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1
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Skamagki M, Zhang C, Hacisuleyman E, Galleti G, Wu C, Vinagolu RK, Cha H, Ata D, Kim J, Weiskittel T, Diop M, Aung T, Del Latto M, Kim AS, Li Z, Miele M, Zhao R, Tang LH, Hendrickson RC, Romesser PB, Smith JJ, Giannakakou P, Darnell RB, Bott MJ, Li H, Kim K. Aging-dependent dysregulation of EXOSC2 is maintained in cancer as a dependency. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.04.04.647279. [PMID: 40236131 PMCID: PMC11996493 DOI: 10.1101/2025.04.04.647279] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/17/2025]
Abstract
Reprogramming of aged donor tissue cells into induced pluripotent stem cells (A-iPSC) preserved the epigenetic memory of aged-donor tissue, defined as genomic instability and poor tissue differentiation in our previous study. The unbalanced expression of RNA exosome subunits affects the RNA degradation complex function and is associated with geriatric diseases including premature aging and cancer progression. We hypothesized that the age-dependent progressive subtle dysregulation of EXOSC2 (exosome component 2) causes the aging traits (abnormal cell cycle and poor tissue differentiation). We used embryonic stem cells as a tool to study EXOSC2 function as the aging trait epigenetic memory determined in A-iPSC because these aging traits could not be studied in senesced aged cells or immortalized cancer cells. We found that the regulatory subunit of PP2A phosphatase, PPP2R5E, is a key target of EXOSC2 and this regulation is preserved in stem cells and cancer.
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2
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Lorentzon E, Lee J, Masaryk J, Keuenhof K, Karlsson N, Galipaud C, Madsen R, Höög JL, Levin DE, Tamás MJ. Direct binding of arsenicals to nuclear transport factors disrupts nucleocytoplasmic transport. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.13.632748. [PMID: 39868121 PMCID: PMC11761705 DOI: 10.1101/2025.01.13.632748] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/28/2025]
Abstract
Human exposure to arsenicals is associated with devastating diseases such as cancer and neurodegeneration. At the same time, arsenic-based drugs are used as therapeutic agents. The ability of arsenic to directly bind to proteins is correlated with its toxic and therapeutic effects highlighting the importance of elucidating arsenic-protein interactions. In this study, we took a proteomic approach and identified 174 proteins that bind to arsenic in Saccharomyces cerevisiae. Proteins involved in nucleocytoplasmic transport were markedly enriched among the arsenic-binding proteins, and we demonstrate that arsenic-binding to nuclear import factors results in their relocation from the nuclear envelope and subsequent aggregation in the cytosol. Similarly, nuclear pore proteins that make up the nuclear pore complex mislocalized and aggregated in arsenic-exposed cells. Consequently, arsenic was shown to inhibit nuclear protein import and export. We propose a model in which arsenic-binding to nuclear transport factors leads to their mislocalization and aggregation, which disrupts nucleocytoplasmic transport and causes arsenic sensitivity.
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Affiliation(s)
- Emma Lorentzon
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
| | - Jongmin Lee
- Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA
| | - Jakub Masaryk
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
| | - Katharina Keuenhof
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
| | - Nora Karlsson
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
| | - Charlotte Galipaud
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
| | - Rebecca Madsen
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
| | - Johanna L. Höög
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
| | - David E. Levin
- Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA
| | - Markus J. Tamás
- Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, S-405 30 Göteborg, Sweden
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3
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Visinoni F, Royle W, Scholey R, Hu Y, Timouma S, Zeef L, Louis EJ, Delneri D. Impact of inter-species hybridisation on antifungal drug response in the Saccharomyces genus. BMC Genomics 2024; 25:1165. [PMID: 39623390 PMCID: PMC11610120 DOI: 10.1186/s12864-024-11009-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2024] [Accepted: 11/07/2024] [Indexed: 12/06/2024] Open
Abstract
BACKGROUND Antifungal drug resistance presents one of the major concerns for global public health, and hybridization allows the development of high fitness organisms that can better survive in restrictive conditions or in presence of antifungal agents. Hence, understanding how allelic variation can influence antifungal susceptibility in hybrid organisms is important for the development of targeted treatments. Here, we exploited recent advances in multigenerational breeding of hemiascomycete hybrids to study the impact of hybridisation on antifungal resistance and identify quantitative trait loci responsible for the phenotype. RESULTS The offspring of Saccharomyces cerevisiae x S. kudriavzevii hybrids were screened in the presence of six antifungal drugs and revealed a broad phenotypic diversity across the progeny. QTL analysis was carried out comparing alleles between pools of high and low fitness offspring, identifying hybrid-specific genetic regions involved in resistance to fluconazole, micafungin and flucytosine. We found both drug specific and pleiotropic regions, including 41 blocks containing genes not previously associated with resistance phenotypes. We identified linked genes that influence the same trait, namely a hybrid specific 'super' QTL, and validated, via reciprocal hemizygosity analysis, two causal genes, BCK2 and DNF1. The co-location of genes with similar phenotypic impact supports the notion of an adaption process that limits the segregation of advantageous alleles via recombination. CONCLUSIONS This study demonstrates the value of QTL studies to elucidate the hybrid-specific mechanisms of antifungal susceptibility. We also show that an inter-species hybrid model system in the Saccharomyces background, can help to decipher the trajectory of antifungal drug resistance in pathogenic hybrid lineages.
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Affiliation(s)
- Federico Visinoni
- Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester, M13 9PT, UK
| | - William Royle
- Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester, M13 9PT, UK
| | - Rachel Scholey
- Bioinformatics Core Facility, University of Manchester, Manchester, M13 9PT, UK
| | - Yue Hu
- Phenotypeca Limited, BioCity Nottingham, Nottingham, NG1 1GF, UK
| | - Soukaina Timouma
- Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester, M13 9PT, UK
| | - Leo Zeef
- Bioinformatics Core Facility, University of Manchester, Manchester, M13 9PT, UK
| | - Edward J Louis
- Phenotypeca Limited, BioCity Nottingham, Nottingham, NG1 1GF, UK
| | - Daniela Delneri
- Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
- Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester, M13 9PT, UK.
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4
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Chen JK, Merrick KA, Kong YW, Izrael-Tomasevic A, Eng G, Handly ED, Patterson JC, Cannell IG, Suarez-Lopez L, Hosios AM, Dinh A, Kirkpatrick DS, Yu K, Rose CM, Hernandez JM, Hwangbo H, Palmer AC, Vander Heiden MG, Yilmaz ÖH, Yaffe MB. An RNA damage response network mediates the lethality of 5-FU in colorectal cancer. Cell Rep Med 2024; 5:101778. [PMID: 39378883 PMCID: PMC11514606 DOI: 10.1016/j.xcrm.2024.101778] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2024] [Revised: 07/15/2024] [Accepted: 09/16/2024] [Indexed: 10/10/2024]
Abstract
5-fluorouracil (5-FU), a major anti-cancer therapeutic, is believed to function primarily by inhibiting thymidylate synthase, depleting deoxythymidine triphosphate (dTTP), and causing DNA damage. Here, we show that clinical combinations of 5-FU with oxaliplatin or irinotecan show no synergy in human colorectal cancer (CRC) trials and sub-additive killing in CRC cell lines. Using selective 5-FU metabolites, phospho- and ubiquitin proteomics, and primary human CRC organoids, we demonstrate that 5-FU-mediated CRC cell killing primarily involves an RNA damage response during ribosome biogenesis, causing lysosomal degradation of damaged rRNAs and proteasomal degradation of ubiquitinated ribosomal proteins. Tumor types clinically responsive to 5-FU treatment show upregulated rRNA biogenesis while 5-FU clinically non-responsive tumor types do not, instead showing greater sensitivity to 5-FU's DNA damage effects. Finally, we show that treatments upregulating ribosome biogenesis, including KDM2A inhibition, promote RNA-dependent cell killing by 5-FU, demonstrating the potential for combinatorial targeting of this ribosomal RNA damage response for improved cancer therapy.
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Affiliation(s)
- Jung-Kuei Chen
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Karl A Merrick
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Yi Wen Kong
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | | | - George Eng
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Erika D Handly
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Jesse C Patterson
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Ian G Cannell
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Lucia Suarez-Lopez
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Aaron M Hosios
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Anh Dinh
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | | | - Kebing Yu
- Genentech Biotechnology company, South San Francisco, CA 94080, USA
| | | | - Jonathan M Hernandez
- Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Haeun Hwangbo
- Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Pharmacology, Computational Medicine Program, UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Adam C Palmer
- Department of Pharmacology, Computational Medicine Program, UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Matthew G Vander Heiden
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA 02215, USA
| | - Ömer H Yilmaz
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Michael B Yaffe
- Center for Precision Cancer Medicine, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Surgery, Beth Israel Medical Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
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5
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Szachnowski U, Sallou O, Boudet M, Bretaudeau A, Wery M, Morillon A, Primig M. The 5-Fluorouracil RNA Expression Viewer (5-FU R) Facilitates Interpreting the Effects of Drug Treatment and RRP6 Deletion on the Transcriptional Landscape in Yeast. Yeast 2024; 41:629-640. [PMID: 39345013 DOI: 10.1002/yea.3982] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2024] [Accepted: 09/11/2024] [Indexed: 10/01/2024] Open
Abstract
Saccharomyces cerevisiae is an excellent model to study the effect of external cues on cell division and stress response. 5-Fluorocuracil (5-FU) has been used to treat solid tumors since several decades. The drug was initially designed to interfere with DNA replication but was later found to exert its antiproliferative effect also via RNA-dependent processes. Since 5-FU inhibits the activity of the 3'-5'-exoribonuclease Rrp6 in yeast and mammals, earlier work has compared the effect of 5-FU treatment and RRP6 deletion at the transcriptome level in diploid synchronized yeast cells. To facilitate interpreting the expression data we have developed an improved 5-Fluorouracil RNA (5-FUR) expression viewer. Users can access information via genome coordinates and systematic or standard names for mRNAs and Xrn1-dependent-, stable-, cryptic-, and meiotic unannotated transcripts (XUTs, SUTs, CUTs, and MUTs). Normalized log2-transformed or linear data can be displayed as filled diagrams, line graphs or color-coded heatmaps. The expression data are useful for researchers interested in processes such as cell cycle regulation, mitotic repression of meiotic genes, the effect of 5-FU treatment and Rrp6 deficiency on the transcriptome and expression profiles of sense/antisense loci that encode overlapping transcripts. The viewer is accessible at http://5fur.genouest.org.
