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The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition*

  • Yanhong Pang
    Affiliations
    From the Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden,
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  • Sriram Kurella
    Affiliations
    From the Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden,
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  • Cécile Voisset
    Affiliations
    INSERM UMR 1078, Université de Bretagne Occidentale, Faculté de Médecine et des Sciences de la Santé, Etablissement Français du Sang (EFS) Bretagne, Centre Hospitalier Régional Universitaire (CHRU) de Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, F-29200 Brest, France,
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  • Dibyendu Samanta
    Footnotes
    Affiliations
    Department of Biophysics, Molecular Biology and Genetics, Calcutta University, Kolkata-700009, India
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  • Debapriya Banerjee
    Affiliations
    From the Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden,
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  • Ariane Schabe
    Footnotes
    Affiliations
    From the Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden,
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  • Chanchal Das Gupta
    Affiliations
    Department of Biophysics, Molecular Biology and Genetics, Calcutta University, Kolkata-700009, India
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  • Hervé Galons
    Affiliations
    Laboratoire de Chimie Organique 2, CNRS UMR 8601, Université Paris Descartes, 75270 Paris Cedex 6, France
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  • Marc Blondel
    Affiliations
    INSERM UMR 1078, Université de Bretagne Occidentale, Faculté de Médecine et des Sciences de la Santé, Etablissement Français du Sang (EFS) Bretagne, Centre Hospitalier Régional Universitaire (CHRU) de Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, F-29200 Brest, France,
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  • Suparna Sanyal
    Correspondence
    To whom correspondence should be addressed. Tel.: 46-18-471-4220; Fax: 46-18-471-4262;.
    Affiliations
    From the Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden,
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  • Author Footnotes
    * This work was supported by the Swedish Research Council (individual grants from the M and NT sections, VR-SIDA (Swedish Research Link), and a Linnaeus grant to the Uppsala RNA Research Center); the Carl Tryggers Stiftelse; a postdoctoral stipendium from the Wenner Gren Stiftelse (to D. B.); the Knut and Wallice Wallenberg Foundation (to RiboCORE), Vinnova/DBT (India), and the SSF-Dalen (Sweden-France Bilateral Collaboration) Program (to S. S.); a scholarship from the Chinese Scholarship Council (to Y. P.); and INSERM, CRITT Santé Bretagne (Région Bretagne), and ANR “Blanche” of the French Government (to M. B. and C. V.).
    1 Present Address: Dept. of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.
    2 Present Address: Max-Delbr”ck-Centrum for Molekular Medicine, Robert-Rössle-Str. 10, 13125 Berlin-Buch, Germany.
Open AccessPublished:May 14, 2013DOI:https://doi.org/10.1074/jbc.M113.466748
      Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR.
      Background: 6-Aminophenanthridine (6AP) is an inhibitor of the protein folding activity of the ribosome (PFAR).
      Results: The protein substrates and 6AP bind at common sites on rRNA; mutations at those sites abolish binding and inhibit PFAR.
      Conclusion: 6AP competitively obstructs the protein-binding sites and thereby inhibits PFAR.
      Significance: We have clarified the mechanism by which 6AP inhibits PFAR.

