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Spectroscopic and DFT studies on 6-Aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Sanyal)
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, Structure and Molecular Biology. (Sanyal group)
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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.

Place, publisher, year, edition, pages
2014. Vol. 97, 194-199 p.
National Category
Natural Sciences
Research subject
Biochemistry; Physics with specialization in Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-218782DOI: 10.1016/j.biochi.2013.10.012ISI: 000331410500022PubMedID: 24184272OAI: oai:DiVA.org:uu-218782DiVA: diva2:697263
Available from: 2014-02-17 Created: 2014-02-17 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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Supervisors
Available from: 2015-12-22 Created: 2015-11-25 Last updated: 2016-01-13

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Banerjee, DebapriyaVovusha, HakkimPang, YanhongSanyal, BiplabSanyal, Suparna

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