Dual Insecticidal Effects of Adenanthera pavonina Kunitz-Type Inhibitor on Plodia interpunctella is Mediated by Digestive Enzymes Inhibition and Chitin-Binding Properties.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

Expression in Escherichia coli of cysteine protease inhibitors from cowpea (Vigna unguiculata): The crystal structure of a single-domain cystatin gives insights on its thermal and pH stability.

Two cysteine proteinase inhibitors from cowpea, VuCys1 and VuCys2, were produced in E. coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100°C for up to 60min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after VuCys1 and VuCys2 were subjected to these treatments. The crystal structure of VuCys1 was determined at a resolution of 1.95Å, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in VuCys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of VuCys1.

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2.

Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 µM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.