      Introduction

      It has been shown over the past 2 decades that the ribosome is able to refold ∼20 different proteins in vitro (
      • Bera A.K.
      • Das B.
      • Chattopadhyay S.
      • DasGupta C.
      Protein folding by ribosome and its RNA.
      ,
      • Das B.
      • Chattopadhyay S.
      • Bera A.K.
      • DasGupta C.
      In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver–the role of the 50S particle and its 23S rRNA.
      ,
      • Argent R.H.
      • Parrott A.M.
      • Day P.J.
      • Roberts L.M.
      • Stockley P.G.
      • Lord J.M.
      • Radford S.E.
      Ribosome-mediated folding of partially unfolded ricin A-chain.
      ,
      • Das D.
      • Das A.
      • Samanta D.
      • Ghosh J.
      • Dasgupta S.
      • Bhattacharya A.
      • Basu A.
      • Sanyal S.
      • Das Gupta C.
      Role of the ribosome in protein folding.
      ,
      • Kudlicki W.
      • Coffman A.
      • Kramer G.
      • Hardesty B.
      Ribosomes and ribosomal RNA as chaperones for folding of proteins.
      ). The protein folding activity of the ribosome (PFAR)
      The abbreviations used are:PFAR, protein folding activity of the ribosome; 6AP, 6-aminophenanthridine; HCA, human carbonic anhydrase.
      is not restricted to any particular species or groups of organisms because ribosomes from various sources have been shown to possess this activity (
      • Bera A.K.
      • Das B.
      • Chattopadhyay S.
      • DasGupta C.
      Protein folding by ribosome and its RNA.
      ,
      • Das B.
      • Chattopadhyay S.
      • Bera A.K.
      • DasGupta C.
      In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver–the role of the 50S particle and its 23S rRNA.
      ,
      • Sulijoadikusumo I.
      • Horikoshi N.
      • Usheva A.
      Another function for the mitochondrial ribosomal RNA: protein folding.
      ). Also, the protein substrates of PFAR are not limited to a specific protein family; proteins from diverse sources with various properties can be folded by ribosomes (
      • Das D.
      • Das A.
      • Samanta D.
      • Ghosh J.
      • Dasgupta S.
      • Bhattacharya A.
      • Basu A.
      • Sanyal S.
      • Das Gupta C.
      Role of the ribosome in protein folding.
      ). The active site for PFAR lies in the large subunit of the ribosome (50S in bacteria and 60S in eukaryotes) and, similar to peptidyl transferase activity, involves rRNA (
      • Das B.
      • Chattopadhyay S.
      • Bera A.K.
      • DasGupta C.
      In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver–the role of the 50S particle and its 23S rRNA.
      ,
      • Chattopadhyay S.
      • Das B.
      • DasGupta C.
      Reactivation of denatured proteins by 23S ribosomal RNA: role of domain V.
      ). In fact, both of these crucial functions of the ribosome share the same active center, i.e. domain V of the 23S rRNA in bacteria and the 25S/28S rRNA in eukaryotes (
      • Chattopadhyay S.
      • Das B.
      • DasGupta C.
      Reactivation of denatured proteins by 23S ribosomal RNA: role of domain V.
      ,
      • Sanyal S.C.
      • Pal S.
      • Chowdhury S.
      • DasGupta C.
      23S rRNA assisted folding of cytoplasmic malate dehydrogenase is distinctly different from its self-folding.
      ,
      • Pal D.
      • Chattopadhyay S.
      • Chandra S.
      • Sarkar D.
      • Chakraborty A.
      • Das Gupta C.
      Reactivation of denatured proteins by domain V of bacterial 23S rRNA.
      ,
      • Pal S.
      • Chandra S.
      • Chowdhury S.
      • Sarkar D.
      • Ghosh A.N.
      • Das Gupta C.
      Complementary role of two fragments of domain V of 23 S ribosomal RNA in protein folding.
      ). The same domain from the mitochondrial ribosome also displays activity in refolding proteins (
      • Sulijoadikusumo I.
      • Horikoshi N.
      • Usheva A.
      Another function for the mitochondrial ribosomal RNA: protein folding.
      ,
      • Das A.
      • Ghosh J.
      • Bhattacharya A.
      • Samanta D.
      • Das D.
      • Das Gupta C.
      Involvement of mitochondrial ribosomal proteins in ribosomal RNA-mediated protein folding.
      ). This RNA domain (referred hereafter as domain V rRNA) is usually free from any ribosomal protein and lies in the subunit interface of the 70S/80S ribosome. However, upon splitting of the ribosomal subunits, it is exposed on the surface of the large subunit. Thus, in vitro, 50S/60S ribosomal subunits show a more pronounced protein folding activity than the fully assembled 70S/80S ribosomes (
      • Basu A.
      • Samanta D.
      • Bhattacharya A.
      • Das A.
      • Das D.
      • DasGupta C.
      Protein folding following synthesis in vitro and in vivo: association of newly synthesized protein with 50S subunit of E. coli ribosome.
      ,
      • Basu A.
      • Samanta D.
      • Das D.
      • Chowdhury S.
      • Bhattacharya A.
      • Ghosh J.
      • Das A.
      • DasGupta C.
      In vitro protein folding by E. coli ribosome: unfolded protein splitting 70S to interact with 50S subunit.
      ).
