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Variations in cyclotide expression in viola species
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
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2004 (English)In: Journal of natural products (Print), ISSN 0163-3864, E-ISSN 1520-6025, Vol. 67, no 5, 806-10 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2004. Vol. 67, no 5, 806-10 p.
Keyword [en]
Amino Acid Sequence, Climate, Databases; Protein, Models; Molecular, Molecular Sequence Data, Peptides; Cyclic/*chemistry/*genetics, Plant Proteins/*chemistry/*genetics, Sequence Alignment, Sequence Analysis; Protein, Sequence Homology; Amino Acid, Support; Non-U.S. Gov't, Sweden, Variation (Genetics), Viola/*chemistry
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-66803DOI: 10.1021/np034068ePubMedID: 15165141OAI: oai:DiVA.org:uu-66803DiVA: diva2:94714
Available from: 2004-09-14 Created: 2004-09-14 Last updated: 2017-11-28Bibliographically approved
In thesis
1. Cytotoxic Cyclotides: Structure, Activity, and Mode of Action
Open this publication in new window or tab >>Cytotoxic Cyclotides: Structure, Activity, and Mode of Action
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyclotides are small cyclic plant proteins, and this thesis addresses their cytotoxic structure-activity properties and their mode of action on human cancer cell lines.

Cyclotides were isolated from Viola odorata and Viola tricolor; three novel cyclotide sequences and two known sequences, but of new origin, were identified using mass spectrometry, amino acid analysis, and Edman degradation. The cyclotide structure includes three disulphide bonds in a knotted arrangement, which forces hydrophobic amino acid residues to be exposed on the surface of the molecule; 3-D homology models of cyclotides have revealed an amphipathic surface and charged residues located at similar positions in the molecules.

The charged amino acid residues were shown to play a key role in the cytotoxicity of the cyclotide cycloviolacinO2 on a human lymphoma cell line. Methylation of Glu caused a dramatic change in cytotoxicity, lowering the potency 48 times, whereas concealing the charge of Arg with 1,2-cyclohexanedione caused virtually no change in potency. Acetylation of the two Lys caused a 3-fold reduction in potency, and masking all positive charges caused a 7-fold reduction. Additionally, disturbing the amphipathic structure by reducing and alkylating the disulphide bonds abolished the cytotoxicity.

The time dependency of cytotoxicity and cell gross morphology after cyclotide exposure were investigated on the lymphoma cell line. Cells exposed to 4 µM of cycloviolacinO2 showed necrotic characteristics, such as membrane disintegration, within 5 min; a membrane disruptive effect of cycloviolacinO2 was also observed in a functional assay based on liposomes at a peptide-to-lipid molar ratio of 6.5.

The anti-tumour properties of cycloviolacinO2 were evaluated on three human cancer cell lines using the hollow fibre assay in vitro and in vivo. The cyclotide exhibited potent anti-tumour activity in the micro-molar concentration range on all cell lines in vitro, but no effect on tumour growth could be established in vivo.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 19
Keyword
Pharmacognosy, cyclotide, cytotoxic, cancer, cyclic cystine knot, Viola, Violaceae, liposome, hollow fibre, homology modelling, mass spectrometry, amphipathic, structure–activity, tumour, membrane disruption, necrosis, Farmakognosi
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-6028 (URN)91-554-6378-9 (ISBN)
Public defence
2005-11-18, Room C4:301, BMC, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2005-10-28 Created: 2005-10-28 Last updated: 2009-10-07Bibliographically approved
2. Structure and Activity of Circular Plant Proteins: Cytotoxic Effects of Viola Cyclotides
Open this publication in new window or tab >>Structure and Activity of Circular Plant Proteins: Cytotoxic Effects of Viola Cyclotides
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications.

The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line.

Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 64
Keyword
Pharmacognosy, cyclotide, cytotoxic, anti-tumour, macrocyclic, cyclic cystine knot, Alpine violet, Sweet violet, Viola, Violaceae, chemical modifications, structure-activity studies, mass spectroscopy, cDNA clones, Farmakognosi
Identifiers
urn:nbn:se:uu:diva-8283 (URN)978-91-554-7003-6 (ISBN)
Public defence
2007-11-30, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2009-10-07Bibliographically approved

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Svangård, ErikaHerrmann, AndersGöransson, UlfBohlin, Lars

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