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Affiliation(s)
| | | | - Mateo Boudet
- GenOuest, IRISA, Campus de Beaulieu, Rennes, France
| | | | - Maxime Wery
- Institut Curie, Sorbonne Université, Paris, France
| | | | - Michael Primig
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail), Rennes, France
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6
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Scholes AN, Stuecker TN, Hood SE, Locke CJ, Stacy CL, Zhang Q, Lewis JA. Natural variation in yeast reveals multiple paths for acquiring higher stress resistance. BMC Biol 2024; 22:149. [PMID: 38965504 PMCID: PMC11225312 DOI: 10.1186/s12915-024-01945-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Accepted: 06/26/2024] [Indexed: 07/06/2024] Open
Abstract
BACKGROUND Organisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated "cross protection" mechanisms, where mild 'primary' doses of one stress can enhance tolerance to severe doses of a different 'secondary' stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature. RESULTS During the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general. CONCLUSIONS Ultimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.
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Affiliation(s)
- Amanda N Scholes
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
- Interdisciplinary Graduate Program in Cell and Molecular Biology, University of Arkansas, Fayetteville, AR, USA
| | - Tara N Stuecker
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
| | - Stephanie E Hood
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
| | - Cader J Locke
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
| | - Carson L Stacy
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
- Interdisciplinary Graduate Program in Cell and Molecular Biology, University of Arkansas, Fayetteville, AR, USA
- Department of Mathematical Sciences, University of Arkansas, Fayetteville, AR, USA
| | - Qingyang Zhang
- Department of Mathematical Sciences, University of Arkansas, Fayetteville, AR, USA
| | - Jeffrey A Lewis
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA.
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7
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Separovich RJ, Karakatsanis NM, Gao K, Fuh D, Hamey JJ, Wilkins MR. Proline-directed yeast and human MAP kinases phosphorylate the Dot1p/DOT1L histone H3K79 methyltransferase. FEBS J 2024; 291:2590-2614. [PMID: 38270553 DOI: 10.1111/febs.17062] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Revised: 10/24/2023] [Accepted: 01/12/2024] [Indexed: 01/26/2024]
Abstract
Disruptor of telomeric silencing 1 (Dot1p) is an exquisitely conserved histone methyltransferase and is the sole enzyme responsible for H3K79 methylation in the budding yeast Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are almost entirely unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that mitogen-activated protein kinase HOG1 (Hog1p) is a bona fide kinase of the Dot1p methyltransferase. In vitro kinase assays showed that Hog1p phosphorylates Dot1p at multiple sites, including at several proline-adjacent sites that are consistent with known Hog1p substrate preferences. The activity of Hog1p was specifically enhanced at these proline-adjacent sites on Dot1p upon Hog1p activation by the osmostress-responsive MAP kinase kinase PBS2 (Pbs2p). Genomic deletion of HOG1 reduced phosphorylation at specific sites on Dot1p in vivo, providing further evidence for Hog1p kinase activity on Dot1p in budding yeast cells. Phenotypic analysis of knockout and phosphosite mutant yeast strains revealed the importance of Hog1p-catalysed phosphorylation of Dot1p for cellular responses to ultraviolet-induced DNA damage. In mammalian systems, this kinase-substrate relationship was found to be conserved: human DOT1L (the ortholog of yeast Dot1p) can be phosphorylated by the proline-directed kinase p38β (also known as MAPK11; the ortholog of yeast Hog1p) at multiple sites in vitro. Taken together, our findings establish Hog1p and p38β as newly identified upstream kinases of the Dot1p/DOT1L H3K79 methyltransferase enzymes in eukaryotes.
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Affiliation(s)
- Ryan J Separovich
- Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
| | - Nicola M Karakatsanis
- Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
| | - Kelley Gao
- Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
| | - David Fuh
- Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
| | - Joshua J Hamey
- Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
| | - Marc R Wilkins
- Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
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8
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Charital S, Shunmugam S, Dass S, Alazzi AM, Arnold CS, Katris NJ, Duley S, Quansah NA, Pierrel F, Govin J, Yamaryo-Botté Y, Botté CY. The acyl-CoA synthetase TgACS1 allows neutral lipid metabolism and extracellular motility in Toxoplasma gondii through relocation via its peroxisomal targeting sequence (PTS) under low nutrient conditions. mBio 2024; 15:e0042724. [PMID: 38501871 PMCID: PMC11005404 DOI: 10.1128/mbio.00427-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2024] [Accepted: 02/23/2024] [Indexed: 03/20/2024] Open
Abstract
Apicomplexa parasites cause major diseases such as toxoplasmosis and malaria that have major health and economic burdens. These unicellular pathogens are obligate intracellular parasites that heavily depend on lipid metabolism for the survival within their hosts. Their lipid synthesis relies on an essential combination of fatty acids (FAs) obtained from both de novo synthesis and scavenging from the host. The constant flux of scavenged FA needs to be channeled toward parasite lipid storage, and these FA storages are timely mobilized during parasite division. In eukaryotes, the utilization of FA relies on their obligate metabolic activation mediated by acyl-co-enzyme A (CoA) synthases (ACSs), which catalyze the thioesterification of FA to a CoA. Besides the essential functions of FA for parasite survival, the presence and roles of ACS are yet to be determined in Apicomplexa. Here, we identified TgACS1 as a Toxoplasma gondii cytosolic ACS that is involved in FA mobilization in the parasite specifically during low host nutrient conditions, especially in extracellular stages where it adopts a different localization. Heterologous complementation of yeast ACS mutants confirmed TgACS1 as being an Acyl-CoA synthetase of the bubble gum family that is most likely involved in β-oxidation processes. We further demonstrate that TgACS1 is critical for gliding motility of extracellular parasite facing low nutrient conditions, by relocating to peroxisomal-like area.IMPORTANCEToxoplasma gondii, causing human toxoplasmosis, is an Apicomplexa parasite and model within this phylum that hosts major infectious agents, such as Plasmodium spp., responsible for malaria. The diseases caused by apicomplexans are responsible for major social and economic burdens affecting hundreds of millions of people, like toxoplasmosis chronically present in about one-third of the world's population. Lack of efficient vaccines, rapid emergence of resistance to existing treatments, and toxic side effects of current treatments all argue for the urgent need to develop new therapeutic tools to combat these diseases. Understanding the key metabolic pathways sustaining host-intracellular parasite interactions is pivotal to develop new efficient ways to kill these parasites. Current consensus supports parasite lipid synthesis and trafficking as pertinent target for novel treatments. Many processes of this essential lipid metabolism in the parasite are not fully understood. The capacity for the parasites to sense and metabolically adapt to the host physiological conditions has only recently been unraveled. Our results clearly indicate the role of acyl-co-enzyme A (CoA) synthetases for the essential metabolic activation of fatty acid (FA) used to maintain parasite propagation and survival. The significance of our research is (i) the identification of seven of these enzymes that localize at different cellular areas in T. gondii parasites; (ii) using lipidomic approaches, we show that TgACS1 mobilizes FA under low host nutrient content; (iii) yeast complementation showed that acyl-CoA synthase 1 (ACS1) is an ACS that is likely involved in peroxisomal β-oxidation; (iv) the importance of the peroxisomal targeting sequence for correct localization of TgACS1 to a peroxisomal-like compartment in extracellular parasites; and lastly, (v) that TgACS1 has a crucial role in energy production and extracellular parasite motility.
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Affiliation(s)
- Sarah Charital
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Serena Shunmugam
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Sheena Dass
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Anna Maria Alazzi
- Team Govin, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Christophe-Sébastien Arnold
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Nicholas J. Katris
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Samuel Duley
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Nyamekye A. Quansah
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Fabien Pierrel
- Université Grenoble Alpes, CNRS, Grenoble INP, TIMC-IMAG, Grenoble, France
| | - Jérôme Govin
- Team Govin, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Yoshiki Yamaryo-Botté
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
| | - Cyrille Y. Botté
- Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Grenoble, France
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9
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Gupta R, Singh M, Pathania R. Chemical genetic approaches for the discovery of bacterial cell wall inhibitors. RSC Med Chem 2023; 14:2125-2154. [PMID: 37974958 PMCID: PMC10650376 DOI: 10.1039/d3md00143a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2023] [Accepted: 08/10/2023] [Indexed: 11/19/2023] Open
Abstract
Antimicrobial resistance (AMR) in bacterial pathogens is a worldwide health issue. The innovation gap in discovering new antibiotics has remained a significant hurdle in combating the AMR problem. Currently, antibiotics target various vital components of the bacterial cell envelope, nucleic acid and protein biosynthesis machinery and metabolic pathways essential for bacterial survival. The critical role of the bacterial cell envelope in cell morphogenesis and integrity makes it an attractive drug target. While a significant number of in-clinic antibiotics target peptidoglycan biosynthesis, several components of the bacterial cell envelope have been overlooked. This review focuses on various antibacterial targets in the bacterial cell wall and the strategies employed to find their novel inhibitors. This review will further elaborate on combining forward and reverse chemical genetic approaches to discover antibacterials that target the bacterial cell envelope.
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Affiliation(s)
- Rinki Gupta
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee Roorkee - 247 667 Uttarakhand India
| | - Mangal Singh
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee Roorkee - 247 667 Uttarakhand India
| | - Ranjana Pathania
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee Roorkee - 247 667 Uttarakhand India
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10
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Chen JK, Merrick KA, Kong YW, Izrael-Tomasevic A, Eng G, Handly ED, Patterson JC, Cannell IG, Suarez-Lopez L, Hosios AM, Dinh A, Kirkpatrick DS, Yu K, Rose CM, Hernandez JM, Hwangbo H, Palmer AC, Vander Heiden MG, Yilmaz ÖH, Yaffe MB. An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.28.538590. [PMID: 37162991 PMCID: PMC10168374 DOI: 10.1101/2023.04.28.538590] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/11/2023]
Abstract
5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.
