Bioactive Macrocyclic Cysteine-rich Peptides: Insights on Sequence, Structure and Application of Cyclotides

Abstract

Cyclotides are a unique class of gene-encoded, ribosomally synthesized, macrocyclic peptides (26-37 residues) produced in several plant species. They form a cyclic cystine knot (CCK) motif comprising of six disulfide bonds and circular backbone, and can be divided into two main subfamilies i.e. Möbius and Bracelet, with Möbius cyclotides containing a cis-proline residue in the loop 5 region. The transcriptome assembly of Clitoria ternatea was performed to facilitate gene mining of cyclotide precursors arising in four tissues (pod, stem, leaf and flower). This resulted in the identification of 71 precursor genes, of which 26 are novel cyclotide sequences. Differential expression analysis revealed that numerous cyclotide genes were differentially expressed across the tissues. Moreover, we have also proposed a model of cyclotide biosynthesis, wherein we followed the presence and expression of several oxidative folding and processing enzymes. Additionally, an assortment of cyclotides was detected across five tissues using proteomics method. Notable variations in LC-MS/MS product ion distributions were observed in cyclotides belonging to the two structural subfamilies based on the number and positions of prolines. Distinct MS/MS patterns determined by Xxx-Pro bond fragmentation of prototypical cyclotides was used as a diagnostic to rapidly sequence two novel cyclotides, ctr pep 30 and ctr pep 43. Furthermore, our in-house disulfide bond database - DSDBASE2.0 was updated to include the latest PDB entries (January 2021 release) and multiple tools were incorporated for structure prediction of disulfide-rich peptides (http://caps.ncbs.res.in/dsdbase2). It is now possible to also obtain threedimensional models of disulfide-rich proteins using an independent algorithm - RANMOD. Finally, we cyclotides’ ability as an inhibitor of β-amyloid (Aβ) fibrils was investigated and demonstrated using an animal model of Alzheimer’s disease. We describe how these peptides protect against β-amyloid toxicity and oxidative stress using different transgenic Caenorhabditis elegans strains. Collectively, the findings in this thesis provide insights into the diversity and characterization of cyclotides from Clitoria ternatea and highlight their future applications as therapeutic lead molecules, especially as an inhibitor of Aβ fibrils.