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Protein Folding Activity of the Ribosome and Its Implication in Prion Processes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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 [en]
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: urn:nbn:se:uu:diva-267737ISBN: 978-91-554-9429-2 (print)OAI: oai:DiVA.org:uu-267737DiVA: diva2:874134
Public defence
2016-01-28, B22, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2015-12-22 Created: 2015-11-25 Last updated: 2016-01-13
List of papers
1. Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding
Open this publication in new window or tab >>Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding
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2011 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 93, no 6, 1047-1054 p.Article in journal (Refereed) Published
Abstract [en]

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (GAP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as 'ribosomal folding modulators' or RFMs) from both bacteria Escherichia con and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.

Keyword
Protein folding, Prion, Ribosome, Antiprion drugs, Carbonic anhydrase
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-154955 (URN)10.1016/j.biochi.2011.03.002 (DOI)000290974300009 ()21396977 (PubMedID)
Available from: 2011-06-14 Created: 2011-06-14 Last updated: 2017-12-11Bibliographically approved
2. The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition
Open this publication in new window or tab >>The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition
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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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-205006 (URN)10.1074/jbc.M113.466748 (DOI)000321335800043 ()
Available from: 2013-08-13 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
3. Spectroscopic and DFT studies on 6-Aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA
Open this publication in new window or tab >>Spectroscopic and DFT studies on 6-Aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA
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2014 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 97, 194-199 p.Article in journal (Refereed) Published
Abstract [en]

6-Aminophenanthridine (6AP), a plant alkaloid possessing antiprion activity, inhibits ribosomal RNA dependent protein folding activity of the ribosome (referred as PFAR). We have compared 6AP and its three derivatives 6AP8Cl, 6AP8CF3 and 6APi for their activity in inhibition of PFAR. Since PFAR inhibition requires 6AP and its derivatives to bind to the ribosomal RNA (rRNA), we have measured the binding affinity of these molecules to domain V of 23S rRNA using fluorescence spectroscopy. Our results show that similar to the antiprion activity, both the inhibition of PFAR and the affinity towards rRNA follow the order 6AP8CF3 > 6AP8Cl > 6AP, while 6APi is totally inactive. To have a molecular insight for the difference in activity despite similarities in structure, we have calculated the nucleus independent chemical shift using first principles density functional theory. The result suggests that the deviation of planarity in 6APi and steric hindrance from its bulky side chain are the probable reasons which prevent it from interacting with rRNA. Finally, we suggest a probable mode of action of 6AP, 6AP8CF3 and 6AP8Cl towards rRNA.

National Category
Natural Sciences
Research subject
Biochemistry; Physics with specialization in Biophysics
Identifiers
urn:nbn:se:uu:diva-218782 (URN)10.1016/j.biochi.2013.10.012 (DOI)000331410500022 ()24184272 (PubMedID)
Available from: 2014-02-17 Created: 2014-02-17 Last updated: 2017-12-06Bibliographically approved
4. Prevention of HET-s prion aggregation by ribosomal RNA
Open this publication in new window or tab >>Prevention of HET-s prion aggregation by ribosomal RNA
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The role of nucleic acids in prion aggregation / disaggregation has remained unclear.  Here, using HET-s prion from fungi Podospora anserina as a model system, we have studied the role of ribosomal RNA (rRNA) in its aggregation process in an unbiased manner. Our results show that rRNA, in particular domain V of rRNA from the large subunit of the ribosome, substantially prevents beta-amyloid aggregation of the HET-s prion in a concentration dependent manner. The sites of interaction of the HET-s prion on domain V rRNA have been identified, which overlap with the sites previously identified for the protein folding activity of the ribosome. This study provides a missing link for the role of rRNA based folding activity of the ribosome in the context of prion propagation.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-267736 (URN)
Available from: 2015-11-25 Created: 2015-11-25 Last updated: 2016-01-13

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