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Affiliation(s)
- Jung-Kuei Chen
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Karl A. Merrick
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Yi Wen Kong
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | | | - George Eng
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Erika D. Handly
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Jesse C. Patterson
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Ian G. Cannell
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Lucia Suarez-Lopez
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Aaron M. Hosios
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Anh Dinh
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | | | - Kebing Yu
- Genentech Biotechnology company, South San Francisco, CA 94080, USA
| | | | - Jonathan M. Hernandez
- Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Haeun Hwangbo
- Curriculum in Bioinformatics and Computational Biology, UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Pharmacology, Computational Medicine Program, and UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Adam C. Palmer
- Department of Pharmacology, Computational Medicine Program, and UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Matthew G. Vander Heiden
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA 02215, USA
| | - Ömer H. Yilmaz
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Michael B. Yaffe
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Department of Surgery, Beth Israel Medical Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
- Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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11
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Chen SAA, Kern AF, Ang RML, Xie Y, Fraser HB. Gene-by-environment interactions are pervasive among natural genetic variants. CELL GENOMICS 2023; 3:100273. [PMID: 37082145 PMCID: PMC10112290 DOI: 10.1016/j.xgen.2023.100273] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/05/2022] [Revised: 10/09/2022] [Accepted: 01/31/2023] [Indexed: 04/22/2023]
Abstract
Gene-by-environment (GxE) interactions, in which a genetic variant's phenotypic effect is condition specific, are fundamental for understanding fitness landscapes and evolution but have been difficult to identify at the single-nucleotide level. Although many condition-specific quantitative trait loci (QTLs) have been mapped, these typically contain numerous inconsequential variants in linkage, precluding understanding of the causal GxE variants. Here, we introduce BARcoded Cas9 retron precise parallel editing via homology (CRISPEY-BAR), a high-throughput precision genome editing strategy, and use it to map GxE interactions of naturally occurring genetic polymorphisms impacting yeast growth. We identified hundreds of GxE variants within condition-specific QTLs, revealing unexpected genetic complexity. Moreover, we found that 93.7% of non-neutral natural variants within ergosterol biosynthesis pathway genes showed GxE interactions, including many impacting antifungal drug resistance through diverse molecular mechanisms. In sum, our results suggest an extremely complex, context-dependent fitness landscape characterized by pervasive GxE interactions while also demonstrating massively parallel genome editing as an effective means for investigating this complexity.
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Affiliation(s)
- Shi-An A. Chen
- Department of Biology, Stanford University, Stanford, CA 94305, USA
| | - Alexander F. Kern
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Roy Moh Lik Ang
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Yihua Xie
- Department of Biology, Stanford University, Stanford, CA 94305, USA
| | - Hunter B. Fraser
- Department of Biology, Stanford University, Stanford, CA 94305, USA
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12
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Islam MD, Harrison BD, Li JJY, McLoughlin AG, Court DA. Do mitochondria use efflux pumps to protect their ribosomes from antibiotics? MICROBIOLOGY (READING, ENGLAND) 2023; 169:001272. [PMID: 36748523 PMCID: PMC9993110 DOI: 10.1099/mic.0.001272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Fungal environments are rich in natural and engineered antimicrobials, and this, combined with the fact that fungal genomes are rich in coding sequences for transporters, suggests that fungi are an intriguing group in which to search for evidence of antimicrobial efflux pumps in mitochondria. Herein, the range of protective mechanisms used by fungi against antimicrobials is introduced, and it is hypothesized, based on the susceptibility of mitochondrial and bacterial ribosomes to the same antibiotics, that mitochondria might also contain pumps that efflux antibiotics from these organelles. Preliminary evidence of ethidium bromide efflux is presented and several candidate efflux pumps are identified in fungal mitochondrial proteomes.
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Affiliation(s)
- Md Deen Islam
- Department of Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
| | - Brian D Harrison
- Department of Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
| | - Judy J-Y Li
- Department of Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
| | - Austein G McLoughlin
- Department of Microbiology, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
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13
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Vanderwaeren L, Dok R, Voordeckers K, Nuyts S, Verstrepen KJ. Saccharomyces cerevisiae as a Model System for Eukaryotic Cell Biology, from Cell Cycle Control to DNA Damage Response. Int J Mol Sci 2022; 23:11665. [PMID: 36232965 PMCID: PMC9570374 DOI: 10.3390/ijms231911665] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 09/26/2022] [Accepted: 09/28/2022] [Indexed: 11/08/2022] Open
Abstract
The yeast Saccharomyces cerevisiae has been used for bread making and beer brewing for thousands of years. In addition, its ease of manipulation, well-annotated genome, expansive molecular toolbox, and its strong conservation of basic eukaryotic biology also make it a prime model for eukaryotic cell biology and genetics. In this review, we discuss the characteristics that made yeast such an extensively used model organism and specifically focus on the DNA damage response pathway as a prime example of how research in S. cerevisiae helped elucidate a highly conserved biological process. In addition, we also highlight differences in the DNA damage response of S. cerevisiae and humans and discuss the challenges of using S. cerevisiae as a model system.
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Affiliation(s)
- Laura Vanderwaeren
- Laboratory of Experimental Radiotherapy, Department of Oncology, KU Leuven, 3000 Leuven, Belgium
- Laboratory of Genetics and Genomics, Centre for Microbial and Plant Genetics, Department M2S, KU Leuven, 3001 Leuven, Belgium
- Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, 3001 Leuven, Belgium
| | - Rüveyda Dok
- Laboratory of Experimental Radiotherapy, Department of Oncology, KU Leuven, 3000 Leuven, Belgium
| | - Karin Voordeckers
- Laboratory of Genetics and Genomics, Centre for Microbial and Plant Genetics, Department M2S, KU Leuven, 3001 Leuven, Belgium
- Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, 3001 Leuven, Belgium
| | - Sandra Nuyts
- Laboratory of Experimental Radiotherapy, Department of Oncology, KU Leuven, 3000 Leuven, Belgium
- Department of Radiation Oncology, Leuven Cancer Institute, University Hospitals Leuven, 3000 Leuven, Belgium
| | - Kevin J. Verstrepen
- Laboratory of Genetics and Genomics, Centre for Microbial and Plant Genetics, Department M2S, KU Leuven, 3001 Leuven, Belgium
- Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, 3001 Leuven, Belgium
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14
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Abstract
The last several decades have witnessed a surge in drug-resistant fungal infections that pose a serious threat to human health. While there is a limited arsenal of drugs that can be used to treat systemic infections, scientific advances have provided renewed optimism for the discovery of novel antifungals. The development of chemical-genomic assays using Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of molecules in a living cell. Advances in molecular biology techniques have enabled complementary assays to be developed in fungal pathogens, including Candida albicans and Cryptococcus neoformans. These approaches enable the identification of target genes for drug candidates, as well as genes involved in buffering drug target pathways. Here, we examine yeast chemical-genomic assays and highlight how such resources can be utilized to predict the mechanisms of action of compounds, to study virulence attributes of diverse fungal pathogens, and to bolster the antifungal pipeline.
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Affiliation(s)
- Nicole Robbins
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada;
| | - Leah E Cowen
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada;
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15
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Dhakal S, Macreadie I. The Use of Yeast in Biosensing. Microorganisms 2022; 10:1772. [PMID: 36144374 PMCID: PMC9505958 DOI: 10.3390/microorganisms10091772] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Revised: 08/24/2022] [Accepted: 08/30/2022] [Indexed: 11/18/2022] Open
Abstract
Yeast has been used as a model for several diseases as it is the simplest unicellular eukaryote, safe and easy to culture and harbors most of the fundamental processes that are present in almost all higher eukaryotes, including humans. From understanding the pathogenesis of disease to drug discovery studies, yeast has served as an important biosensor. It is not only due to the conservation of genetics, amenable modification of its genome and easily accessible analytical methods, but also some characteristic features such as its ability to survive with defective mitochondria, making it a highly flexible microbe for designing whole-cell biosensing systems. The aim of this review is to report on how yeasts have been utilized as biosensors, reporting on responses to various stimuli.
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Affiliation(s)
| | - Ian Macreadie
- School of Science, RMIT University, Bundoora, VIC 3083, Australia
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16
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Chauvin A, Bergeron D, Vencic J, Lévesque D, Paquette B, Scott MS, Boisvert FM. Downregulation of KRAB zinc finger proteins in 5-fluorouracil resistant colorectal cancer cells. BMC Cancer 2022; 22:363. [PMID: 35379199 PMCID: PMC8981854 DOI: 10.1186/s12885-022-09417-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Accepted: 03/15/2022] [Indexed: 12/23/2022] Open
Abstract
Radio-chemotherapy with 5-flu orouracil (5-FU) is the standard of care treatment for patients with colorectal cancer, but it is only effective for a third of them. Despite our understanding of the mechanism of action of 5-FU, drug resistance remains a significant limitation to the clinical use of 5-FU, as both intrinsic and acquired chemoresistance represents the major obstacles for the success of 5-FU-based chemotherapy. In order to identify the mechanism of acquired resistance, 5-FU chemoresistance was induced in CRC cell lines by passaging cells with increasing concentrations of 5-FU. To study global molecular changes, quantitative proteomics and transcriptomics analyses were performed on these cell lines, comparing the resistant cells as well as the effect of chemo and radiotherapy. Interestingly, a very high proportion of downregulated genes were annotated as transcription factors coding for Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), the largest family of transcriptional repressors. Among nearly 350 KRAB-ZFPs, almost a quarter were downregulated after the induction of a 5-FU-resistance including a common one between the three CRC cell lines, ZNF649, whose role is still unknown. To confirm the observations of the proteomic and transcriptomic approaches, the abundance of 20 different KZFPs and control mRNAs was validated by RT-qPCR. In fact, several KZFPs were no longer detectable using qPCR in cell lines resistant to 5-FU, and the KZFPs that were downregulated only in one or two cell lines showed similar pattern of expression as measured by the omics approaches. This proteomic, transcriptomic and genomic analysis of intrinsic and acquired resistance highlights a possible new mechanism involved in the cellular adaptation to 5-FU and therefore identifies potential new therapeutic targets to overcome this resistance.
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Affiliation(s)
- Anaïs Chauvin
- Department of Immunology and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada
| | - Danny Bergeron
- Department of Biochemistry and Functional Genomics, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada
| | - Jean Vencic
- Department of Biochemistry and Functional Genomics, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada
| | - Dominique Lévesque
- Department of Immunology and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada
| | - Benoit Paquette
- Department of Nuclear Medicine and Radiobiology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada
| | - Michelle S Scott
- Department of Biochemistry and Functional Genomics, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada
| | - François-Michel Boisvert
- Department of Immunology and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
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17
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Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast. METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2022; 2394:377-432. [PMID: 35094338 DOI: 10.1007/978-1-0716-1811-0_21] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Genetic code expansion has allowed for extraordinary advances in enhancing protein chemical diversity and functionality, but there remains a critical need for understanding and engineering genetic code expansion systems for improved efficiency. Incorporation of noncanonical amino acids (ncAAs) at stop codons provides a site-specific method for introducing unique chemistry into proteins, though often at reduced yields compared to wild-type proteins. A powerful platform for ncAA incorporation supports both the expression and evaluation of chemically diverse proteins for a broad range of applications. In yeast, ncAAs have been used to study dynamic cellular processes such as protein-protein interactions and also allow for exploration of eukaryotic-specific biology such as epigenetics. Furthermore, yeast display is an advantageous technology for engineering and screening the properties of proteins in high throughput. The protocols presented in this chapter describe detailed methods for the yeast-based genetic encoding of ncAAs in proteins intracellularly or on the yeast surface. In addition, methods are presented for modifying proteins on the yeast surface using bioorthogonal chemical reactions and evaluating reaction efficiency. Finally, protocols are included for the preparation of libraries that involve genetic code expansion. Libraries of proteins that contain ncAAs or libraries of the cellular machinery required to encode ncAAs can be constructed and screened in high throughput for many biological and chemical applications. Efficient incorporation of ncAAs facilitates elucidation of fundamental eukaryotic biology and advances tools for enzyme and genome engineering to evolve host cells that are better able to accommodate alternative genetic codes.
