake assay showed that hypoculoside disrupted the plasma membrane integrity of budding yeast cells. Interestingly, the glycosidic moiety of hypoculoside is required for its deleterious effect on growth, vacuoles and plasma membrane of budding yeast cells.
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http://dx.doi.org/10.1038/s41598-018-35979-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345779PMC
January 2019

Expression of a Functional IL-2 Receptor in Vascular Smooth Muscle Cells.

J Immunol 2019 02 31;202(3):694-703. Epub 2018 Dec 31.

Department of Neuroscience, Cell Biology, and Physiology, Wright State University, Dayton, OH 45435;

Many nonlymphoid cell types express at least two, if not all three, subunits of the IL-2R; although, compared with lymphocytes, relatively little is known about how IL-2 affects the function of nonlymphoid cells. The limited information available suggests that IL-2 has a substantial impact on cells such as gastrointestinal epithelial cells, endothelial cells, and fibroblasts. In a previous report from our laboratory, we noted that IL-2 and IL-2Rβ-deficient mice lose smooth muscle cells over time, eventually resulting in aneurysmal aortas and ectatic esophagi. This finding, combined with our work showing that IL-2 surrounds vascular smooth muscle cells by association with perlecan, led us to ask whether vascular smooth muscle cells express an IL-2R. Toward this end, we reported the expression of IL-2Rβ on human and murine vascular smooth muscle cells. We now report that vascular smooth muscle cells express all three subunits of the IL-2R, and that expression of IL-2Rα varies with vascular smooth muscle cell phenotype. Furthermore, we show that, through a functional IL-2R, IL-2 initiates signaling pathways and impacts vascular smooth muscle cell function. Finally, we demonstrate that IL-2 expression increases upon initiation of conditions that promote intimal hyperplasia, suggesting a mechanism by which the IL-2/IL-2R system may impact this widespread vascular pathology.
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http://dx.doi.org/10.4049/jimmunol.1701151DOI Listing
February 2019

The 160K Natural Organism Library, a unique resource for natural products research.

Nat Biotechnol 2018 07;36(7):570-573

Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore.

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http://dx.doi.org/10.1038/nbt.4187DOI Listing
July 2018

Structure-activity relationship studies of ultra-short peptides with potent activities against fluconazole-resistant Candida albicans.

Eur J Med Chem 2018 Apr 9;150:479-490. Epub 2018 Mar 9.

Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos #03-01, Singapore, 138669, Singapore. Electronic address:

Vulvovaginal candidiasis (VVC) is a genital fungal infection afflicting approximately 75% of women globally and is primarily caused by the yeast Candida albicans. The extensive use of fluconazole, the first-line antifungal drug of choice, has led to the emergence of fluconazole-resistant C. albicans, creating a global clinical concern. This, coupled to the lack of new antifungal drugs entering the market over the past decade, has made it imperative for the introduction of new antifungal drug classes. Peptides with antifungal properties are deemed potential drug candidates due to their rapid membrane-disrupting mechanism of action. By specifically targeting and rapidly disrupting fungal membranes, they reduce the chances of resistance development and treatment duration. In a previous screening campaign involving an antimicrobial peptide library, we identified an octapeptide (IKIKIKIK-NH) with potent activity against C. albicans. Herein, we report a structure-activity relationship study on this peptide with the aim of designing a more potent peptide for further development. The lead peptide was then tested against a panel of fluconazole-resistant C. albicans, subjected to a fungicidal/static determination assay, a human dermal fibroblast viability assay and a homozygous profiling assay to gain insights into its mechanism of action and potential for further development as a topical antifungal agent.
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http://dx.doi.org/10.1016/j.ejmech.2018.03.027DOI Listing
April 2018

A yeast two-hybrid system for the screening and characterization of small-molecule inhibitors of protein-protein interactions identifies a novel putative Mdm2-binding site in p53.

BMC Biol 2017 Nov 9;15(1):108. Epub 2017 Nov 9.

