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Insights into cyclotide evolution from transcriptomic analyses and studies on their distribution in violets
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. (Pharmacognosy)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cyclotides are a family of plant-derived proteins that are characterized by a cyclic backbone and a knotted disulfide topology. Their cyclic cystine knot (CCK) motif makes them exceptionally resistant to thermal, chemical, and enzymatic degradation. Cyclotides exert much of their biological activity via interactions with cell membranes. In this work, we explore cyclotide evolution by sequencing and analyzing the transcriptomes of Viola albida var. takahashii, V. mandshurica, V. orientalis, V. verecunda and V. acuminata, in combination with reported precursor sequences. The distribution of the cyclotide precursors in the genus Viola was analyzed in relation to the infrageneric phylogeny, providing insights into the overall distribution of cyclotides within the genus. The structures and activities of predicted cyclotides are linked to the evolution of their precursors and the associated selective pressures. Furthermore, sequence variations between related precursors were generalized to deduce evolutionary trends, and to shed light on the evolution into novel mature cyclotides with the emergence of new functions. 

National Category
Evolutionary Biology
URN: urn:nbn:se:uu:diva-292958OAI: oai:DiVA.org:uu-292958DiVA: diva2:927084
Available from: 2016-05-11 Created: 2016-05-11 Last updated: 2016-07-26Bibliographically approved
In thesis
1. Cyclotides evolve: Studies on their natural distribution, structural diversity, and activity
Open this publication in new window or tab >>Cyclotides evolve: Studies on their natural distribution, structural diversity, and activity
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cyclotides are a family of naturally occurring peptides characterized by cyclic cystine knot (CCK) structural motif, which comprises a cyclic head-to-tail backbone featuring six conserved cysteine residues that form three disulfide bonds. This unique structural motif makes cyclotides exceptionally resistant to chemical, thermal and enzymatic degradation. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects.

The cyclotides found in plants exhibit considerable sequence and structural diversity, which can be linked to their evolutionary history and that of their host plants. To clarify the evolutionary link between sequence diversity and the distribution of individual cyclotides across the genus Viola, selected known cyclotides were classified using signature sequences within their precursor proteins. By mapping the classified sequences onto the phylogenetic system of Viola, we traced the flow of cyclotide genes over evolutionary history and were able to estimate the prevalence of cyclotides in this genus. In addition, the structural diversity of the cyclotides was related to specific features of the sequences of their precursor proteins, their evolutionary selection and expression levels.

A number of studies have suggested that the biological activities of the cyclotides are due to their ability to interact with and disrupt biological membranes. To better explain this behavior, quantitative structure-activity relationship (QSAR) models were developed to link the cyclotides’ biological activities to the membrane-interactive physicochemical properties of their molecular surfaces. Both scalar quantities (such as molecular surface areas) and moments (such as the distributions of specific properties over the molecular surface) were systematically taken into account in the development of these models. This approach allows the physicochemical properties of cyclotides to be geometrically interpreted, facilitating the development of guidelines for drug design using cyclotide scaffolds.

Finally, an optimized microwave-assisted Fmoc-SPSS procedure for the total synthesis of cyclotides was developed. Microwave irradiation is used to accelerate and improve all the key steps in cyclotide synthesis, including the assembly of the peptide backbone by Fmoc-SPPS, the cleavage of the protected peptide, and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 71 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 218
cyclotide, cyclic cystine knot, evolution, peptide synthesis, chemical ligation, QSAR, Viola, Violaceae, phylogeny
National Category
Mathematics Organic Chemistry Physical Chemistry Evolutionary Biology
Research subject
urn:nbn:se:uu:diva-292668 (URN)978-91-554-9604-3 (ISBN)
Public defence
2016-06-10, B/C4:301, BMC, Husargatan 3, Uppsala, 09:00 (English)
Available from: 2016-05-24 Created: 2016-05-06 Last updated: 2016-06-15

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Park, SungkyuJacobsson, ErikBacklund, AndersGöransson, Ulf
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