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18
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Zhang T, Li YN, Li X, Gu W, Moeketsi EK, Zhou R, Zheng X, Zhang Z, Zhang H. The Peroxisomal-CoA Synthetase MoPcs60 Is Important for Fatty Acid Metabolism and Infectious Growth of the Rice Blast Fungus. FRONTIERS IN PLANT SCIENCE 2022; 12:811041. [PMID: 35154208 PMCID: PMC8826238 DOI: 10.3389/fpls.2021.811041] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Accepted: 12/01/2021] [Indexed: 06/14/2023]
Abstract
Fatty acid metabolism is important for the maintenance of fatty acid homeostasis. Free fatty acids, which are toxic in excess, are activated by esterification with coenzyme A (CoA) and then subjected to β-oxidization. Fatty acid β-oxidation-related genes play critical roles in the development and virulence of several phytopathogens. In this study, we identified and characterized a peroxisomal-CoA synthetase in the rice blast fungus Magnaporthe oryzae, MoPCS60, which is a homolog of PCS60 in budding yeast. MoPCS60 was highly expressed during the conidial and early infectious stages and was induced under oleate treatment. Targeted deletion of MoPCS60 resulted in a significant reduction in growth rate when oleate and olive oil were used as the sole carbon sources. Compared with the wild-type strain Guy11, the ΔMopcs60 mutant exhibited fewer peroxisomes, more lipid droplets, and decreased pathogenicity. The distribution of MoPcs60 varied among developmental stages and was mainly localized to peroxisomes in the hyphae, conidia, and appressoria when treated with oleate. Our results suggest that MoPcs60 is a key peroxisomal-CoA synthetase involved in fatty acid β-oxidation and pathogenicity in rice blast fungi.
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19
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di Punzio G, Gilberti M, Baruffini E, Lodi T, Donnini C, Dallabona C. A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool. Int J Mol Sci 2021; 22:ijms222212223. [PMID: 34830106 PMCID: PMC8621932 DOI: 10.3390/ijms222212223] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Revised: 11/08/2021] [Accepted: 11/09/2021] [Indexed: 12/30/2022] Open
Abstract
Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.
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20
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Halder V, McDonnell B, Uthayakumar D, Usher J, Shapiro RS. Genetic interaction analysis in microbial pathogens: unravelling networks of pathogenesis, antimicrobial susceptibility and host interactions. FEMS Microbiol Rev 2021; 45:fuaa055. [PMID: 33145589 DOI: 10.1093/femsre/fuaa055] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Accepted: 10/16/2020] [Indexed: 12/13/2022] Open
Abstract
Genetic interaction (GI) analysis is a powerful genetic strategy that analyzes the fitness and phenotypes of single- and double-gene mutant cells in order to dissect the epistatic interactions between genes, categorize genes into biological pathways, and characterize genes of unknown function. GI analysis has been extensively employed in model organisms for foundational, systems-level assessment of the epistatic interactions between genes. More recently, GI analysis has been applied to microbial pathogens and has been instrumental for the study of clinically important infectious organisms. Here, we review recent advances in systems-level GI analysis of diverse microbial pathogens, including bacterial and fungal species. We focus on important applications of GI analysis across pathogens, including GI analysis as a means to decipher complex genetic networks regulating microbial virulence, antimicrobial drug resistance and host-pathogen dynamics, and GI analysis as an approach to uncover novel targets for combination antimicrobial therapeutics. Together, this review bridges our understanding of GI analysis and complex genetic networks, with applications to diverse microbial pathogens, to further our understanding of virulence, the use of antimicrobial therapeutics and host-pathogen interactions. .
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Affiliation(s)
- Viola Halder
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
| | - Brianna McDonnell
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
| | - Deeva Uthayakumar
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
| | - Jane Usher
- Medical Research Council Centre for Medical Mycology, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
| | - Rebecca S Shapiro
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
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21
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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:e0246264. [PMID: 33861751 PMCID: PMC8051802 DOI: 10.1371/journal.pone.0246264] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Accepted: 01/17/2021] [Indexed: 11/19/2022] Open
Abstract
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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22
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Lee S, Nam M, Lee AR, Baek ST, Kim MJ, Kim JS, Kong AH, Lee M, Lee SJ, Kim SY, Kim DU, Hoe KL. Genetic alterations in Wnt family of genes and their putative association with head and neck squamous cell carcinoma. Genomics Inform 2021; 19:e39. [PMID: 35172472 PMCID: PMC8752990 DOI: 10.5808/gi.21049] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 10/15/2021] [Indexed: 11/24/2022] Open
Abstract
Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) ‘biological process’ terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7 breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including ‘cell cycle’ (cdc2, rik1, pas1, and leo1), ‘signaling’ (sck2, oga1, and cki3), and ‘vesicle-mediated transport’ (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the ‘signaling’ GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.
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Affiliation(s)
- Sol Lee
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Miyoung Nam
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Ah-Reum Lee
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Seung-Tae Baek
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Min Jung Kim
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Ju Seong Kim
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Andrew Hyunsoo Kong
- Morrissey College of Arts and Sciences, Boston College, Boston 02467, MA, USA
| | - Minho Lee
- Department of Life Science, Dongguk University-Seoul, Goyang 10326, Korea
| | - Sook-Jeong Lee
- Department of Bioactive Material Science, Jeonbuk National University, Jeonju 54896, Korea
| | - Seon-Young Kim
- Personalized Medicine Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Korea
| | - Dong-Uk Kim
- Rare Disease Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Korea
| | - Kwang-Lae Hoe
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea.,Korea Research Institute of Chemistry & Technology, Daejeon 34141, Korea
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23
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Lee S, Nam M, Lee AR, Lee J, Woo J, Kang NS, Balupuri A, Lee M, Kim SY, Ro H, Choi YW, Kim DU, Hoe KL. Systematic Target Screening Revealed That Tif302 Could Be an Off-Target of the Antifungal Terbinafine in Fission Yeast. Biomol Ther (Seoul) 2021; 29:234-247. [PMID: 33223513 PMCID: PMC7921855 DOI: 10.4062/biomolther.2020.166] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2020] [Revised: 10/12/2020] [Accepted: 10/13/2020] [Indexed: 11/22/2022] Open
Abstract
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, spt7, spt20, and elp2] and 4 essential genes (tif302, rpl2501, rpl31, and erg1). Expectedly, their erg1 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 erg1 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 erg1 transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of erg1 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 tif302 heterozygous strain was attributed to both decreased erg1 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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Affiliation(s)
- Sol Lee
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Miyoung Nam
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Ah-Reum Lee
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Jaewoong Lee
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Jihye Woo
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Nam Sook Kang
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Anand Balupuri
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Minho Lee
- Department of Life Science, Dongguk University-Seoul, Goyang 10326, Republic of Korea
| | - Seon-Young Kim
- Personalized Genomic Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
| | - Hyunju Ro
- Department of Biological Science, College of Bioscience & Biotechnology, Chungnam National University, Daejeon 34134, Republic of Korea
| | | | - Dong-Uk Kim
- Rare Disease Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
| | - Kwang-Lae Hoe
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea
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24
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Chung WH. Pleiotropic Effects of Caffeine Leading to Chromosome Instability and Cytotoxicity in Eukaryotic Microorganisms. J Microbiol Biotechnol 2021; 31:171-180. [PMID: 33397827 PMCID: PMC9706025 DOI: 10.4014/jmb.2011.11042] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 12/20/2020] [Accepted: 11/22/2020] [Indexed: 12/15/2022]
Abstract
Caffeine, a methylxanthine analog of purine bases, is a compound that is largely consumed in beverages and medications for psychoactive and diuretic effects and plays many beneficial roles in neuronal stimulation and enhancement of anti-tumor immune responses by blocking adenosine receptors in higher organisms. In single-cell eukaryotes, however, caffeine somehow impairs cellular fitness by compromising cell wall integrity, inhibiting target of rapamycin (TOR) signaling and growth, and overriding cell cycle arrest caused by DNA damage. Among its multiple inhibitory targets, caffeine specifically interacts with phosphatidylinositol 3-kinase (PI3K)-related kinases causing radiosensitization and cytotoxicity via specialized intermediate molecules. Caffeine potentiates the lethality of cells in conjunction with several other stressors such as oxidants, irradiation, and various toxic compounds through largely unknown mechanisms. In this review, recent findings on caffeine effects and cellular detoxification schemes are highlighted and discussed with an emphasis on the inhibitory interactions between caffeine and its multiple targets in eukaryotic microorganisms such as budding and fission yeasts.
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Affiliation(s)
- Woo-Hyun Chung
- College of Pharmacy, Duksung Women’s University, Seoul 0369, Republic of Korea,Innovative Drug Center, Duksung Women’s University, Seoul 01369, Republic of Korea,Corresponding author Phone: +82-2-901-8737 Fax: +82-2-901-8386 E-mail:
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25
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Tulha J, Amorim-Rodrigues M, Esquembre LA, Rauch S, Tamás MJ, Lucas C. Physical, genetic and functional interactions between the eisosome protein Pil1 and the MBOAT O-acyltransferase Gup1. FEMS Yeast Res 2020; 21:6045508. [PMID: 33355361 DOI: 10.1093/femsyr/foaa070] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Accepted: 12/21/2020] [Indexed: 11/14/2022] Open
Abstract
The Saccharomyces cerevisiae MBOAT O-acyltransferase Gup1 is involved in many processes, including cell wall and membrane composition and integrity, and acetic acid-induced cell death. Gup1 was previously shown to interact physically with the mitochondrial membrane VDAC (Voltage-Dependent Anion Channel) protein Por1 and the ammonium transceptor Mep2. By co-immunoprecipitation, the eisosome core component Pil1 was identified as a novel physical interaction partner of Gup1. The expression of PIL1 and Pil1 protein levels were found to be unaffected by GUP1 deletion. In ∆gup1 cells, Pil1 was distributed in dots (likely representing eisosomes) in the membrane, identically to wt cells. However, ∆gup1 cells presented 50% less Pil1-GFP dots/eisosomes, suggesting that Gup1 is important for eisosome formation. The two proteins also interact genetically in the maintenance of cell wall integrity, and during arsenite and acetic acid exposure. We show that Δgup1 Δpil1 cells take up more arsenite than wt and are extremely sensitive to arsenite and to acetic acid treatments. The latter causes a severe apoptotic wt-like cell death phenotype, epistatically reverting the ∆gup1 necrotic type of death. Gup1 and Pil1 are thus physically, genetically and functionally connected.