Bioinformatics Institute, 30 Biopolis Street, #07-01, Matrix, Singapore, 138671, Singapore.

Background: Protein-protein interactions (PPIs) are fundamental to the growth and survival of cells and serve as excellent targets to develop inhibitors of biological processes such as host-pathogen interactions and cancer cell proliferation. However, isolation of PPI inhibitors is extremely challenging. While several in vitro assays to screen for PPI inhibitors are available, they are often expensive, cumbersome, and require large amounts of purified protein. In contrast, limited in vivo assays are available to screen for small-molecule inhibitors of PPI.

Methods: We have engineered a yeast strain that is suitable for screening of small-molecule inhibitors of protein-protein interaction using the Yeast 2-hybrid Assay. We have optimised and validated the assay using inhibitors of the p53-Mdm2 interaction and identified a hitherto unreported putative Mdm2-binding domain in p53.

Results: We report a significantly improved and thoroughly validated yeast two-hybrid (Y2H) assay that can be used in a high throughput manner to screen for small-molecule PPI inhibitors. Using the p53-Mdm2 interaction to optimize the assay, we show that the p53-Mdm2 inhibitor nutlin-3 is a substrate for the yeast ATP-binding cassette (ABC) transporter Pdr5. By deleting nine ABC transporter-related genes, we generated a ABC9Δ yeast strain that is highly permeable to small molecules. In the ABC9Δ strain, p53-Mdm2 interaction inhibitors, like AMG232 and MI-773, completely inhibited the p53-Mdm2 interaction at nanomolar concentrations in the Y2H assay. In addition, we identified a conserved segment in the core DNA-binding domain of p53 that facilitates stable interaction with Mdm2 in yeast cells and in vitro.

Conclusion: The Y2H assay can be utilized for high-throughput screening of small-molecule inhibitors of PPIs and to identify domains that stabilize PPIs.
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http://dx.doi.org/10.1186/s12915-017-0446-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680816PMC
November 2017

PP2A(Cdc55)'s role in reductional chromosome segregation during achiasmate meiosis in budding yeast is independent of its FEAR function.

Sci Rep 2016 07 26;6:30397. Epub 2016 Jul 26.

Bioinformatics Institute (A*STAR), 30 Biopolis Street, #07-01 Matrix, 138671, Singapore.

PP2A(Cdc55) is a highly conserved serine-threonine protein phosphatase that is involved in diverse cellular processes. In budding yeast, meiotic cells lacking PP2A(Cdc55) activity undergo a premature exit from meiosis I which results in a failure to form bipolar spindles and divide nuclei. This defect is largely due to its role in negatively regulating the Cdc Fourteen Early Anaphase Release (FEAR) pathway. PP2A(Cdc55) prevents nucleolar release of the Cdk (Cyclin-dependent kinase)-antagonising phosphatase Cdc14 by counteracting phosphorylation of the nucleolar protein Net1 by Cdk. CDC55 was identified in a genetic screen for monopolins performed by isolating suppressors of spo11Δ spo12Δ lethality suggesting that Cdc55 might have a role in meiotic chromosome segregation. We investigated this possibility by isolating cdc55 alleles that suppress spo11Δ spo12Δ lethality and show that this suppression is independent of PP2A(Cdc55)'s FEAR function. Although the suppressor mutations in cdc55 affect reductional chromosome segregation in the absence of recombination, they have no effect on chromosome segregation during wild type meiosis. We suggest that Cdc55 is required for reductional chromosome segregation during achiasmate meiosis and this is independent of its FEAR function.
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http://dx.doi.org/10.1038/srep30397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960654PMC
July 2016

Identification of a cytotoxic form of dimeric interleukin-2 in murine tissues.

PLoS One 2014 14;9(7):e102191. Epub 2014 Jul 14.

Department of Neuroscience, Cell Biology, and Physiology, Boonshoft School of Medicine Wright State University, Dayton, Ohio, United States of America.

