A largely underexplored class of bioactive peptides are the ribosomally synthesized and post-translationally modified peptides (RiPPs), also known as ribosomal natural products. Such peptides have been evolutionarily optimized over millions of years for the purposes of regulating specific enzymes and pathways, ion channels and receptors. Cyclotides are one such unique class of gene-encoded, ribosomally synthesized macrocyclic peptides (26-37 residues) produced in several plant species as a form of their defence strategy (1). In addition to the end-to-end circular backbone, they form a cyclic cystine knot (CCK) arrangement comprising a conserved six cysteine framework (Cys I-IV, II-V, III-VI). Structurally, cyclotides can be divided into two main subfamilies i.e. MÃÂöbius and Bracelet. The main difference is that MÃÂöbius cyclotides contain a cis-proline residue in the loop 5 region, whereas this is absent in the Bracelet subtype.
Using RNA-Seq data, the de novo assembly of Clitoria ternatea (butterfly pea) transcriptome 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 cyclotides, of which 26 are novel cyclotide sequences (2). Differential expression analysis revealed that numerous cyclotide genes were highly expressed in one tissue, but were minimally expressed/absent in the others. Additionally, we have also proposed a model of cyclotide biosynthesis, wherein we followed the presence and expression of oxidative folding and processing enzymes, such as asparaginyl endopeptidases (AEP), protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIase), responsible for proper folding and processing of cyclotides in the plant cell.
The next objective was to understand the proteome-level expression of cyclotides in C. ternatea using high-resolution mass-spectrometry (3). An assortment of cyclotides was detected and variations were observed across the different tissues (pod, stem, leaf, flower and root). Notable variations in LC-MS/MS product ion distributions were observed in cyclotides belonging to the two structural subfamilies (MÃÂöbius and Bracelet) based on the number and positions of prolines. Distinct MS/MS fragmentation patterns determined by Xxx-Pro bond fragmentation of prototypical cyclotides was used as a diagnostic to rapidly identify and sequence two novel cyclotides, ctr pep 30 and ctr pep 43. Extensive LC-MS/MS analysis also revealed that cyclotides in C. ternatea adopt a few novel structural arrangements.
Furthermore, an in-house disulfide bond database - DSDBASE2.0 was updated to include the latest PDB entries and multiple tools were incorporated in the webserver for structure prediction of disulfide-rich peptides such as that of the cyclotides (http://caps.ncbs.res.in/dsdbase2). DSDBASE is a database on disulfide bonds in proteins that provides information on native disulfides and those which are stereochemically possible between pairs of residues in a protein (4). The database has been updated to include 153,944 PDB entries, 216,096 native and 20,153,850 modelled disulfide bond segments. Additionally, it is now possible to obtain three-dimensional models of disulfide-rich small proteins using an independent algorithm - RANMOD, that generates and examines random, but allowed backbone conformations to the query polypeptide (5). I used the RANMOD program to model the cyclic conformations adopted by several cyclotide sequences.
Finally, we focussed on understanding the potential of cyclotides as therapeutic leads, specifically against neurodegenerative diseases. CyclotidesâÃÂàability as an inhibitor of ÃÂò-amyloid (AÃÂò) fibrils was investigated and demonstrated using an animal model of AlzheimerâÃÂÃÂs disease (6). We describe how these peptides protect against ÃÂò-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 across multiple tissues of the butterfly pea plant and highlight their future applications as therapeutic lead molecules, especially as an inhibitor of AÃÂò fibrils.
References:
1. De Veer, S. J., Kan, M. W., and Craik, D. J. (2019). Cyclotides: From Structure to Function. Chem. Rev. 119, 12375-12421.
2. Kalmankar N. V., Venkatesan R., Balaram P. and Sowdhamini R. (2020). Transcriptomic profiling of the medicinal plant Clitoria ternatea: Identification of potential genes in cyclotide biosynthesis. Scientific Reports. 10, 12658.
3. Kalmankar N. V.*, Balaram P.* and Venkatesan R.* (2021). Mass spectrometric analysis of cyclotides from Clitoria ternatea: Xxx-Pro bond fragmentation as convenient diagnostic of Pro residue positioning. Chemistry - an Asian Journal. 16, 2920-2931.
4. Vinayagam, A., Pugalenthi, G., Rajesh, R., and Sowdhamini, R. (2004). DSDBASE: A consortium of native and modelled disulphide bonds in proteins. Nucleic Acids Res. 32(Database issue):D200-202.
5. Sowdhamini, R., Ramakrishnan, C., and Balaram, P. (1993). Modelling multiple disulphide loop containing polypeptides by random conformation generation. The test cases of ÃÂñ-conotoxin gi and edothelin I. Protein Eng. Des. Sel. 6, 873âÃÂÃÂ882.
6. Kalmankar N. V., Hari H., Sowdhamini R and Venkatesan R. (2021). Disulfide-rich, cyclic peptides from Clitoria ternatea protect against ÃÂò-amyloid toxicity and oxidative stress in transgenic Caenorhabditis elegans. Journal of Medicinal Chemistry. 64, 11, 7422âÃÂÃÂ7433.