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Affiliation(s)
- Joana Tulha
- Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal
| | - Mariana Amorim-Rodrigues
- Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal.,Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Campus de Gualtar 4710-057 Braga, Portugal
| | - Lidia Alejo Esquembre
- Department of Chemistry and Molecular Biology, University of Gothenburg, Kemihuset 412 96 Gothenburg, Sweden
| | - Sebastien Rauch
- Water Environment Technology, Department of Architecture and Civil and Environmental Engineering, Chalmers University of Technology, S-412 96 Gothenburg, Sweden
| | - Markus J Tamás
- Department of Chemistry and Molecular Biology, University of Gothenburg, Kemihuset 412 96 Gothenburg, Sweden
| | - Cândida Lucas
- Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal.,Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Campus de Gualtar 4710-057 Braga, Portugal
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26
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SIR2 Expression Noise Can Generate Heterogeneity in Viability but Does Not Affect Cell-to-Cell Epigenetic Silencing of Subtelomeric URA3 in Yeast. G3-GENES GENOMES GENETICS 2020; 10:3435-3443. [PMID: 32727919 PMCID: PMC7466964 DOI: 10.1534/g3.120.401589] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Chromatin structure clearly modulates gene expression noise, but the reverse influence has never been investigated, namely how the cell-to-cell expression heterogeneity of chromatin modifiers may generate variable rates of epigenetic modification. Sir2 is a well-characterized histone deacetylase of the Sirtuin family. It strongly influences chromatin silencing, especially at telomeres, subtelomeres and rDNA. This ability to influence epigenetic landscapes makes it a good model to study the largely unexplored interplay between gene expression noise and other epigenetic processes leading to phenotypic diversification. Here, we addressed this question by investigating whether noise in the expression of SIR2 was associated with cell-to-cell heterogeneity in the frequency of epigenetic silencing at subtelomeres in Saccharomyces cerevisiae Using cell sorting to isolate subpopulations with various expression levels, we found that heterogeneity in the cellular concentration of Sir2 does not lead to heterogeneity in the epigenetic silencing of subtelomeric URA3 between these subpopulations. We also noticed that SIR2 expression noise can generate cell-to-cell variability in viability, with lower levels being associated with better viability. This work shows that SIR2 expression fluctuations are not sufficient to generate cell-to-cell heterogeneity in the epigenetic silencing of URA3 at subtelomeres in Saccharomyces cerevisiae but can strongly affect cellular viability.
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27
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Xue A, Robbins N, Cowen LE. Advances in fungal chemical genomics for the discovery of new antifungal agents. Ann N Y Acad Sci 2020; 1496:5-22. [PMID: 32860238 DOI: 10.1111/nyas.14484] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 08/09/2020] [Accepted: 08/13/2020] [Indexed: 12/15/2022]
Abstract
Invasive fungal infections have escalated from a rare curiosity to a major cause of human mortality around the globe. This is in part due to a scarcity in the number of antifungal drugs available to combat mycotic disease, making the discovery of novel bioactive compounds and determining their mode of action of utmost importance. The development and application of chemical genomic assays using the model yeast Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of diverse molecules in a living cell. Furthermore, complementary assays are continually being developed in fungal pathogens, most notably Candida albicans and Cryptococcus neoformans, to elucidate compound mechanism of action directly in the pathogen of interest. Collectively, the suite of chemical genetic assays that have been developed in multiple fungal species enables the identification of candidate drug target genes, as well as genes involved in buffering drug target pathways, and genes involved in general cellular responses to small molecules. In this review, we examine current yeast chemical genomic assays and highlight how such resources provide powerful tools that can be utilized to bolster the antifungal pipeline.
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Affiliation(s)
- Alice Xue
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Nicole Robbins
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Leah E Cowen
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
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28
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Ruta LL, Farcasanu IC. Saccharomyces cerevisiae and Caffeine Implications on the Eukaryotic Cell. Nutrients 2020; 12:nu12082440. [PMID: 32823708 PMCID: PMC7468979 DOI: 10.3390/nu12082440] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 08/07/2020] [Accepted: 08/10/2020] [Indexed: 02/07/2023] Open
Abstract
Caffeine-a methylxanthine analogue of the purine bases adenine and guanine-is by far the most consumed neuro-stimulant, being the active principle of widely consumed beverages such as coffee, tea, hot chocolate, and cola. While the best-known action of caffeine is to prevent sleepiness by blocking the adenosine receptors, caffeine exerts a pleiotropic effect on cells, which lead to the activation or inhibition of various cell integrity pathways. The aim of this review is to present the main studies set to investigate the effects of caffeine on cells using the model eukaryotic microorganism Saccharomyces cerevisiae, highlighting the caffeine synergy with external cell stressors, such as irradiation or exposure to various chemical hazards, including cigarette smoke or chemical carcinogens. The review also focuses on the importance of caffeine-related yeast phenotypes used to resolve molecular mechanisms involved in cell signaling through conserved pathways, such as target of rapamycin (TOR) signaling, Pkc1-Mpk1 mitogen activated protein kinase (MAPK) cascade, or Ras/cAMP protein kinase A (PKA) pathway.
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29
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Zhu P, Li M, Yan C, Sun J, Peng M, Huang Z, Shi P. Aspirin Causes Lipid Accumulation and Damage to Cell Membrane by Regulating DCI1/ OLE1 in Saccharomyces cerevisiae. Microb Drug Resist 2020; 26:857-868. [PMID: 32049589 DOI: 10.1089/mdr.2019.0200] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Aspirin is one of the most commonly used nonsteroidal anti-inflammatory drugs. Various potential pharmacological effects of aspirin, such as anticancer, antibacterial activity, and prolonging life expectancy have been discovered. However, the mechanism of aspirin is not fully elucidated. Herein, the effects of aspirin on fatty acid metabolism in yeast cell model Saccharomyces cerevisiae were studied. The results showed that aspirin can induce lipid accumulation and reduce the unsaturated fat index in cells. The assessment of cell membrane integrity demonstrated that aspirin caused damage to the cell membrane. These effects of aspirin were attributed to the alterations of the expression of DCI1 and OLE1. Similarly, aspirin was able to cause lipid accumulation and damage to the cell membrane by interfering with the expression of OLE1 in Candida albicans. These findings are expected to improve current understanding of the mode of action of aspirin and provide a novel strategy for antifungal drug design. Graphical abstract [Figure: see text].
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Affiliation(s)
- Pan Zhu
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
| | - Ming Li
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
| | - Chongjia Yan
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
| | - Jing Sun
- Qinghai Key Laboratory of Qinghai-Tibet Plateau Biological Resources, Northwest Institute of Plateau Biology, the Chinese Academy of Sciences, Xining, Qinghai, China
| | - Min Peng
- Qinghai Key Laboratory of Qinghai-Tibet Plateau Biological Resources, Northwest Institute of Plateau Biology, the Chinese Academy of Sciences, Xining, Qinghai, China
| | - Zhiwei Huang
- Key Lab of Eco-Textile, Ministry of Education, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China
| | - Ping Shi
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
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30
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Abstract
To survive under unpredictable conditions, all organisms must adapt to stressors by regulating adaptive cellular responses. Arrestin proteins are conserved regulators of adaptive cellular responses in eukaryotes. Studies that have been limited to mammals and model fungi have demonstrated that the disruption of arrestin-regulated pathways is detrimental for viability. The human fungal pathogen Cryptococcus neoformans causes more than 180,000 infection-related deaths annually, especially among immunocompromised patients. In addition to being genetically tractable, C. neoformans has a small arrestin family of four members, lending itself to a comprehensive characterization of its arrestin family. This study serves as a functional analysis of arrestins in a pathogen, particularly in the context of fungal fitness and virulence. We investigate the functions of one arrestin protein, Ali1, and define its novel contributions to cytokinesis. We additionally explore the virulence contributions of the C. neoformans arrestin family and find that they contribute to disease establishment and progression. Arrestins, a structurally specialized and functionally diverse group of proteins, are central regulators of adaptive cellular responses in eukaryotes. Previous studies on fungal arrestins have demonstrated their capacity to modulate diverse cellular processes through their adaptor functions, facilitating the localization and function of other proteins. However, the mechanisms by which arrestin-regulated processes are involved in fungal virulence remain unexplored. We have identified a small family of four arrestins, Ali1, Ali2, Ali3, and Ali4, in the human fungal pathogen Cryptococcus neoformans. Using complementary microscopy, proteomic, and reverse genetics techniques, we have defined a role for Ali1 as a novel contributor to cytokinesis, a fundamental cell cycle-associated process. We observed that Ali1 strongly interacts with proteins involved in lipid synthesis, and that ali1Δ mutant phenotypes are rescued by supplementation with lipid precursors that are used to build cellular membranes. From these data, we hypothesize that Ali1 contributes to cytokinesis by serving as an adaptor protein, facilitating the localization of enzymes that modify the plasma membrane during cell division, specifically the fatty acid synthases Fas1 and Fas2. Finally, we assessed the contributions of the C. neoformans arrestin family to virulence to better understand the mechanisms by which arrestin-regulated adaptive cellular responses influence fungal infection. We observed that the C. neoformans arrestin family contributes to virulence, and that the individual arrestin proteins likely fulfill distinct functions that are important for disease progression.
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31
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Colic M, Hart T. Chemogenetic interactions in human cancer cells. Comput Struct Biotechnol J 2019; 17:1318-1325. [PMID: 31921397 PMCID: PMC6945272 DOI: 10.1016/j.csbj.2019.09.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2019] [Revised: 09/24/2019] [Accepted: 09/25/2019] [Indexed: 12/26/2022] Open
Abstract
Chemogenetic profiling enables the identification of genes that enhance or suppress the phenotypic effect of chemical compounds. Using this approach in cancer therapies could improve our ability to predict the response of specific tumor genotypes to chemotherapeutic agents, thus accelerating the development of personalized drug therapy. In the not so distant past, this strategy was only applied in model organisms because there was no feasible technology to thoroughly exploit desired genetic mutations and their impact on drug efficacy in human cells. Today, with the advent of CRISPR gene-editing technology and its application to pooled library screens in mammalian cells, chemogenetic screens are performed directly in human cell lines with high sensitivity and specificity. Chemogenetic profiling provides insights into drug mechanism-of-action, genetic vulnerabilities, and resistance mechanisms, all of which will help to accurately deliver the right drug to the right target in the right patient while minimizing side effects.
