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Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin

Nature Chemical Biology volume 6, pages 209–217 (2010)Cite this article

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Abstract

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

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Figure 1
Figure 2: Inhibition of protein translation by LTM and isomigrastatin.
Figure 3: Effects of LTM and cycloheximide on translation elongation in vitro and in vivo.
Figure 4: Effects of LTM and cycloheximide on different steps of translation elongation.
Figure 5: Footprinting analysis revealed the common binding sites of LTM and cycloheximide at the E-site of the larger ribosome subunit.
Figure 6: Mechanistic models for inhibition of translation elongation by CHX and LTM.

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References

  1. Poehlsgaard, J. & Douthwaite, S. The bacterial ribosome as a target for antibiotics. Nat. Rev. Microbiol. 3, 870–881 (2005).

    Article  CAS  Google Scholar 

  2. Obrig, T.G., Culp, W.J., McKeehan, W.L. & Hardesty, B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J. Biol. Chem. 246, 174–181 (1971).

    CAS  PubMed  Google Scholar 

  3. Pestova, T.V. & Hellen, C.U. Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA. Genes Dev. 17, 181–186 (2003).

    Article  CAS  Google Scholar 

  4. Ju, J., Lim, S.K., Jiang, H., Seo, J.W. & Shen, B. Iso-migrastatin congeners from Streptomyces platensis and generation of a glutarimide polyketide library featuring the dorrigocin, lactimidomycin, migrastatin, and NK30424 scaffolds. J. Am. Chem. Soc. 127, 11930–11931 (2005).

    Article  CAS  Google Scholar 

  5. Sugawara, K. et al. Lactimidomycin, a new glutarimide group antibiotic. Production, isolation, structure and biological activity. J. Antibiot. (Tokyo) 45, 1433–1441 (1992).

    Article  CAS  Google Scholar 

  6. Gaul, C. et al. The migrastatin family: discovery of potent cell migration inhibitors by chemical synthesis. J. Am. Chem. Soc. 126, 11326–11337 (2004).

    Article  CAS  Google Scholar 

  7. Shan, D. et al. Synthetic analogues of migrastatin that inhibit mammary tumor metastasis in mice. Proc. Natl. Acad. Sci. USA 102, 3772–3776 (2005).

    Article  CAS  Google Scholar 

  8. Kadam, S. & McAlpine, J.B. Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. III. Biological properties and mechanism of action. J. Antibiot. (Tokyo) 47, 875–880 (1994).

    Article  CAS  Google Scholar 

  9. Karwowski, J.P. et al. Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. I. Taxonomy of the producing organism, fermentation and biological activity. J. Antibiot. (Tokyo) 47, 862–869 (1994).

    Article  CAS  Google Scholar 

  10. Ju, J., Lim, S.K., Jiang, H. & Shen, B. Migrastatin and dorrigocins are shunt metabolites of iso-migrastatin. J. Am. Chem. Soc. 127, 1622–1623 (2005).

    Article  CAS  Google Scholar 

  11. Feng, Z. et al. Engineered production of iso-migrastatin in heterologous Streptomyces hosts. Bioorg. Med. Chem. 17, 2147–2153 (2009).

    Article  CAS  Google Scholar 

  12. Nakae, K. et al. Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929–43F1. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. (Tokyo) 53, 1130–1136 (2000a).

    Article  CAS  Google Scholar 

  13. Nakae, K. et al. Migrastatin, a novel 14-membered lactone from Streptomyces sp. MK929–43F1. J. Antibiot. (Tokyo) 53, 1228–1230 (2000).

    Article  CAS  Google Scholar 

  14. Fried, H.M. & Warner, J.R. Molecular cloning and analysis of yeast gene for cycloheximide resistance and ribosomal protein L29. Nucleic Acids Res. 10, 3133–3148 (1982).

    Article  CAS  Google Scholar 

  15. Kaufer, N.F., Fried, H.M., Schwindinger, W.F., Jasin, M. & Warner, J.R. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 11, 3123–3135 (1983).

    Article  CAS  Google Scholar 

  16. Planta, R.J. & Mager, W.H. The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Yeast 14, 471–477 (1998).

    Article  CAS  Google Scholar 

  17. Stevens, D.R., Atteia, A., Franzen, L.G. & Purton, S. Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii. Mol. Gen. Genet. 264, 790–795 (2001).

    Article  CAS  Google Scholar 

  18. Kawai, S. et al. Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts. J. Bacteriol. 174, 254–262 (1992).

    Article  CAS  Google Scholar 

  19. Robert, F. et al. Altering chemosensitivity by modulating translation elongation. PLoS One 4, e5428 (2009).

    Article  Google Scholar 

  20. Tai, P.C., Wallace, B.J. & Davis, B.D. Selective action of erythromycin on initiating ribosomes. Biochemistry 13, 4653–4659 (1974).

    Article  CAS  Google Scholar 

  21. Anthony, D.D. & Merrick, W.C. Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J. Biol. Chem. 267, 1554–1562 (1992).

    CAS  PubMed  Google Scholar 

  22. Jan, E. & Sarnow, P. Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus. J. Mol. Biol. 324, 889–902 (2002).

    Article  CAS  Google Scholar 

  23. Novac, O., Guenier, A.S. & Pelletier, J. Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res. 32, 902–915 (2004).