      Despite a series of in vitro demonstrations of PFAR, a question still remains open in the field. Is PFAR functional in the modern cells, or is it an evolutionary relic representing function of an ancient protein production machine? Although there are few reports of PFAR in living bacterial cells (
      • Chattopadhyay S.
      • Pal S.
      • Pal D.
      • Sarkar D.
      • Chandra S.
      • Das Gupta C.
      Protein folding in Escherichia coli: role of 23S ribosomal RNA.
      ,
      • Tribouillard-Tanvier D.
      • Dos Reis S.
      • Gug F.
      • Voisset C.
      • Béringue V.
      • Sabate R.
      • Kikovska E.
      • Talarek N.
      • Bach S.
      • Huang C.
      • Desban N.
      • Saupe S.J.
      • Supattapone S.
      • Thuret J.Y.
      • Chédin S.
      • Vilette D.
      • Galons H.
      • Sanyal S.
      • Blondel M.
      Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs.
      ), the in vivo context of PFAR has not been fully established. However, one recent finding has linked PFAR to living cells and also associated it with diseases of higher eukaryotes. It has been shown that the two unrelated compounds 6-aminophenanthridine (6AP) and guanabenz acetate, with demonstrated activity against yeast ([PSI+] and [URE3]) and mammalian prions, bind to rRNA and inhibit PFAR (
      • Tribouillard-Tanvier D.
      • Dos Reis S.
      • Gug F.
      • Voisset C.
      • Béringue V.
      • Sabate R.
      • Kikovska E.
      • Talarek N.
      • Bach S.
      • Huang C.
      • Desban N.
      • Saupe S.J.
      • Supattapone S.
      • Thuret J.Y.
      • Chédin S.
      • Vilette D.
      • Galons H.
      • Sanyal S.
      • Blondel M.
      Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs.
      ,
      • Bach S.
      • Talarek N.
      • Andrieu T.
      • Vierfond J.M.
      • Mettey Y.
      • Galons H.
      • Dormont D.
      • Meijer L.
      • Cullin C.
      • Blondel M.
      Isolation of drugs active against mammalian prions using a yeast-based screening assay.
      ,
      • Tribouillard-Tanvier D.
      • Béringue V.
      • Desban N.
      • Gug F.
      • Bach S.
      • Voisset C.
      • Galons H.
      • Laude H.
      • Vilette D.
      • Blondel M.
      Antihypertensive drug guanabenz is active in vivo against both yeast and mammalian prions.
      ). The correlation between the antiprion activity of these two drugs and their ability to specifically inhibit PFAR suggests that PFAR could be involved in the establishment or maintenance of the prion processes in cells. This notion was further reinforced by the discovery that a 6AP derivative called 6APi, in which the 6-amino group of 6AP is substituted with 2-butan-1-ol, was inactive in both the reversion of the prion phenotype in vivo and inhibition of PFAR in vitro (
      • Tribouillard-Tanvier D.
      • Dos Reis S.
      • Gug F.
      • Voisset C.
      • Béringue V.
      • Sabate R.
      • Kikovska E.
      • Talarek N.
      • Bach S.
      • Huang C.
      • Desban N.
      • Saupe S.J.
      • Supattapone S.
      • Thuret J.Y.
      • Chédin S.
      • Vilette D.
      • Galons H.
      • Sanyal S.
      • Blondel M.
      Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs.
      ). In a different context, PFAR was suggested to be involved in another amyloid-based disease, oculopharyngeal muscular dystrophy, which is an inherited myodegenerative disease caused by the aggregation of PABPN1 protein into amyloid fibers within the nucleus of muscle cells (
      • Barbezier N.
      • Chartier A.
      • Bidet Y.
      • Buttstedt A.
      • Voisset C.
      • Galons H.
      • Blondel M.
      • Schwarz E.
      • Simonelig M.
      Antiprion drugs 6-aminophenanthridine and guanabenz reduce PABPN1 toxicity and aggregation in oculopharyngeal muscular dystrophy.
      ). Thus, even though the involvement of PFAR in prion processes has yet to be directly demonstrated, 6AP and guanabenz acetate constitute valuable tools for studying PFAR (
      • Voisset C.
      • Thuret J.Y.
      • Tribouillard-Tanvier D.
      • Saupe S.J.
      • Blondel M.
      Tools for the study of ribosome-borne protein folding activity.
      ).
      In this work, we elucidated how 6AP inhibits PFAR. Using UV cross-linking followed by primer extension, we determined that the protein substrates of PFAR and 6AP (but not the inactive analog 6APi) interacted with largely overlapping sites of domain V of 23S rRNA. Mutations in the interaction sites not only abolished or changed the interaction map of both the protein substrates and 6AP but also decreased the protein folding activity of domain V of rRNA from both Escherichia coli and Saccharomyces cerevisiae. Moreover, we determined that 6AP did not affect the kinetics of PFAR but reduced the yield of the refolded protein. Our results led to a simple model for PFAR inhibition by 6AP.

      Acknowledgments

      We acknowledge Lars Hellman (Uppsala University) for allowing us to use the UV cross-linker. We also thank Susan Liebman (University of Illinois, Chicago, IL) for providing the construct for 25S rRNA. Xueliang Ge and Petar Kovachev are acknowledged for help in manuscript preparation.

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