Interleukin-2 (IL-2) is a multi-faceted cytokine, known for promoting proliferation, survival, and cell death depending on the cell type and state. For example, IL-2 facilitates cell death only in activated T cells when antigen and IL-2 are abundant. The availability of IL-2 clearly impacts this process. Our laboratory recently demonstrated that IL-2 is retained in blood vessels by heparan sulfate, and that biologically active IL-2 is released from vessel tissue by heparanase. We now demonstrate that heparanase digestion also releases a dimeric form of IL-2 that is highly cytotoxic to cells expressing the IL-2 receptor. These cells include "traditional" IL-2 receptor-bearing cells such as lymphocytes, as well as those less well known for IL-2 receptor expression, such as epithelial and smooth muscle cells. The morphologic changes and rapid cell death induced by dimeric IL-2 imply that cell death is mediated by disruption of membrane permeability and subsequent necrosis. These findings suggest that IL-2 has a direct and unexpectedly broad influence on cellular homeostatic mechanisms in both immune and non-immune systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102191PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097599PMC
November 2015

The Rim15-endosulfine-PP2ACdc55 signalling module regulates entry into gametogenesis and quiescence via distinct mechanisms in budding yeast.

PLoS Genet 2014 Jun 26;10(6):e1004456. Epub 2014 Jun 26.

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

Quiescence and gametogenesis represent two distinct survival strategies in response to nutrient starvation in budding yeast. Precisely how environmental signals are sensed by yeast cells to trigger quiescence and gametogenesis is not fully understood. A conserved signalling module consisting of Greatwall kinase, Endosulfine and Protein Phosphatase PP2ACdc55 proteins regulates entry into mitosis in Xenopus egg extracts and meiotic maturation in flies. We report here that an analogous signalling module consisting of the serine-threonine kinase Rim15, the Endosulfines Igo1 and Igo2 and the Protein Phosphatase PP2ACdc55, regulates entry into both quiescence and gametogenesis in budding yeast. PP2ACdc55 inhibits entry into gametogenesis and quiescence. Rim15 promotes entry into gametogenesis and quiescence by converting Igo1 into an inhibitor of PP2ACdc55 by phosphorylating at a conserved serine residue. Moreover, we show that the Rim15-Endosulfine-PP2ACdc55 pathway regulates entry into quiescence and gametogenesis by distinct mechanisms. In addition, we show that Igo1 and Igo2 are required for pre-meiotic autophagy but the lack of pre-meiotic autophagy is insufficient to explain the sporulation defect of igo1Δ igo2Δ cells. We propose that the Rim15-Endosulfine-PP2ACdc55 signalling module triggers entry into quiescence and gametogenesis by regulating dephosphorylation of distinct substrates.
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http://dx.doi.org/10.1371/journal.pgen.1004456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072559PMC
June 2014

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions.

PLoS Genet 2013 26;9(12):e1004071. Epub 2013 Dec 26.

MRC Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom.

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4(Eme1). Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe.
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http://dx.doi.org/10.1371/journal.pgen.1004071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873251PMC
August 2014

CDK-dependent nuclear localization of B-cyclin Clb1 promotes FEAR activation during meiosis I in budding yeast.

PLoS One 2013 1;8(11):e79001. Epub 2013 Nov 1.

University of Warwick, Coventry, United Kingdom.

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079001PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815228PMC
August 2014

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

PLoS Genet 2013 4;9(7):e1003610. Epub 2013 Jul 4.

University of Warwick, Coventry, United Kingdom.

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.
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http://dx.doi.org/10.1371/journal.pgen.1003610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701701PMC
January 2014

DNA polymerase α (swi7) and the flap endonuclease Fen1 (rad2) act together in the S-phase alkylation damage response in S. pombe.

PLoS One 2012 11;7(10):e47091. Epub 2012 Oct 11.

Division of Biomedical Cell Biology, Warwick Medical School, Gibbet Hill Campus, University of Warwick, Coventry, United Kingdom.

