Discovery and mode of action of cyclotides

Ravipati, Anjaneya (2016). Discovery and mode of action of cyclotides PhD Thesis, Institute for Molecular Bioscience, The University of Queensland. doi:10.14264/uql.2016.392

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Author Ravipati, Anjaneya
Thesis Title Discovery and mode of action of cyclotides
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
DOI 10.14264/uql.2016.392
Publication date 2016-07-01
Thesis type PhD Thesis
Supervisor David Craik
Total pages 163
Language eng
Subjects 0699 Other Biological Sciences
0301 Analytical Chemistry
Formatted abstract
Cyclotides are plant derived macrocyclic peptides featuring a cyclic cystine knot (CCK) formed by six cysteine residues on a head-to-tail cyclic backbone. Their unique structural features make them exceptionally stable against thermal, enzymatic or chemical degradation. Naturally isolated cyclotides have many pharmaceutically important activities, including uterotonic, anti-HIV, antimicrobial and anticancer properties. The combination of structural features and biological activities make cyclotides an attractive framework in drug design applications.

To date over 300 cyclotides have been discovered in five plant families: Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae and Solanaceae. However, their origin and distribution in the plant kingdom remains unclear. In this thesis a total of 206 plants belonging to 46 different plant families have been screened for expression of cyclotides. In this screening program, 50 novel cyclotides were discovered from 31 plant species belonging to the Rubiaceae and Violaceae families. Interestingly, all the Violaceae species possess cyclotides supporting the hypothesis that the Violaceae is a rich source of cyclotides. As a matter of fact, cyclotides have been found in every plant species belonging to the Violaceae family screened so far.

It is noteworthy that a novel suite of Lys-rich cyclotides were discovered from Melicytus chathamicus and M. latifolius belonging to the Violaceae family which are endemic to remote islands of Australia and New Zealand. Unlike generic cyclotides, Lys-rich cyclotides possess higher positive charge, which correlates with their earlier retention times in RP-HPLC indicative of lower hydrophobic properties.

The broad spectrum of biological activities of cyclotides suggests the involvement of a common mechanism of action. Earlier studies have demonstrated that cyclotides exert biological activities through interaction with biological membranes. In order to understand the mode of action of novel cyclotides, membrane binding, and membrane permeabilization properties were evaluated on model membranes using a range of biophysical techniques including surface plasmon resonance (SPR), fluorescence spectroscopy and cryo-transmission electron microscopy (cryo-TEM). Regardless of their sequence diversity all of the cyclotides showed affinity towards phospholipids containing a phosphatidylethanolamine (PE) headgroup, which is in agreement with previous studies. In comparison to typical cyclotides, Lys-rich cyclotides display relatively lower haemolytic activities and correspondingly these peptides also showed lower affinities with model membranes. These results support the theory that membrane binding affinities influence the biological activities.

The spectroscopic studies clearly show that cyclotides do target and disrupt membranes. However, the complete membrane disruption mechanism has not been evaluated yet. In this thesis, cyclotide-membrane interactions were investigated using cryo-TEM imaging. The results suggest that the membrane disruption mechanism is sequence dependent.

Overall, the thesis has provided an increased understanding of the natural diversity of cyclotides and has added to our understanding of their mode of action. In particular, the discovery of a novel family of Lys-rich cyclotides suggests that there might be other new types of cyclotides yet to be discovered. Hence, discovery and characterization of cyclotides in this field of research has much to offer.
Keyword Cyclotide
Mass spectrometry (MS)
Cyclotide-membrane interactions
Peptide sequencing
Cryo-TEM imaging
Surface plasmon resonance (SPR)

Document type: Thesis
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Created: Thu, 16 Jun 2016, 16:07:26 EST by Anjaneya Ravipati on behalf of Learning and Research Services (UQ Library)