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Affiliation(s)
- Medina Colic
- Department of Bioinformatics and Computational Biology and Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- UTHealth Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Traver Hart
- Department of Bioinformatics and Computational Biology and Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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32
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Kim DU, Lee M, Han S, Nam M, Lee S, Lee J, Woo J, Kim D, Hoe KL. Optimization of a microarray for fission yeast. Genomics Inform 2019; 17:e28. [PMID: 31610624 PMCID: PMC6808646 DOI: 10.5808/gi.2019.17.3.e28] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Accepted: 06/28/2019] [Indexed: 11/30/2022] Open
Abstract
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℃ 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℃) was optimal for cultivation instead of a normal temperature (30℃) 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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Affiliation(s)
- Dong-Uk Kim
- Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Korea
| | - Minho Lee
- Catholic Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea
| | - Sangjo Han
- Data Analytics CoE, SK Telecom, Seongnam 13595, Korea
| | - Miyoung Nam
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Sol Lee
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Jaewoong Lee
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Jihye Woo
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
| | - Dongsup Kim
- Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST), Daejeon 34141, Korea
| | - Kwang-Lae Hoe
- Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea
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33
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Shah P, Wu WS, Chen CS. Systematical Analysis of the Protein Targets of Lactoferricin B and Histatin-5 Using Yeast Proteome Microarrays. Int J Mol Sci 2019; 20:ijms20174218. [PMID: 31466342 PMCID: PMC6747642 DOI: 10.3390/ijms20174218] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2019] [Revised: 08/23/2019] [Accepted: 08/23/2019] [Indexed: 12/12/2022] Open
Abstract
Antimicrobial peptides (AMPs) have potential antifungal activities; however, their intracellular protein targets are poorly reported. Proteome microarray is an effective tool with high-throughput and rapid platform that systematically identifies the protein targets. In this study, we have used yeast proteome microarrays for systematical identification of the yeast protein targets of Lactoferricin B (Lfcin B) and Histatin-5. A total of 140 and 137 protein targets were identified from the triplicate yeast proteome microarray assays for Lfcin B and Histatin-5, respectively. The Gene Ontology (GO) enrichment analysis showed that Lfcin B targeted more enrichment categories than Histatin-5 did in all GO biological processes, molecular functions, and cellular components. This might be one of the reasons that Lfcin B has a lower minimum inhibitory concentration (MIC) than Histatin-5. Moreover, pairwise essential proteins that have lethal effects on yeast were analyzed through synthetic lethality. A total of 11 synthetic lethal pairs were identified within the protein targets of Lfcin B. However, only three synthetic lethal pairs were identified within the protein targets of Histatin-5. The higher number of synthetic lethal pairs identified within the protein targets of Lfcin B might also be the reason for Lfcin B to have lower MIC than Histatin-5. Furthermore, two synthetic lethal pairs were identified between the unique protein targets of Lfcin B and Histatin-5. Both the identified synthetic lethal pairs proteins are part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex that regulates gene expression via histone modification. Identification of synthetic lethal pairs between Lfcin B and Histatin-5 and their involvement in the same protein complex indicated synergistic combination between Lfcin B and Histatin-5. This hypothesis was experimentally confirmed by growth inhibition assay.
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Affiliation(s)
- Pramod Shah
- Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli 32001, Taiwan
- Department of Biomedical Science and Engineering, National Central University, Jhongli 32001, Taiwan
| | - Wei-Sheng Wu
- Department of Electrical Engineering, National Cheng Kung University, Tainan City 701, Taiwan
| | - Chien-Sheng Chen
- Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli 32001, Taiwan.
- Department of Biomedical Science and Engineering, National Central University, Jhongli 32001, Taiwan.
- Department of Food Safety/Hygiene and Risk Management, College of Medicine, National Cheng Kung University, Tainan City 701, Taiwan.
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34
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Awad D, Prattes M, Kofler L, Rössler I, Loibl M, Pertl M, Zisser G, Wolinski H, Pertschy B, Bergler H. Inhibiting eukaryotic ribosome biogenesis. BMC Biol 2019; 17:46. [PMID: 31182083 PMCID: PMC6558755 DOI: 10.1186/s12915-019-0664-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Accepted: 05/14/2019] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND Ribosome biogenesis is a central process in every growing cell. In eukaryotes, it requires more than 250 non-ribosomal assembly factors, most of which are essential. Despite this large repertoire of potential targets, only very few chemical inhibitors of ribosome biogenesis are known so far. Such inhibitors are valuable tools to study this highly dynamic process and elucidate mechanistic details of individual maturation steps. Moreover, ribosome biogenesis is of particular importance for fast proliferating cells, suggesting its inhibition could be a valid strategy for treatment of tumors or infections. RESULTS We systematically screened ~ 1000 substances for inhibitory effects on ribosome biogenesis using a microscopy-based screen scoring ribosomal subunit export defects. We identified 128 compounds inhibiting maturation of either the small or the large ribosomal subunit or both. Northern blot analysis demonstrates that these inhibitors cause a broad spectrum of different rRNA processing defects. CONCLUSIONS Our findings show that the individual inhibitors affect a wide range of different maturation steps within the ribosome biogenesis pathway. Our results provide for the first time a comprehensive set of inhibitors to study ribosome biogenesis by chemical inhibition of individual maturation steps and establish the process as promising druggable pathway for chemical intervention.
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Affiliation(s)
- Dominik Awad
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
- Present address: Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Michael Prattes
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
| | - Lisa Kofler
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
| | - Ingrid Rössler
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
| | - Mathias Loibl
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
| | - Melanie Pertl
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
| | - Gertrude Zisser
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
| | - Heimo Wolinski
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria
| | - Brigitte Pertschy
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria.
| | - Helmut Bergler
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/EG, A-8010, Graz, Austria.
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Xie B, Becker E, Stuparevic I, Wery M, Szachnowski U, Morillon A, Primig M. The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast. RNA Biol 2019; 16:727-741. [PMID: 30760080 PMCID: PMC6546400 DOI: 10.1080/15476286.2019.1581596] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2018] [Revised: 01/16/2019] [Accepted: 02/06/2019] [Indexed: 10/27/2022] Open
Abstract
5-fluorouracil (5-FU) was isolated as an inhibitor of thymidylate synthase, which is important for DNA synthesis. The drug was later found to also affect the conserved 3'-5' exoribonuclease EXOSC10/Rrp6, a catalytic subunit of the RNA exosome that degrades and processes protein-coding and non-coding transcripts. Work on 5-FU's cytotoxicity has been focused on mRNAs and non-coding transcripts such as rRNAs, tRNAs and snoRNAs. However, the effect of 5-FU on long non-coding RNAs (lncRNAs), which include regulatory transcripts important for cell growth and differentiation, is poorly understood. RNA profiling of synchronized 5-FU treated yeast cells and protein assays reveal that the drug specifically inhibits a set of cell cycle regulated genes involved in mitotic division, by decreasing levels of the paralogous Swi5 and Ace2 transcriptional activators. We also observe widespread accumulation of different lncRNA types in treated cells, which are typically present at high levels in a strain lacking EXOSC10/Rrp6. 5-FU responsive lncRNAs include potential regulatory antisense transcripts that form double-stranded RNAs (dsRNAs) with overlapping sense mRNAs. Some of these transcripts encode proteins important for cell growth and division, such as the transcription factor Ace2, and the RNA exosome subunit EXOSC6/Mtr3. In addition to revealing a transcriptional effect of 5-FU action via DNA binding regulators involved in cell cycle progression, our results have implications for the function of putative regulatory lncRNAs in 5-FU mediated cytotoxicity. The data raise the intriguing possibility that the drug deregulates lncRNAs/dsRNAs involved in controlling eukaryotic cell division, thereby highlighting a new class of promising therapeutical targets.
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Affiliation(s)
- Bingning Xie
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)- UMR_S 1085, Rennes, France
| | - Emmanuelle Becker
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)- UMR_S 1085, Rennes, France
- Univ Rennes, Inria, CNRS, IRISA F-35000, Rennes, France
| | - Igor Stuparevic
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)- UMR_S 1085, Rennes, France
| | - Maxime Wery
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, PSL UniversityCNRS UMR 3244, Université Pierre et Marie Curie, Paris, France
| | - Ugo Szachnowski
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, PSL UniversityCNRS UMR 3244, Université Pierre et Marie Curie, Paris, France
| | - Antonin Morillon
- ncRNA, Epigenetic and Genome Fluidity, Institut Curie, PSL UniversityCNRS UMR 3244, Université Pierre et Marie Curie, Paris, France
| | - Michael Primig
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)- UMR_S 1085, Rennes, France
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Tulha J, Lucas C. Saccharomyces cerevisiae mitochondrial Por1/yVDAC1 (voltage-dependent anion channel 1) interacts physically with the MBOAT O-acyltransferase Gup1/HHATL in the control of cell wall integrity and programmed cell death. FEMS Yeast Res 2019; 18:5089977. [PMID: 30184078 DOI: 10.1093/femsyr/foy097] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2018] [Accepted: 08/31/2018] [Indexed: 02/06/2023] Open
Abstract
Gup1 is the yeast counterpart of the high eukaryotes HHATL. This and the close homologue Gup2/HHAT regulate the Hedgehog morphogenic, developmental pathway. In yeasts, a similar paracrine pathway is not known though the Δgup1 mutant is associated with morphology and proliferation/death processes. As a first step toward identifying the actual molecular/enzymatic function of Gup1, this work identified by co-immunoprecipitation the yeast mitochondria membrane VDAC1/Por1 as a physical partner of Gup1. Gup1 locates in the ER and the plasma membrane. It was now confirmed to further locate, as Por1, in the mitochondrial sub-cellular fraction. The yeast Por1-Gup1 association was found important for (i) the sensitivity to cell wall perturbing agents and high temperature, (ii) the differentiation into structured colonies, (iii) the size achieved by multicellular aggregates/mats and (iv) acetic-acid-induced Programmed Cell Death. Moreover, the absence of Gup1 increased the levels of POR1 mRNA, while decreasing the amounts of intracellular Por1, which was concomitantly previously known to be secreted by the mutant but not by wt. Additionally, Por1 patchy distribution in the mitochondrial membrane was evened. Results suggest that Por1 and Gup1 collaborate in the control of colony morphology and mat development, but more importantly of cellular integrity and death.
