Abstract
Linking bioactive compounds to their cellular targets is a central challenge in chemical biology. Here we report the mode of action of theonellamides, bicyclic peptides derived from marine sponges. We generated a chemical-genomic profile of theonellamide F using a collection of fission yeast strains in which each open reading frame (ORF) is expressed under the control of an inducible promoter. Clustering analysis of the Gene Ontology (GO) terms associated with the genes that alter drug sensitivity suggested a mechanistic link between theonellamide and 1,3-β-D-glucan synthesis. Indeed, theonellamide F induced overproduction of 1,3-β-D-glucan in a Rho1-dependent manner. Subcellular localization and in vitro binding assays using a fluorescent theonellamide derivative revealed that theonellamides specifically bind to 3β-hydroxysterols, including ergosterol, and cause membrane damage. The biological activity of theonellamides was alleviated in mutants defective in ergosterol biosynthesis. Theonellamides thus represent a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-β-D-glucan synthesis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Baltz, R.H. Daptomycin: mechanisms of action and resistance, and biosynthetic engineering. Curr. Opin. Chem. Biol. 13, 144–151 (2009).
Denning, D.W. Echinocandin antifungal drugs. Lancet 362, 1142–1151 (2003).
Rovira, P., Mascarell, L. & Truffa-Bachi, P. The impact of immunosuppressive drugs on the analysis of T-cell activation. Curr. Med. Chem. 7, 673–692 (2000).
Dawson, S., Malkinson, J.P., Paumier, D. & Searcey, M. Bisintercalator natural products with potential therapeutic applications: isolation, structure determination, synthetic and biological studies. Nat. Prod. Rep. 24, 109–126 (2007).
Kong, D. et al. Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity. Cancer Res. 65, 9047–9055 (2005).
Vera, M.D. & Joullie, M.M. Natural products as probes of cell biology: 20 years of didemnin research. Med. Res. Rev. 22, 102–145 (2002).
Matsunaga, S. & Fusetani, N. Nonribosomal peptides from marine sponges. Curr. Org. Chem. 7, 945–966 (2003).
Matsunaga, S., Fusetani, N., Hashimoto, K. & Walchli, M. Theonellamide-F - a novel antifungal bicyclic peptide from a marine sponge Theonella sp. J. Am. Chem. Soc. 111, 2582–2588 (1989).
Matsunaga, S. & Fusetani, N. Theonellamides A–E, cytotoxic bicyclic peptides, from a marine sponge Theonella sp. J. Org. Chem. 60, 1177–1181 (1995).
Bewley, C.A. & Faulkner, D.J. Theonegramide, an antifungal glycopeptide from the Philippine lithistid sponge Theonella swinhoei. J. Org. Chem. 59, 4849–4852 (1994).
Schmidt, E.W., Bewley, C.A. & Faulkner, D.J. Theopalauamide, a bicyclic glycopeptide from filamentous bacterial symbionts of the lithistid sponge Theonella swinhoei from Palau and Mozambique. J. Org. Chem. 63, 1254–1258 (1998).
Wada, S., Matsunaga, S., Fusetani, N. & Watabe, S. Interaction of cytotoxic bicyclic peptides, theonellamides A and F, with glutamate dehydrogenase and 17beta-hydroxysteroid dehydrogenase IV. Mar. Biotechnol. (NY) 2, 285–292 (2000).
Matsuyama, A. et al. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol. 24, 841–847 (2006).
Shirai, A. et al. Global analysis of gel mobility of proteins and its use in target identification. J. Biol. Chem. 283, 10745–10752 (2008).
Kanoh, N., Honda, K., Simizu, S., Muroi, M. & Osada, H. Photo-cross-linked small-molecule affinity matrix for facilitating forward and reverse chemical genetics. Angew. Chem. Int. Edn Engl. 44, 3559–3562 (2005).
Hughes, T.R. et al. Functional discovery via a compendium of expression profiles. Cell 102, 109–126 (2000).
Arellano, M. et al. Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity. J. Cell Sci. 112, 3569–3578 (1999).
Tomishima, M. et al. FK463, a novel water-soluble echinocandin lipopeptide: synthesis and antifungal activity. J. Antibiot. (Tokyo) 52, 674–676 (1999).
Deng, L. et al. Phosphatidylinositol-4-phosphate 5-kinase regulates fission yeast cell integrity through a phospholipase C-mediated protein kinase C-independent pathway. J. Biol. Chem. 280, 27561–27568 (2005).
Kippert, F. & Lloyd, D. The aniline blue fluorochrome specifically stains the septum of both live and fixed Schizosaccharomyces pombe cells. FEMS Microbiol. Lett. 132, 215–219 (1995).
Arellano, M., Duran, A. & Perez, P. Rho 1 GTPase activates the (1–3)beta-D-glucan synthase and is involved in Schizosaccharomyces pombe morphogenesis. EMBO J. 15, 4584–4591 (1996).
Arellano, M., Duran, A. & Perez, P. Localisation of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organisation of the actin cytoskeleton. J. Cell Sci. 110, 2547–2555 (1997).
Nakano, K., Arai, R. & Mabuchi, I. The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe. Genes Cells 2, 679–694 (1997).
Sayers, L.G. et al. Rho-dependence of Schizosaccharomyces pombe Pck2. Genes Cells 5, 17–27 (2000).
Takeda, T., Kawate, T. & Chang, F. Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeast. Nat. Cell Biol. 6, 1142–1144 (2004).
Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).
Iwaki, T. et al. Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe. Microbiology 154, 830–841 (2008).
Drabikowski, W., Lagwińska, E. & Sarzala, M.G. Filipin as a fluorescent probe for location of cholesterol in membranes of fragmented sarcoplasmic reticulum. Biochim. Biophys. Acta 291, 61–70 (1973).
Wachtler, V., Rajagopalan, S. & Balasubramanian, M.K. Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 116, 867–874 (2003).
Codlin, S., Haines, R.L. & Mole, S.E. btn1 affects endocytosis, polarization of sterol-rich membrane domains and polarized growth in Schizosaccharomyces pombe. Traffic 9, 936–950 (2008).
Ishiguro, J. & Kobayashi, W. An actin point-mutation neighboring the 'hydrophobic plug' causes defects in the maintenance of cell polarity and septum organization in the fission yeast Schizosaccharomyces pombe. FEBS Lett. 392, 237–241 (1996).
Villar-Tajadura, M.A. et al. Rga2 is a Rho2 GAP that regulates morphogenesis and cell integrity in S. pombe. Mol. Microbiol. 70, 867–881 (2008).
Chang, E.C. et al. Cooperative interaction of S. pombe proteins required for mating and morphogenesis. Cell 79, 131–141 (1994).
Iwaki, N., Karatsu, K. & Miyamoto, M. Role of guanine nucleotide exchange factors for Rho family GTPases in the regulation of cell morphology and actin cytoskeleton in fission yeast. Biochem. Biophys. Res. Commun. 312, 414–420 (2003).
Hampsey, M. A review of phenotypes in Saccharomyces cerevisiae. Yeast 13, 1099–1133 (1997).
Garton, S., Michaelson, L.V., Beaudoin, F., Beale, M.H. & Napier, J.A. The dihydroceramide desaturase is not essential for cell viability in Schizosaccharomyces pombe. FEBS Lett. 538, 192–196 (2003).
Takeo, K. et al. Rapid, extensive and reversible vacuolation of Schizosaccharomyces pombe induced by amphotericin B. FEMS Microbiol. Lett. 108, 265–269 (1993).
Piel, J. Bacterial symbionts: prospects for the sustainable production of invertebrate-derived pharmaceuticals. Curr. Med. Chem. 13, 39–50 (2006).
Ho, C.H. et al. A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds. Nat. Biotechnol. 27, 369–377 (2009).
Bolard, J. How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim. Biophys. Acta 864, 257–304 (1986).
Shogomori, H. & Kobayashi, T. Lysenin: a sphingomyelin specific pore-forming toxin. Biochim. Biophys. Acta 1780, 612–618 (2008).
Matsuyama, A. et al. pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast. Yeast 21, 1289–1305 (2004).
Gachet, Y. & Hyams, J.S. Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis. J. Cell Sci. 118, 4231–4242 (2005).
Acknowledgements
We thank C. Boone (Univ. Toronto) for sharing unpublished results and discussions, A. Fujie (Astellas Pharma) for kind gifts of FK463 and cispentacin, K. Takegawa (Kyushu Univ.) for erg deletion strains, T. Kuno (Kobe Univ.) for the bgs1 mutant and pck deletion strains and K. Nakano (University of Tsukuba) for rho1-expression plasmids. We also thank J. Ishiguro (Konan Univ.) for the act1/cps8 mutant strain, which was provided through the Yeast Genetic Resource Center (YGRC), N. Kanoh, S. Shimizu and H. Osada (RIKEN Advanced Science Institute) for helping in the preparation of TNM affinity beads, J. Ochi (Kyoto Univ.) for technical assistance and J. Piotrowski (RIKEN Advanced Science Institute) for critical reading of the manuscript. We are grateful to the RIKEN Brain Science Institute's Research Resource Center for mass spectrometry. This work was supported in part by the New Energy and Industrial Technology Development Organization Project on Development of Basic Technology to Control Biological Systems Using Chemical Compounds, a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Chemical Genomics Research Project, RIKEN Advanced Science Institute.
Author information
Authors and Affiliations
Contributions
M.Y. is responsible for project planning and experimental design, with support from K.I., H. Kawasaki, H. Kakeya and T.K.; S.N. performed most of the experiments; Y.A. assisted in vitro sterol binding experiments; M.H. assisted chemical-genomic screen; A.M. and A.S. prepared the yeast strain collection; S.M. prepared theonellamides.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Methods, Supplementary Figures 1–20, Supplementary Tables 1–3 and Supplementary Datasets 1–8 (PDF 2119 kb)
Rights and permissions
About this article
Cite this article
Nishimura, S., Arita, Y., Honda, M. et al. Marine antifungal theonellamides target 3β-hydroxysterol to activate Rho1 signaling. Nat Chem Biol 6, 519–526 (2010). https://doi.org/10.1038/nchembio.387
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.387
This article is cited by
-
Marine natural products targeting the eukaryotic cell membrane
The Journal of Antibiotics (2021)
-
Biodistribution of arctigenin-loaded nanoparticles designed for multimodal imaging
Journal of Nanobiotechnology (2017)
-
Cholesterol impairment contributes to neuroserpin aggregation
Scientific Reports (2017)
-
Susceptibility of outer hair cells to cholesterol chelator 2-hydroxypropyl-β-cyclodextrine is prestin-dependent
Scientific Reports (2016)
-
Functional genomics to uncover drug mechanism of action
Nature Chemical Biology (2015)