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The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 26, 19081-19089 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2013. Vol. 288, no 26, 19081-19089 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-205006DOI: 10.1074/jbc.M113.466748ISI: 000321335800043OAI: oai:DiVA.org:uu-205006DiVA: diva2:640512
Available from: 2013-08-13 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Protein Folding Activity of the Ribosome and Its Implication in Prion Processes
Open this publication in new window or tab >>Protein Folding Activity of the Ribosome and Its Implication in Prion Processes
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

How the linear protein chains fold into their three-dimensional active conformation is one of the remaining puzzles of modern science. Other than molecular chaperones, ribosome - the cellular protein synthesis machinery, has also been implicated in protein folding. The active site of protein folding activity of the ribosome (PFAR) is in the domain V of the main RNA component of the large ribosomal subunit, which also constitutes the peptidyl transferase center.

We have characterized the mechanism of PFAR using ribosomes or ribosome-borne folding modulators (RFMs) and human carbonic anhydrase I (HCA) as a model system. RFMs from all three kingdoms of life showed PFAR.  By multiple addition of the denatured protein in the refolding assay we demonstrate that the RFMs can recycle efficiently to assist refolding of a new batch of denatured protein. The turnover of the RFMs, which includes release of the protein substrate, takes milliseconds. Furthermore, fast kinetics of HCA refolding suggests that an early folding intermediate is the substrate for PFAR. Our results demonstrate for the first time that PFAR is catalytic.

It was shown that two anti-prion drugs 6AP and GA specifically inhibit PFAR by binding to the domain V of the 23S / 25S rRNA. Using UV-crosslinking followed by primer extension we have identified the interaction sites of 6AP on domain V of 23S rRNA, which overlap with the protein binding sites, and are sensitive to mutagenesis. We find that 6AP and GA inhibit PFAR by direct competition with the substrate protein for the binding sites. Also, 6AP derivatives inhibit PFAR in the same order as their antiprion activity, 6AP8CF3 > 6AP8Cl > 6AP > 6APi. These results suggest involvement of PFAR in prion processes.

To clarify the role of PFAR in prion processes, we studied HET-s prion aggregation in the presence of domain V/ IV/II of rRNA. The rRNAs, especially domain V rRNA not only reduced HET-s aggregation, but also changed the morphology of the HET-s fibrils, which became shorter and less compact. These results show that PFAR actively prevents large amyloid aggregation and thus, possibly influence prion propagation. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1327
Keyword
Ribosome, Protein folding, Prion disease, Antiprion drug, Competitive inhibition, PFAR, Amyloid
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-267737 (URN)978-91-554-9429-2 (ISBN)
Public defence
2016-01-28, B22, Husargatan 3, Uppsala, 13:00 (English)
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Available from: 2015-12-22 Created: 2015-11-25 Last updated: 2016-01-13

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Pang, YanhongBanerjee, DebapriyaSanyal, Suparna

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