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Affiliation(s)
- Joana Tulha
- Centre of Molecular and Environmental Biology (CBMA), University of Minho, 4710-054 Braga, Portugal
| | - Cândida Lucas
- Centre of Molecular and Environmental Biology (CBMA), University of Minho, 4710-054 Braga, Portugal.,Institute of Science and Innovation on Bio-sustainability (IB-S), University of Minho, 4710-054 Braga, Portugal
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Can Saccharomyces cerevisiae keep up as a model system in fungal azole susceptibility research? Drug Resist Updat 2019; 42:22-34. [PMID: 30822675 DOI: 10.1016/j.drup.2019.02.002] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2018] [Revised: 01/30/2019] [Accepted: 02/11/2019] [Indexed: 12/14/2022]
Abstract
The difficulty of manipulation and limited availability of genetic tools for use in many pathogenic fungi hamper fast and adequate investigation of cellular metabolism and consequent possibilities for antifungal therapies. S. cerevisiae is a model organism that is used to study many eukaryotic systems. In this review, we analyse the potency and relevance of this model system in investigating fungal susceptibility to azole drugs. Although many of the concepts apply to multiple pathogenic fungi, for the sake of simplicity, we will focus on the validity of using S. cerevisiae as a model organism for two Candida species, C. albicans and C. glabrata. Apart from the general benefits, we explore how S. cerevisiae can specifically be used to improve our knowledge on azole drug resistance and enables fast and efficient screening for novel drug targets in combinatorial therapy. We consider the shortcomings of the model system, yet conclude that it is still opportune to use S. cerevisiae as a model system for pathogenic fungi in this era.
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38
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Lee AR, Lee SJ, Lee M, Nam M, Lee S, Choi J, Lee HJ, Kim DU, Hoe KL. Editor's Highlight: A Genome-wide Screening of Target Genes Against Silver Nanoparticles in Fission Yeast. Toxicol Sci 2019; 161:171-185. [PMID: 29294138 PMCID: PMC5837777 DOI: 10.1093/toxsci/kfx208] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
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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Affiliation(s)
- Ah-Reum Lee
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Sook-Jeong Lee
- Department of Bioactive Material Science, Chonbuk National University, Jeonju 54896, Republic of Korea
| | - Minho Lee
- Catholic Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
| | - Miyoung Nam
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Sol Lee
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Jian Choi
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Hye-Jin Lee
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea
| | - Dong-Uk Kim
- Department of Aging Research Center, KRIBB, Daejeon 34141, Republic of Korea
| | - Kwang-Lae Hoe
- Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea
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Pavlinov I, Gerlach EM, Aldrich LN. Next generation diversity-oriented synthesis: a paradigm shift from chemical diversity to biological diversity. Org Biomol Chem 2019; 17:1608-1623. [PMID: 30328455 DOI: 10.1039/c8ob02327a] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Diversity-oriented synthesis adds biological performance as a new diversity element.
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Affiliation(s)
- Ivan Pavlinov
- University of Illinois at Chicago
- Department of Chemistry
- 845 West Taylor Street
- USA
| | - Erica M. Gerlach
- University of Illinois at Chicago
- Department of Chemistry
- 845 West Taylor Street
- USA
| | - Leslie N. Aldrich
- University of Illinois at Chicago
- Department of Chemistry
- 845 West Taylor Street
- USA
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40
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Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus. Antimicrob Agents Chemother 2018; 63:AAC.01615-18. [PMID: 30397071 DOI: 10.1128/aac.01615-18] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Accepted: 10/31/2018] [Indexed: 12/24/2022] Open
Abstract
Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in Aspergillus fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high-throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show that heterozygous cyp51A and arf2 null mutants have reduced fitness in the presence of itraconazole and brefeldin A, respectively, and a heterozygous atp17 null mutant is resistant to brefeldin A.
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41
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Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ. Antimicrob Agents Chemother 2018; 62:AAC.01231-18. [PMID: 30297366 PMCID: PMC6256756 DOI: 10.1128/aac.01231-18] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2018] [Accepted: 09/01/2018] [Indexed: 12/26/2022] Open
Abstract
To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.
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42
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Prescott TAK, Jaeg T, Hoepfner D. Yeast Chemogenomic Profiling Reveals Iron Chelation To Be the Principle Cell Inhibitory Mode of Action of Gossypol. J Med Chem 2018; 61:7381-7386. [PMID: 30016095 DOI: 10.1021/acs.jmedchem.8b00692] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Gossypol is an inhibitor of eukaryotic cells with an undetermined mode of action. Here we show that the chemogenomic profile of gossypol is strikingly similar to that of the iron chelators deferasirox and desferricoprogen. Iron import channels Fet1 and Fet3 are prominent in all three profiles. Furthermore, yeast inhibited by gossypol and deferasirox is rescued by the addition of Fe2+. We propose that Fe2+ chelation is in fact the principle mode of action of gossypol.
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Affiliation(s)
| | - Tiphaine Jaeg
- Developmental & Molecular Pathways , Novartis Institutes for BioMedical Research, Novartis Pharma AG , Fabrikstrasse 22 , CH-4056 Basel , Switzerland
| | - Dominic Hoepfner
- Developmental & Molecular Pathways , Novartis Institutes for BioMedical Research, Novartis Pharma AG , Fabrikstrasse 22 , CH-4056 Basel , Switzerland
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43
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Canestrari E, Paroo Z. Ribonucleases as Drug Targets. Trends Pharmacol Sci 2018; 39:855-866. [PMID: 30144949 DOI: 10.1016/j.tips.2018.07.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2018] [Revised: 07/19/2018] [Accepted: 07/20/2018] [Indexed: 12/26/2022]
Abstract
Across disease indications, there is immediate need for new drug targets. Target scarcity is reflected in a growing number of same-target drugs of marginal clinical value. Advances in RNA mechanisms of disease are revealing a windfall of targets for nucleic acids therapeutics. However, nucleic acids remain limited as pharmaceutical agents. Because enzymes are predominant drug targets, ribonucleases represent an established target class to capitalize on RNA mechanisms of disease. Analysis of the human proteome identified 122 ribonucleases. This small ribonucleome mediates the biosynthetic and catabolic processing of a large transcriptome. Thus, ribonucleases represent critical signaling targets. Similar to kinases, proteases, and epigenetic enzymes, ribonucleases are rational targets for development of therapies with novel mechanisms, expanding treatment options for improved patient outcomes.
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Affiliation(s)
- Emanuele Canestrari
- Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Zain Paroo
- Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA.
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44
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Garcia C, Burgain A, Chaillot J, Pic É, Khemiri I, Sellam A. A phenotypic small-molecule screen identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell invasion. Sci Rep 2018; 8:11559. [PMID: 30068935 PMCID: PMC6070544 DOI: 10.1038/s41598-018-29973-8] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2018] [Accepted: 07/23/2018] [Indexed: 12/15/2022] Open
Abstract
A poorly exploited paradigm in the antimicrobial therapy field is to target virulence traits for drug development. In contrast to target-focused approaches, antivirulence phenotypic screens enable identification of bioactive molecules that induce a desirable biological readout without making a priori assumption about the cellular target. Here, we screened a chemical library of 678 small molecules against the invasive hyphal growth of the human opportunistic yeast Candida albicans. We found that a halogenated salicylanilide (N1-(3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide) and one of its analogs, Niclosamide, an FDA-approved anthelmintic in humans, exhibited both antifilamentation and antibiofilm activities against C. albicans and the multi-resistant yeast C. auris. The antivirulence activity of halogenated salicylanilides were also expanded to C. albicans resistant strains with different resistance mechanisms. We also found that Niclosamide protected the intestinal epithelial cells against invasion by C. albicans. Transcriptional profiling of C. albicans challenged with Niclosamide exhibited a signature that is characteristic of the mitochondria-to-nucleus retrograde response. Our chemogenomic analysis showed that halogenated salicylanilides compromise the potential-dependant mitochondrial protein translocon machinery. Given the fact that the safety of Niclosamide is well established in humans, this molecule could represent the first clinically approved antivirulence agent against a pathogenic fungus.
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Affiliation(s)
- Carlos Garcia
- CHU de Québec Research Center (CHUQ), Université Laval, Quebec City, QC, Canada
| | - Anaïs Burgain
- CHU de Québec Research Center (CHUQ), Université Laval, Quebec City, QC, Canada
| | - Julien Chaillot
- CHU de Québec Research Center (CHUQ), Université Laval, Quebec City, QC, Canada
| | - Émilie Pic
- CHU de Québec Research Center (CHUQ), Université Laval, Quebec City, QC, Canada
| | - Inès Khemiri
- CHU de Québec Research Center (CHUQ), Université Laval, Quebec City, QC, Canada
| | - Adnane Sellam
- CHU de Québec Research Center (CHUQ), Université Laval, Quebec City, QC, Canada.
- Department of Microbiology-Infectious Disease and Immunology, Faculty of Medicine, Université Laval, Quebec City, QC, Canada.
- Big Data Research Centre (BDRC-UL), Université Laval, Faculty of Sciences and engineering, Quebec City, QC, Canada.
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45
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Esher SK, Ost KS, Kohlbrenner MA, Pianalto KM, Telzrow CL, Campuzano A, Nichols CB, Munro C, Wormley FL, Alspaugh JA. Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition. PLoS Pathog 2018; 14:e1007126. [PMID: 29864141 PMCID: PMC6002136 DOI: 10.1371/journal.ppat.1007126] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2018] [Revised: 06/14/2018] [Accepted: 05/29/2018] [Indexed: 11/19/2022] Open
Abstract
The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.
