@article {108045, title = {DSDBASE 2.0: updated version of DiSulphide dataBASE, a database on disulphide bonds in proteins.}, journal = {Database (Oxford)}, volume = {2022}, year = {2022}, month = {2022 Mar 01}, abstract = {

Disulphide bonds are stabilizing crosslinks in proteins and serve to enhance their thermal stability. In proteins that are small and rich in disulphide bonds, they could be the major determining factor for the choice of conformational state since their constraints on appropriate backbone conformation can be substantial. Such crosslinks and their positional conservation could itself enable protein family and functional association. Despite the importance of the field, there is no comprehensive database on disulphide crosslinks that is available to the public. Herein we provide information on disulphides in DSDBASE2.0, an updated and significantly expanded database that is freely available, fully annotated and manually curated database on native and modelled disulphides. The web interface also provides several useful computational tools that have been specifically developed for proteins containing disulphide crosslinks. The modelling of disulphide crosslinks is performed using stereochemical criteria, coded within our Modelling of Disulphides in Proteins (MODIP) algorithm. The inclusion of modelled disulphides potentially enhances the loop database substantially, thereby permitting the recognition of compatible polypeptide segments that could serve as templates for immediate modelling. The DSDBASE2.0 database has been updated to include 153,944 PDB entries, 216,096 native and 20,153,850 modelled disulphide bond segments from PDB January 2021 release. The current database also provides a resource to user-friendly search for multiple disulphide bond containing loops, along with annotation of their function using GO and subcellular localization of the query. Furthermore, it is possible to obtain the three-dimensional models of disulphide-rich small proteins using an independent algorithm, RANMOD, that generates and examines random, but allowed backbone conformations of the polypeptide. DSDBASE2.0 still remains the largest open-access repository that organizes all disulphide bonds of proteins on a single platform. The database can be accessed from http://caps.ncbs.res.in/dsdbase2.

}, issn = {1758-0463}, doi = {10.1093/database/baac005}, author = {Kalmankar, Neha V and Pavalam, Murugavel and Indrakumar, Sowmya and Srinivasan, Narayanaswamy and Sowdhamini, Ramanathan} } @article {118434, title = {Effects of a plant cyclotide on conformational dynamics and destabilization of β-amyloid fibrils through molecular dynamics simulations.}, journal = {Front Mol Biosci}, volume = {9}, year = {2022}, month = {2022}, pages = {986704}, abstract = {

Aggregation of β-amyloid (Aβ) peptide is one of the hallmarks of Alzheimer{\textquoteright}s disease (AD) which results in chronic and progressive neurodegeneration of the brain. A recent study by our group have shown the ability of cyclic disulfide-rich peptides ("cyclotides") isolated from a medicinal plant, , to inhibit the aggregation of Aβ peptides and reduce oxidative stress caused by reactive oxygen species using models of transgenic . In the present study, through extensive computational docking and multi-ns molecular dynamics (MD) simulation, we evaluated if cyclotides can stably bind to Aβ molecules and/or destabilize the Aβ fibril by preventing conformational changes from α-helical to β-sheet rich structures. We demonstrate that cyclotides bind effectively and stably to different forms of Aβ structures hydrogen bonding and hydrophobic interactions. One of the conserved hydrophobic interface residues, Tyr10 was mutated to Ala and the impact of this virtual mutation was estimated by additional MD simulations for the wild-type (WT) and mutant protein-peptide complexes. A detailed MD simulation analyses revealed that cyclotides form hydrogen bonds with the toxic amyloid assemblies thereby weakening the inter-strand hydrogen bonds between the Aβ peptide. The φ-ѱ distribution map of residues in the cyclotide binding pocket that ideally adopt β-sheet conformation show deviation towards right-handed ɑ-helical (ɑ) conformation. This effect was similar to that observed for the Tyr10Ala mutant and doubly so, for the cyclotide bound form. It is therefore possible to hypothesise that the opening up of amyloid β-sheet is due to an unfolding process occurring in the Aβ caused by cyclotide binding and inhibition. Our current findings provide novel structural insights on the mode of interaction between cyclotides and Aβ fibrils and describe their anti-amyloid aggregation potential. This sheds light on the future of cyclotide-based drug design against protein aggregation, a hallmark event in many neurodegenerative diseases.

