Assessment of recombinant glutathione-S-transferase (HaGST-8) silica nano-conjugates for effective removal of pesticides.

Diverse glutathione-S-transferases (GSTs) are produced by insect pests including Helicoverpa armigera (HaGSTs) for detoxification of insecticides or xenobiotic compounds that they encounter. In an earlier study, the HaGST-8 gene was isolated from H. armigera larvae exposed to pesticide mixtures and the recombinant protein was expressed in the yeast Pichia pastoris. In this investigation, HaGST-8 was successfully immobilized on glutaraldehyde-activated APTES functionalized silica nanoparticles to obtain SiAPT-HaGST-8 nano-conjugates. Although enzyme activity associated with these conjugates was comparable to that of free HaGST-8, the specific activity of the former was found to be 1.25 times higher than the latter. In comparison with the free enzyme (that demonstrated a pH optimum of 9.0), for the nano-conjugates, the pH range was extended between pH 8.0 to 9.0. The optimum temperature for activity of both forms of the enzyme was found to be 30 °C. Stability of the enzyme was improved from 20 d for free HaGST-8 to 30 d for SiAPT-HaGST-8 nano-conjugates. Some loss in GST activity was detected after every reuse cycle of nano-conjugates and in all, 63% reduction was observed after three cycles. When 3 kinds of pesticides (namely, chlorpyrifos, dichlorvos and cypermethrin) were reacted with SiAPT-HaGST-8, more than 80% reduction in levels were observed. On the basis of the results obtained, the use of such silica nanoparticle-based systems for stable enzyme conjugation followed by effective removal of pesticides from aqueous media is envisaged.

In vitro and in silico studies on membrane interactions of diverse Capsicum annuum flower γ-thionin peptides.

Thionins are small, cysteine-rich peptides that play an important role in plant defense, primarily through their interactions with membranes. Eight novel γ-thionin peptides (CanThio1-8) were isolated from the flower of Capsicum annuum. Sequence analysis revealed that the peptides cluster into three groups. A representative peptide from each group (CanThio1, 2, and 3) was used for experimental characterization. Interestingly, peptides were found to possess some cytotoxic activity against normal human embryonic kidney cell line but higher cytotoxicity against cancer cell line MCF-7. CanThio3 peptide was chosen as a representative peptide to study the molecular mechanism of action on membranes. Microsecond timescale atomistic simulations of CanThio3 were performed in the presence of a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Simulations revealed that CanThio3 interacts with the bilayer and causes lipid thinning in the vicinity. Nonpolar amino acids specific to the α-core region of CanThio3 along with nonpolar residues in the γ-core region are seen to interact with the lipid tails. The differences in the amino acid sequence of CanThio peptides in these regions explain the variability in cytotoxic activities. In summary, our results demonstrate the membrane-mediated activity of a novel series of γ-thionin peptides from C. annuum.

Novel insights into plant defensin ingestion induced metabolic responses in the polyphagous insect pest Helicoverpa armigera.

Lepidopteran insect pest Helicoverpa armigera is one of the most destructive pests of crop plants and several biotechnological approaches are being developed for its control. Plant defensins are small cationic and cysteine-rich peptides that play a role in plant defense. Ingestion of a defensin from Capsicum annuum (CanDef-20) induced a dose-dependent reduction in larval and pupal mass, delayed metamorphosis and also severely reduced fecundity and fertility in H. armigera. To understand the molecular mechanisms of CanDef-20 ingestion-mediated antibiosis in H. armigera larvae, a comparative transcriptomics analysis was carried out. Predominant downregulation of GOs represents serine-type endopeptidases, structural constituents of ribosomes and integral membrane components and differential upregulation of ATP binding, nucleus and translation, while up-regulation of nucleic acid binding represented by transposable elements, were detected. Different isoforms of lipase, serine endopeptidase, glutathione S-transferase, cadherin, alkaline phosphatase and aminopeptidases were found to be upregulated as a compensatory response to CanDef-20 ingestion. In vitro enzyme assays and qPCR analysis of some representative genes associated with vital cellular processes like metamorphosis, food digestion and gut membrane indicated adaptive differential regulations in CanDef-20 fed H. armigera larvae. We conclude that CanDef-20 ingestion affects insect metabolism in a number of ways through its interaction with cell membrane, enzymes, cytoplasmic proteins and triggering transposon mobilization which are linked to growth retardation and adaptive strategies in H. armigera.