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Affiliation(s)
- Shannon K. Esher
- Departments of Molecular Genetics and Microbiology/Medicine, Duke University School of Medicine, Durham, NC, United States of America
| | - Kyla S. Ost
- Departments of Molecular Genetics and Microbiology/Medicine, Duke University School of Medicine, Durham, NC, United States of America
| | - Maria A. Kohlbrenner
- Departments of Molecular Genetics and Microbiology/Medicine, Duke University School of Medicine, Durham, NC, United States of America
| | - Kaila M. Pianalto
- Departments of Molecular Genetics and Microbiology/Medicine, Duke University School of Medicine, Durham, NC, United States of America
| | - Calla L. Telzrow
- Departments of Molecular Genetics and Microbiology/Medicine, Duke University School of Medicine, Durham, NC, United States of America
| | - Althea Campuzano
- Department of Biology, University of Texas at San Antonio, San Antonio, Texas, United States of America
| | - Connie B. Nichols
- Departments of Molecular Genetics and Microbiology/Medicine, Duke University School of Medicine, Durham, NC, United States of America
| | - Carol Munro
- MRC Centre for Medical Mycology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, United Kingdom
| | - Floyd L. Wormley
- Department of Biology, University of Texas at San Antonio, San Antonio, Texas, United States of America
| | - J. Andrew Alspaugh
- Departments of Molecular Genetics and Microbiology/Medicine, Duke University School of Medicine, Durham, NC, United States of America
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Gene dosage effects in yeast support broader roles for the LOG1, HAM1 and DUT1 genes in detoxification of nucleotide analogues. PLoS One 2018; 13:e0196840. [PMID: 29738539 PMCID: PMC5940212 DOI: 10.1371/journal.pone.0196840] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Accepted: 04/21/2018] [Indexed: 12/22/2022] Open
Abstract
Purine and pyrimidine analogues have important uses in chemotherapies against cancer, and a better understanding of the mechanisms that cause resistance to these drugs is therefore of importance in cancer treatment. In the yeast Saccharomyces cerevisiae, overexpression of the HAM1 gene encoding inosine triphosphate pyrophosphatase confers resistance to both the purine analogue 6-N-hydroxylaminopurine (HAP) and the pyrimidine analogue 5-fluorouracil (5-FU) (Carlsson et al., 2013, PLoS One 8, e52094). To find out more about the mechanisms of resistance to nucleotide analogues, and possible interdependencies between purine and pyrimidine analogue resistance mechanisms, we screened a plasmid library in yeast for genes that confer HAP resistance when overexpressed. We cloned four such genes: ADE4, DUT1, APT2, and ATR1. We further looked for genetic interactions between these genes and genes previously found to confer resistance to 5-FU. We found that HMS1, LOG1 (YJL055W), HAM1, and ATR1 confer resistance to both 5-FU and HAP, whereas ADE4, DUT1 and APT2 are specific for HAP resistance, and CPA1 and CPA2 specific for 5-FU resistance. Possible mechanisms for 5-FU and HAP detoxification are discussed based on the observed genetic interactions. Based on the effect of LOG1 against both 5-FU and HAP toxicity, we propose that the original function of the LOG (LONELY GUY) family of proteins likely was to degrade non-canonical nucleotides, and that their role in cytokinin production is a later development in some organisms.
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Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism. Oncotarget 2018; 8:46219-46233. [PMID: 28515355 PMCID: PMC5542262 DOI: 10.18632/oncotarget.17597] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2017] [Accepted: 04/06/2017] [Indexed: 11/25/2022] Open
Abstract
5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in colorectal cancer. Previous studies showed that 5-FU modulates RNA metabolism and mRNA expression. In addition, it has been reported that 5-FU incorporates into the RNAs constituting the translational machinery and that 5-FU affects the amount of some mRNAs associated with ribosomes. However, the impact of 5-FU on translational regulation remains unclear. Using translatome profiling, we report that a clinically relevant dose of 5-FU induces a translational reprogramming in colorectal cancer cell lines. Comparison of mRNA distribution between polysomal and non-polysomal fractions in response to 5-FU treatment using microarray quantification identified 313 genes whose translation was selectively regulated. These regulations were mostly stimulatory (91%). Among these genes, we showed that 5-FU increases the mRNA translation of HIVEP2, which encodes a transcription factor whose translation in normal condition is known to be inhibited by mir-155. In response to 5-FU, the expression of mir-155 decreases thus stimulating the translation of HIVEP2 mRNA. Interestingly, the 5-FU-induced increase in specific mRNA translation was associated with reduction of global protein synthesis. Altogether, these findings indicate that 5-FU promotes a translational reprogramming leading to the increased translation of a subset of mRNAs that involves at least for some of them, miRNA-dependent mechanisms. This study supports a still poorly evaluated role of translational control in drug response.
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Datta S, Jankowicz‐Cieslak J, Nielen S, Ingelbrecht I, Till BJ. Induction and recovery of copy number variation in banana through gamma irradiation and low-coverage whole-genome sequencing. PLANT BIOTECHNOLOGY JOURNAL 2018; 16:1644-1653. [PMID: 29476650 PMCID: PMC6097122 DOI: 10.1111/pbi.12901] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/06/2017] [Revised: 02/02/2018] [Accepted: 02/07/2018] [Indexed: 06/08/2023]
Abstract
Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical mutagen EMS. However, officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic insertions and deletions (indels). Such dosage mutations may be important for generating observable phenotypes in polyploids. In this study, we establish a low-coverage whole-genome sequencing approach in triploid bananas to recover large genomic indels caused by treatment with gamma irradiation. We first evaluated the commercially released mutant cultivar 'Novaria' and found that it harbours multiple predicted deletions, ranging from 0.3 to 3.8 million base pairs (Mbp). In total, predicted deletions span 189 coding regions. To evaluate the feasibility of generating and maintaining new mutations, we developed a pipeline for mutagenesis and screening for copy number variation in Cavendish bananas using the cultivar 'Williams'. Putative mutations were recovered in 70% of lines treated with 20 Gy and 60% of the lines treated with 40 Gy. While deletion events predominate, insertions were identified in 20 Gy-treated material. Based on these results, we believe this approach can be scaled up to support large breeding projects.
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Affiliation(s)
- Sneha Datta
- Plant Breeding and Genetics LaboratoryJoint FAO/IAEA Division of Nuclear Techniques in Food and AgricultureIAEA Laboratories SeibersdorfInternational Atomic Energy AgencyVienna International CentreViennaAustria
| | - Joanna Jankowicz‐Cieslak
- Plant Breeding and Genetics LaboratoryJoint FAO/IAEA Division of Nuclear Techniques in Food and AgricultureIAEA Laboratories SeibersdorfInternational Atomic Energy AgencyVienna International CentreViennaAustria
| | - Stephan Nielen
- Plant Breeding and Genetics LaboratoryJoint FAO/IAEA Division of Nuclear Techniques in Food and AgricultureIAEA Laboratories SeibersdorfInternational Atomic Energy AgencyVienna International CentreViennaAustria
| | - Ivan Ingelbrecht
- Plant Breeding and Genetics LaboratoryJoint FAO/IAEA Division of Nuclear Techniques in Food and AgricultureIAEA Laboratories SeibersdorfInternational Atomic Energy AgencyVienna International CentreViennaAustria
| | - Bradley J. Till
- Plant Breeding and Genetics LaboratoryJoint FAO/IAEA Division of Nuclear Techniques in Food and AgricultureIAEA Laboratories SeibersdorfInternational Atomic Energy AgencyVienna International CentreViennaAustria
- Present address:
Department of Chromosome BiologyUniversity of ViennaA‐1030ViennaAustria
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Zampieri M, Szappanos B, Buchieri MV, Trauner A, Piazza I, Picotti P, Gagneux S, Borrell S, Gicquel B, Lelievre J, Papp B, Sauer U. High-throughput metabolomic analysis predicts mode of action of uncharacterized antimicrobial compounds. Sci Transl Med 2018; 10:eaal3973. [PMID: 29467300 PMCID: PMC6544516 DOI: 10.1126/scitranslmed.aal3973] [Citation(s) in RCA: 84] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2016] [Revised: 04/11/2017] [Accepted: 09/27/2017] [Indexed: 12/19/2022]
Abstract
Rapidly spreading antibiotic resistance and the low discovery rate of new antimicrobial compounds demand more effective strategies for early drug discovery. One bottleneck in the drug discovery pipeline is the identification of the modes of action (MoAs) of new compounds. We have developed a rapid systematic metabolome profiling strategy to classify the MoAs of bioactive compounds. The method predicted MoA-specific metabolic responses in the nonpathogenic bacterium Mycobacterium smegmatis after treatment with 62 reference compounds with known MoAs and different metabolic and nonmetabolic targets. We then analyzed a library of 212 new antimycobacterial compounds with unknown MoAs from a drug discovery effort by the pharmaceutical company GlaxoSmithKline (GSK). More than 70% of these new compounds induced metabolic responses in M. smegmatis indicative of known MoAs, seven of which were experimentally validated. Only 8% (16) of the compounds appeared to target unconventional cellular processes, illustrating the difficulty in discovering new antibiotics with different MoAs among compounds used as monotherapies. For six of the GSK compounds with potentially new MoAs, the metabolome profiles suggested their ability to interfere with trehalose and lipid metabolism. This was supported by whole-genome sequencing of spontaneous drug-resistant mutants of the pathogen Mycobacterium tuberculosis and in vitro compound-proteome interaction analysis for one of these compounds. Our compendium of drug-metabolome profiles can be used to rapidly query the MoAs of uncharacterized antimicrobial compounds and should be a useful resource for the drug discovery community.
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Affiliation(s)
- Mattia Zampieri
- Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.
| | - Balazs Szappanos
- Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland
- Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
| | - Maria Virginia Buchieri
- Mycobacterial Genetics Unit, Institut Pasteur, 25-28 Rue du Docteur Roux, 75015 Paris, France
| | - Andrej Trauner
- Swiss Tropical and Public Health Institute, Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Ilaria Piazza
- Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland
| | - Paola Picotti
- Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland
| | - Sébastien Gagneux
- Swiss Tropical and Public Health Institute, Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Sonia Borrell
- Swiss Tropical and Public Health Institute, Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Brigitte Gicquel
- Mycobacterial Genetics Unit, Institut Pasteur, 25-28 Rue du Docteur Roux, 75015 Paris, France
| | - Joel Lelievre
- Disease of the Developing World, GlaxoSmithKline, Severo Ochoa, Tres Cantos, Madrid 28760, Spain
| | - Balazs Papp
- Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
| | - Uwe Sauer
- Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland
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50
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Abstract
A long-standing challenge in drug development is the identification of the mechanisms of action of small molecules with therapeutic potential. A number of methods have been developed to address this challenge, each with inherent strengths and limitations. We here provide a brief review of these methods with a focus on chemical-genetic methods that are based on systematically profiling the effects of genetic perturbations on drug sensitivity. In particular, application of these methods to mammalian systems has been facilitated by the recent advent of CRISPR-based approaches, which enable one to readily repress, induce, or delete a given gene and determine the resulting effects on drug sensitivity.
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Affiliation(s)
- Marco Jost
- Department
of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute,
Center for RNA Systems Biology, University of California, San Francisco, San
Francisco, California 94158, United States
- Department
of Microbiology and Immunology, University of California, San Francisco, San
Francisco, California 94158, United States
| | - Jonathan S. Weissman
- Department
of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute,
Center for RNA Systems Biology, University of California, San Francisco, San
Francisco, California 94158, United States
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