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047091PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469492PMC
May 2013

How to halve ploidy: lessons from budding yeast meiosis.

Cell Mol Life Sci 2012 Sep 6;69(18):3037-51. Epub 2012 Apr 6.

School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.

Maintenance of ploidy in sexually reproducing organisms requires a specialized form of cell division called meiosis that generates genetically diverse haploid gametes from diploid germ cells. Meiotic cells halve their ploidy by undergoing two rounds of nuclear division (meiosis I and II) after a single round of DNA replication. Research in Saccharomyces cerevisiae (budding yeast) has shown that four major deviations from the mitotic cell cycle during meiosis are essential for halving ploidy. The deviations are (1) formation of a link between homologous chromosomes by crossover, (2) monopolar attachment of sister kinetochores during meiosis I, (3) protection of centromeric cohesion during meiosis I, and (4) suppression of DNA replication following exit from meiosis I. In this review we present the current understanding of the above four processes in budding yeast and examine the possible conservation of molecular mechanisms from yeast to humans.
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http://dx.doi.org/10.1007/s00018-012-0974-9DOI Listing
September 2012

Meiotic nuclear divisions in budding yeast require PP2A(Cdc55)-mediated antagonism of Net1 phosphorylation by Cdk.

J Cell Biol 2011 Jun 20;193(7):1157-66. Epub 2011 Jun 20.

University of Warwick, Coventry CV4 7AL, England, UK.

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2A(Cdc55) activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)-counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2A(Cdc55) is essential for preventing precocious exit from meiosis I.
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http://dx.doi.org/10.1083/jcb.201103019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216327PMC
June 2011

Homolog pairing during meiosis: dyneins on the move.

Authors:
Prakash Arumugam

Cell Cycle 2010 Jun 1;9(11):2060. Epub 2010 Jun 1.

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June 2010

Evaluation of Mycoplasma hyopneumoniae bacterins for porcine torque teno virus DNAs.

Am J Vet Res 2008 Dec;69(12):1601-7

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA.

Objective: To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV).

Sample Population: 22 commercially available M hyopneumoniae bacterins.

Procedures: Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe.

Results: Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1- and genogroup 2-TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays.

Conclusions And Clinical Relevance: Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.
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http://dx.doi.org/10.2460/ajvr.69.12.1601DOI Listing
December 2008

The cohesin ring concatenates sister DNA molecules.

Nature 2008 Jul 2;454(7202):297-301. Epub 2008 Jul 2.

University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK.

Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin. Cohesin's Scc1, Smc1 and Smc3 subunits form a tripartite ring structure, and it has been proposed that cohesin holds sister DNA molecules together by trapping them inside its ring. To test this, we used site-specific crosslinking to create chemical connections at the three interfaces between the three constituent polypeptides of the ring, thereby creating covalently closed cohesin rings. As predicted by the ring entrapment model, this procedure produced dimeric DNA-cohesin structures that are resistant to protein denaturation. We conclude that cohesin rings concatenate individual sister minichromosome DNA molecules.
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http://dx.doi.org/10.1038/nature07098DOI Listing
July 2008

Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge.

Cell 2006 Nov;127(3):523-37

Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

Cohesin is a multisubunit complex that mediates sister-chromatid cohesion. Its Smc1 and Smc3 subunits possess ABC-like ATPases at one end of 50 nm long coiled coils. At the other ends are pseudosymmetrical hinge domains that interact to create V-shaped Smc1/Smc3 heterodimers. N- and C-terminal domains within cohesin's kleisin subunit Scc1 bind to Smc3 and Smc1 ATPase heads respectively, thereby creating a huge tripartite ring. It has been suggested that cohesin associates with chromosomes by trapping DNA within its ring. Opening of the ring due to cleavage of Scc1 by separase destroys sister-chromatid cohesion and triggers anaphase. We show that cohesin's hinges are not merely dimerization domains. They are essential for cohesin's association with chromosomes, which is blocked by artificially holding hinge domains together but not by preventing Scc1's dissociation from SMC ATPase heads. Our results suggest that entry of DNA into cohesin's ring requires transient dissociation of Smc1 and Smc3 hinge domains.
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http://dx.doi.org/10.1016/j.cell.2006.08.048DOI Listing
November 2006

Cohesin's ATPase activity is stimulated by the C-terminal Winged-Helix domain of its kleisin subunit.

