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Cyclotide stability against Pseudomonas aeruginosa and Staphylococcus aureus secreted proteases
Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

Production and release of proteases is among the strategies by which pathogenic bacteria increase their virulence. Moreover, these bacteria are also known to produce a cocktail of enzymes to degrade and inhibit antimicrobial peptides. Cyclotides, on the other hand, are circular plant mini-proteins having a unique cyclic topology (termed as the cyclic cystine knot, CCK) which endows them with exceptional stability. Their ultra-stable framework has attracted interest in drug design as a scaffold to otherwise susceptible drugs including antibacterial peptides. We have determined the stability of three peptides with the CCK motif, i.e., the cyclotides cycloviolacin O2, kalata B1 and MCoTI-II in elastase and aureolysin and also in the supernatant cultures of P. aeruginosa and S. aureus. The cyclotides have resisted enzymatic degradation both from the specific proteases and the bacterial cultures, with MCoTI-II being the least stable of the three. The cyclotides find application in drug engineering as their stable framework can be used as a scaffold or as a template for unstable drugs. The findings of the current study further support this idea; the cyclotide structure can be utilized as a novel strategy in enhancing the stability of antibacterial peptides.

Keyword [en]
cyclotides, cycloviolacin O2, kalata B1, MCoTI-II, Pseudomonas aeruginosa, Staphylococcus aureus, elastase, aureolysin, Viola odorata, Oldeanlandia affinis, Momordica cochinchinensis, protease, serum
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-171141OAI: oai:DiVA.org:uu-171141DiVA: diva2:510406
Available from: 2012-03-16 Created: 2012-03-15 Last updated: 2016-10-05
In thesis
1. Cyclotides: Tuning Parameters Toward Their Use in Drug Design
Open this publication in new window or tab >>Cyclotides: Tuning Parameters Toward Their Use in Drug Design
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyclotides are plant proteins with a unique topology, defined as the cyclic cystine knot motif. The motif endows cyclotides with exceptional chemical and biological stability. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. Hence, cyclotides have become potential candidates in the development of peptide-based drugs; either as scaffolds to stabilize susceptible peptide sequences or as drugs by their own right. In this thesis, important parameters that could be inputs toward this development have been tuned.

An extraction protocol that can be extended to industrial scale production of the cyclotides from natural sources was developed; accordingly, a single maceration with hydroalcoholic solutions of medium polarity represented an optimum extraction method.

Moreover, it was shown that investigating the cyclotide content of cyclotide-bearing plants from diverse environments is a promising approach for extending the knowledge of both structural and biological diversity of these proteins. Five novel cyclotides with new sequence diversity were isolated and characterized from a violet that grows on Ethiopian highlands at an altitude of 3400 m.

One of the areas where the cyclotide framework has attracted interest is the development of stable antimicrobial peptides. A stability study was carried out to determine the stability of the cyclotide framework in a cocktail of bacterial proteases and serum where the native forms of tested cyclotides exhibited high stability profile.

Understanding the modes of cyclotide-cell interaction is certainly an important factor for the potential development of cyclotide-based drugs. Cellular studies were carried out using the comet assay and microautoradiography. A bell-shaped dose response curve was obtained for the DNA damaging effect of the cyclotides in the comet assay, which was the first toxicological assay of its kind on this class of proteins. The microautoradiography study revealed that the cyclotides penetrate into the cells even at cytotoxic concentrations. From previous reports, it was known that the cyclotides interact with membranes; the cellular studies in this thesis added to this knowledge by clearly demonstrating that these proteins have multiple modes of action. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 81 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 156
cyclotide, Viola abyssinica, Viola odorata, Extraction, Violaceae, vaby, cytotoxic, genotoxic, cell penetrating, microautoradiography, LC-MS, comet assay, cycloviolacin O2
National Category
Medical and Health Sciences Natural Sciences
Research subject
urn:nbn:se:uu:diva-169712 (URN)978-91-554-8307-4 (ISBN)
Public defence
2012-04-26, B41, Husargatan 3, Biomedical Center, Uppsala University, Uppsala, 10:15 (English)
Available from: 2012-04-04 Created: 2012-03-05 Last updated: 2012-04-19Bibliographically approved

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Strömstedt, Adam A.
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