    Article  CAS  Google Scholar 

  24. Algire, M.A. & Lorsch, J.R. Where to begin? The mechanism of translation initiation codon selection in eukaryotes. Curr. Opin. Chem. Biol. 10, 480–486 (2006).

    Article  CAS  Google Scholar 

  25. Kapp, L.D. & Lorsch, J.R. The molecular mechanics of eukaryotic translation. Annu. Rev. Biochem. 73, 657–704 (2004).

    Article  CAS  Google Scholar 

  26. Bordeleau, M.E. et al. Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation. Proc. Natl. Acad. Sci. USA 102, 10460–10465 (2005).

    Article  CAS  Google Scholar 

  27. Rodnina, M.V. et al. Mechanism of tRNA translocation on the ribosome. Mol. Biol. (Mosk.) 35, 655–665 (2001).

    Article  CAS  Google Scholar 

  28. Merrick, W.C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56, 291–315 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lorsch, J.R. & Herschlag, D. Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro. EMBO J. 18, 6705–6717 (1999).

    Article  CAS  Google Scholar 

  30. Saini, P., Eyler, D.E., Green, R. & Dever, T.E. Hypusine-containing protein eIF5A promotes translation elongation. Nature 459, 118–121 (2009).

    Article  CAS  Google Scholar 

  31. Holmberg, L., Melander, Y. & Nygard, O. Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ. Nucleic Acids Res. 22, 1374–1382 (1994).

    Article  CAS  Google Scholar 

  32. Chandramouli, P. et al. Structure of the mammalian 80S ribosome at 8.7 A resolution. Structure 16, 535–548 (2008).

    Article  CAS  Google Scholar 

  33. Cannone, J.J. et al. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3, 2 (2002).

    Article  Google Scholar 

  34. Moazed, D. & Noller, H.F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell 57, 585–597 (1989).

    Article  CAS  Google Scholar 

  35. Ledoux, S. & Uhlenbeck, O.C. [3′-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation. Methods 44, 74–80 (2008).

    Article  CAS  Google Scholar 

  36. Lill, R., Robertson, J.M. & Wintermeyer, W. Binding of the 3′ terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation. EMBO J. 8, 3933–3938 (1989).

    Article  CAS  Google Scholar 

  37. Ruggero, D. & Pandolfi, P.P. Does the ribosome translate cancer? Nat. Rev. Cancer 3, 179–192 (2003).

    Article  CAS  Google Scholar 

  38. Rinehart, K.L. Antitumor compounds from tunicates. Med. Res. Rev. 20, 1–27 (2000).

    Article  CAS  Google Scholar 

  39. SirDeshpande, B.V. & Toogood, P.L. Mechanism of protein synthesis inhibition by didemnin B in vitro. Biochemistry 34, 9177–9184 (1995).

    Article  CAS  Google Scholar 

  40. Acker, M.G., Kolitz, S.E., Mitchell, S.F., Nanda, J.S. & Lorsch, J.R. Reconstitution of yeast translation initiation. Methods Enzymol. 430, 111–145 (2007).

    Article  CAS  Google Scholar 

  41. Smith, C.W.J. RNA-Protein Interactions: a Practical Approach (Oxford University Press, 1998).

  42. Holmberg, L., Melander, Y. & Nygard, O. Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes. Nucleic Acids Res. 22, 2776–2783 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We are indebted to J. Boeke (Johns Hopkins University) and J. Warner (Albert Einstein College of Medicine) for the CHX-resistant strains of S. cerevisiae, J. Pelletier (McGill University) for providing us with the HCV and EMCV IRES reporter constructs and P. Sarnow (Stanford University) for providing the CrPV vector. We thank the laboratories of J. Hart, P. Englund, J. Lorsch, S. Sukumar and R. Rao for use of specialized equipment and constructive advice. This work was supported in part by grants from the US National Cancer Institute and the Flight Attendant Medical Research Institute (J.O.L.) and by US National Cancer Institute grants CA106150 and CA113297 (B.S.).

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Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Tilman Schneider-Poetsch, Yongjun Dang, Shridhar Bhat & Jun O Liu

  2. Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin, USA

    Jianhua Ju & Ben Shen

  3. Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Daniel E Eyler & Rachel Green

  4. Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA

    William C Merrick

  5. University of Wisconsin National Cooperative Drug Discovery Group, Madison, Wisconsin, USA

    Ben Shen

  6. Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA

    Ben Shen

  7. Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Jun O Liu

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  1. Tilman Schneider-Poetsch
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Contributions

T.S.-P. and J.O.L. designed the experiments; T.S.-P., D.E.E., Y.D. and S.B. performed the experiments; J.J., W.C.M., R.G. and B.S. contributed reagents; T.S.-P., D.E.E., Y.D., R.G., B.S. and J.O.L. analyzed data and T.S.-P. and J.O.L. wrote the manuscript.

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Correspondence to Jun O Liu.

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Schneider-Poetsch, T., Ju, J., Eyler, D. et al. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6, 209–217 (2010). https://doi.org/10.1038/nchembio.304

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