Curr Biol 2006 Oct;16(20):1998-2008

Research Institute of Molecular Pathology, Dr. Bohr Gasse 7, A-1030 Vienna, Austria.

Background: Cohesin, a multisubunit protein complex conserved from yeast to humans, holds sister chromatids together from the onset of replication to their separation during anaphase. Cohesin consists of four core subunits, namely Smc1, Smc3, Scc1, and Scc3. Smc1 and Smc3 proteins are characterized by 50-nm-long anti-parallel coiled coils flanked by a globular hinge domain and an ABC-like ATPase head domain. Whereas Smc1 and Smc3 heterodimerize via their hinge domains, the kleisin subunit Scc1 connects their ATPase heads, and this results in the formation of a large ring. Biochemical studies suggest that cohesin might trap sister chromatids within its ring, and genetic evidence suggests that ATP hydrolysis is required for the stable association of cohesin with chromosomes. However, the precise role of the ATPase domains remains enigmatic.

Results: Characterization of cohesin's ATPase activity suggests that hydrolysis depends on the binding of ATP to both Smc1 and Smc3 heads. However, ATP hydrolysis at the two active sites is not per se cooperative. We show that the C-terminal winged-helix domain of Scc1 stimulates the ATPase activity of the Smc1/Smc3 heterodimer by promoting ATP binding to Smc1's head. In contrast, we do not detect any effect of Scc1's N-terminal domain on Smc1/Smc3 ATPase activity.

Conclusions: Our studies reveal that Scc1 not only connects the Smc1 and Smc3 ATPase heads but also regulates their ATPase activity.
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http://dx.doi.org/10.1016/j.cub.2006.09.002DOI Listing
October 2006

ATP hydrolysis is required for cohesin's association with chromosomes.

Curr Biol 2003 Nov;13(22):1941-53

Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

Background: A multi-subunit protein complex called cohesin is involved in holding sister chromatids together after DNA replication. Cohesin contains four core subunits: Smc1, Smc3, Scc1, and Scc3. Biochemical studies suggest that Smc1 and Smc3 each form 50 nm-long antiparallel coiled coils (arms) and bind to each other to form V-shaped heterodimers with globular ABC-like ATPases (created by the juxtaposition of N- and C-terminal domains) at their apices. These Smc "heads" are connected by Scc1, creating a tripartite proteinaceous ring.

Results: To investigate the role of Smc1 and Smc3's ATPase domains, we engineered smc1 and smc3 mutations predicted to abolish either ATP binding or hydrolysis. All mutations abolished Smc protein function. The binding of ATP to Smc1, but not Smc3, was essential for Scc1's association with Smc1/3 heterodimers. In contrast, mutations predicted to prevent hydrolysis of ATP bound to either head abolished cohesin's association with chromatin but not Scc1's ability to connect Smc1's head with that of Smc3. Inactivation of the Scc2/4 complex had a similar if not identical effect; namely, the production of tripartite cohesin rings that cannot associate with chromosomes.

Conclusions: Cohesin complexes whose heads have been connected by Scc1 must hydrolyze ATP in order to associate stably with chromosomes. If chromosomal association is mediated by the topological entrapment of DNA inside cohesin's ring, then ATP hydrolysis may be responsible for creating a gate through which DNA can enter. We suggest that ATP hydrolysis drives the temporary disconnection of Scc1 from Smc heads that are needed for DNA entrapment and that this process is promoted by Scc2/4.
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http://dx.doi.org/10.1016/j.cub.2003.10.036DOI Listing
November 2003