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Cyclotides evolve: Studies on their natural distribution, structural diversity, and activity
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 218
Keyword [en]
cyclotide, cyclic cystine knot, evolution, peptide synthesis, chemical ligation, QSAR, Viola, Violaceae, phylogeny
National Category
Mathematics Organic Chemistry Physical Chemistry Evolutionary Biology
Research subject
Pharmacognosy
Identifiers
URN: urn:nbn:se:uu:diva-292668ISBN: 978-91-554-9604-3 (print)OAI: oai:DiVA.org:uu-292668DiVA: diva2:926556
Public defence
2016-06-10, B/C4:301, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2016-05-24 Created: 2016-05-06 Last updated: 2016-06-15
List of papers
1. Insights into cyclotide evolution from transcriptomic analyses and studies on their distribution in violets
Open this publication in new window or tab >>Insights into cyclotide evolution from transcriptomic analyses and studies on their distribution in violets
Show others...
(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
Identifiers
urn:nbn:se:uu:diva-292958 (URN)
Available from: 2016-05-11 Created: 2016-05-11 Last updated: 2016-07-26Bibliographically approved
2. Cyclotide Structure-Activity Relationships: Qualitative and Quantitative Approaches Linking Cytotoxic and Anthelmintic Activity to the Clustering of Physicochemical Forces
Open this publication in new window or tab >>Cyclotide Structure-Activity Relationships: Qualitative and Quantitative Approaches Linking Cytotoxic and Anthelmintic Activity to the Clustering of Physicochemical Forces
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 3, e91430- p.Article in journal (Refereed) Published
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 qualitatively and quantitatively analyze the cytotoxic and anthelmintic membrane activities of cyclotides. The qualitative and quantitative models describe the potency of cyclotides using four simple physicochemical terms relevant to membrane contact. Specifically, surface areas of the cyclotides representing lipophilic and hydrogen bond donating properties were quantified and their distribution across the molecular surface was determined. The resulting quantitative structure-activity relation (QSAR) models suggest that the activity of the cyclotides is proportional to their lipophilic and positively charged surface areas, provided that the distribution of these surfaces is asymmetric. In addition, we qualitatively analyzed the physicochemical differences between the various cyclotide subfamilies and their effects on the cyclotides' orientation on the membrane and membrane activity.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-224356 (URN)10.1371/journal.pone.0091430 (DOI)000333678100003 ()
Available from: 2014-05-13 Created: 2014-05-09 Last updated: 2017-12-05Bibliographically approved
3. Exposing the true bactericidal potency of cyclotides: explained by lipid selectivity, structural characteristics and correlating antimicrobial activities
Open this publication in new window or tab >>Exposing the true bactericidal potency of cyclotides: explained by lipid selectivity, structural characteristics and correlating antimicrobial activities
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cyclotides are a family of plant peptides characterized by a cystine knot embedded in a macrocyclic backbone. They bind to and disrupt phospholipid membranes, which explain their activity against eukaryotic cells and enveloped viruses. In the current study, we show that potent antibacterial activity is frequent among cyclotides as long as the activity inhibiting presence of rich growth media is avoided. For that purpose a modified microdilution assay protocol was developed. This, the largest and most diverse set of cyclotides to be tested for antibacterial and antifungal activity, show that most cyclotides are active in this respect, especially against Gram-negative bacteria. Activity was observed at sub-micromolar concentrations for three of the cyclotides surpassing that of the potent control peptides melittin and LL-37. Noteworthy, two net anionic cyclotides were active on Pseudomonas aeruginosa at low micromolar concentrations. Activity against Staphylococcus aureus or Candida albicans was lower and less frequent. Permeabilizing activity on liposomes of various compositions was compared with effects on bacteria, Candida and lymphoma cells to which physiochemical properties of the cyclotides could be assigned. Analysis of quantitative structure-activity relationship found correlations between molecular patterns, phospholipid specificity and antimicrobial activity. Although certain bracelet cyclotides, with broad-spectrum activity, relied more on electrostatic and hydrophobic parameters for their bioactivity, those with primarily Gram-negative activity, in particular against P. aeruginosa, were achieving membrane binding and disruption in a phospholipid specific manner, namely a high affinity for phosphatidylethanolamine (PE). We conclude that the high PE-selectivity is linked to the potency of antibacterial activity.

Keyword
cyclotide; membrane; phosphatidylethanolamine; cytotoxicity; antibacterial; antifungal; peptide surface chemistry
National Category
Physical Chemistry
Research subject
Pharmacognosy; Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-292756 (URN)
Available from: 2016-05-09 Created: 2016-05-09 Last updated: 2016-07-26Bibliographically approved
4. An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS
Open this publication in new window or tab >>An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS
2010 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 16, no 3, 167-176 p.Article in journal (Refereed) Published
Abstract [en]

Cyclotides are mini-proteins of approximately 30 amino acid residues that have a unique structure consisting of a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. This unique cyclotide structure provides exceptional stability to chemical, enzymatic and thermal treatments and has been implicated as an ideal drug scaffold for the development into agricultural and biotechnological agents. In the current work, we present the first method for microwave assisted Fmoc-SPPS of cyclotides. This protocol adopts a strategy that combines optimized microwave assisted chemical reactions for Fmoc-SPPS of the peptide backbone, 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. To exemplify the utility of this protocol in the synthesis of a wide array of different cyclotide sequences we synthesized representative members from the three cyclotide subfamilies-the Mobius kalata B1, the bracelet cycloviolacin O2 and the trypsin inhibitory MCoTI-II. In addition, a "one pot" reaction promoting both cyclization and oxidative folding of crude peptide thioester was adapted for kalata B1 and MCoTI-II.

Keyword
Cyclotides, Microwave chemistry, Fmoc-SPPS, Circular proteins, Cystine knot, Native chemical ligation
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-134899 (URN)10.1007/s10989-010-9221-0 (DOI)000281682600007 ()
Available from: 2010-12-02 Created: 2010-12-02 Last updated: 2017-05-11Bibliographically approved

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