}, issn = {2296-889X}, doi = {10.3389/fmolb.2022.986704}, author = {Kalmankar, Neha V and Gehi, Bhuvaneshwari Rajendrakumar and Sowdhamini, Ramanathan} } @article {94157, title = {Disulfide-Rich Cyclic Peptides from Protect against β-Amyloid Toxicity and Oxidative Stress in Transgenic .}, journal = {J Med Chem}, year = {2021}, month = {2021 May 28}, abstract = {

Neurotoxic aggregation of β-amyloid (Aβ) peptides is a hallmark of Alzheimer{\textquoteright}s disease and increased reactive oxygen species (ROS) is an associated process. In the present study, we report the neuroprotective effects of disulfide-rich, circular peptides from () (butterfly pea) on Aβ-induced toxicity in transgenic . Cyclotides (\~{}30 amino acids long) are a special class of cyclic cysteine knot peptides. We show that cyclotide-rich fractions from different plant tissues delay Aβ-induced paralysis in the transgenic CL4176 strain expressing the human muscle-specific Aβ gene. They also improved Aβ-induced chemotaxis defects in CL2355 strain expressing Aβ in the neuronal cells. ROS assay suggests that this protection is likely mediated by the inhibition of Aβ oligomerization. Furthermore, Aβ deposits were reduced in the CL2006 strain treated with the fractions. The study shows that cyclotides from could be a source of a novel pharmacophore scaffold against neurodegenerative diseases.

}, issn = {1520-4804}, doi = {10.1021/acs.jmedchem.1c00033}, author = {Kalmankar, Neha V and Hari, Hrudya and Sowdhamini, Ramanathan and Venkatesan, Radhika} } @article {96411, title = {Mass spectrometric analysis of cyclotides from Clitoria ternatea: Xxx-Pro bond fragmentation as convenient diagnostic of Pro residue positioning.}, journal = {Chem Asian J}, year = {2021}, month = {2021 Jul 20}, abstract = {

Cyclotides, a class of macrocyclic plant peptides, characterized by a cyclic backbone and three inter-locking disulfide bonds, may be divided into two structural subfamilies, M{\"o}bius and Bracelet, based on presence or absence of a specific proline residue. The present study describes the suite of cyclotides obtained from Clitoria ternatea , characterized by LC-MS and MS/MS techniques. Notable variations in product ion distributions were observed in cyclotides belonging to different structural subfamilies based on the number and positions of proline residues. For instance, Cter M which is an abundant M{\"o}bius cyclotide in this plant containing three proline residues, displayed distinct b- and y- ion characteristics in the MS/MS spectra compared to Cliotide T1, another commonly identified cyclotide but belonging to the Bracelet subfamily having two proline residues. The distinct fragmentation pattern of prototypical cyclotides of each structural subfamily, determined by Xxx-Pro bond fragmentation, was used to rapidly identify and sequence a novel cyclotide ctr pep 30 from this plant.

}, issn = {1861-471X}, doi = {10.1002/asia.202100585}, author = {Venkatesan, Radhika and Balaram, Padmanabhan and Kalmankar, Neha V} } @article {80094, title = {Transcriptomic profiling of the medicinal plant Clitoria ternatea: identification of potential genes in cyclotide biosynthesis.}, journal = {Sci Rep}, volume = {10}, year = {2020}, month = {2020 Jul 29}, pages = {12658}, abstract = {

Clitoria ternatea a perennial climber of the Fabaceae family, is well known for its agricultural and medical applications. It is also currently the only known member of the Fabaceae family that produces abundant amounts of the ultra-stable macrocyclic peptides, cyclotides, across all tissues. Cyclotides are a class of gene-encoded, disulphide-rich, macrocyclic peptides (26-37 residues) acting as defensive metabolites in several plant species. Previous transcriptomic studies have demonstrated the genetic origin of cyclotides from the Fabaceae plant family to be embedded in the albumin-1 genes, unlike its counterparts in other plant families. However, the complete mechanism of its biosynthesis and the repertoire of enzymes involved in cyclotide folding and processing remains to be understood. In this study, using RNA-Seq data and de novo transcriptome assembly of Clitoria ternatea, we have identified 71 precursor genes of cyclotides. Out of 71 unique cyclotide precursor genes obtained, 51 sequences display unique cyclotide domains, of which 26 are novel cyclotide sequences, arising from four individual tissues. MALDI-TOF mass spectrometry analysis of fractions from different tissue extracts, coupled with precursor protein sequences obtained from transcriptomic data, established the cyclotide diversity in this plant species. Special focus in this study has also been on identifying possible enzymes responsible for proper folding and processing of cyclotides in the cell. Transcriptomic mining for oxidative folding enzymes such as protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIases)/cyclophilins, and their levels of expression are also reported. In particular, it was observed that the CtPDI genes formed plant-specific clusters among PDI genes as compared to those from other plant species. Collectively, this work provides insights into the biogenesis of the medicinally important cyclotides and establishes the expression of certain key enzymes participating in peptide biosynthesis. Also, several novel cyclotide sequences are reported and precursor sequences are analysed in detail. In the absence of a published reference genome, a comprehensive transcriptomics approach was adopted to provide an overview of diverse properties and constituents of C. ternatea.

}, issn = {2045-2322}, doi = {10.1038/s41598-020-69452-7}, author = {Kalmankar, Neha V and Venkatesan, Radhika and Balaram, Padmanabhan and Sowdhamini, Ramanathan} }