Reg-1α/lithostathine, a protein mainly associated with the digestive system, was previously shown to be overexpressed in the pre-clinical stages of Alzheimer’s disease. In vitro, the glycosylated protein was reported to form fibrils at physiological pH following the proteolytic action of trypsin. However, the nature of the protease able to act in the central nervous system is unknown. In the present study, we showed that Reg-1α can be cleaved in vitro by calpain-2, the calcium activated neutral protease, overexpressed in neurodegenerative diseases. Using chemical crosslinking experiments, we found that the two proteins can interact with each other. Identification of the cleavage site using mass spectrometry, between Gln⁴ and Thr⁵, was found in agreement with the in silico prediction of the calpain cleavage site, in a position different from the one reported for trypsin, i.e., Arg¹¹-Ile¹² peptide bond. We showed that the cleavage was impeded by the presence of the neighboring glycosylation of Thr⁵. Moreover, in vitro studies using electron microscopy showed that calpain-cleaved protein does not form fibrils as observed after trypsin cleavage. Collectively, our results show that calpain-2 cleaves Reg-1α in vitro, and that this action is not associated with fibril formation.

Cone snails are carnivorous marine predators that prey on mollusks, worms, or fish. They purposefully inject a highly diversified and peptide-rich venom, which can vary according to the predatory or defensive intended use. Previous studies have shown some correlations between the predation- and defense-evoked venoms and specific sections of the venom gland. In this study, we focus on the characterization of the venom of Cylinder canonicus, a molluscivorous species collected from Mayotte Island. Integrated proteomics and transcriptomics studies allowed for the identification of 108 conotoxin sequences from 24 gene superfamilies, with the most represented sequences belonging to the O1, O2, M, and conkunitzin superfamilies. A comparison of the predatory injected venom and the distal, central, and proximal sections of the venom duct suggests mostly distal origin. Identified conotoxins will contribute to a better understanding of venom–ecology relationships in cone snails and provide a novel resource for potential drug discovery.

Xenorhabdus is an entomopathogenic bacterium involved in a mutualistic relationship with Steinernema nematodes. Xenorhabdus produces a multitude of specialized metabolites by Non-Ribosomal Peptide Synthetases (NRPSs) pathways to mediate bacterial–nematode– insect interactions. PAX cyclolipopeptides are a family of NRP-type molecules whose ecological role remains poorly understood. In this study, the pleiotropic role of PAX peptides in the life cycle of Xenorhabdus nematophila has been investigated. By mass spectrometry analysis, we first demonstrated that PAX peptides were detected from the pathogenic stage up to the necrotrophic stage. We discovered that the bromothymol blue adsorption phenotype historically used to discriminate Xenorhabdus variants was associated with the presence of PAX peptides. We found that PAX peptides were positively involved in biofilm formation and negatively involved in swimming motility. PAX peptides were also shown to promote in vivo the production of infective Steinernema juveniles, suggesting their involvement in the mutualistic relationship between Xenorhabdus and its nematode partner. Finally, we showed that the paxTABC cluster as well as PAX peptide production was conserved across the whole Xenorhabdus genus except in Xenorhabdus poinarii and Xenorhabdus ishibashii . This work has revealed multiple new ecological roles for NRP-type peptides. Importance: Xenorhabdus bacteria are models of particular interest for their mutualistic relationship with Steinernema nematodes and their ability to produce a wide range of natural NRP-type bioactive metabolites. These compounds are mostly studied for their medical or industrial applications, but their ecological role is poorly understood. This study provides a dynamic characterization of PAX cyclolipopeptide presence during Xenorhabdus nematophila life cycle, as well as confirmation of their production by 7 different strains within the Xenorhabdus genus. We revealed new multiple functions for PAX peptides in biofilm formation, swimming motility and juvenile nematode production. A deeper understanding of how PAX peptides interact with the nematode host would provide a better insight into the role of these cyclolipopeptides in bacterial-nematode mutualism.

Xenorhabdus is an entomopathogenic bacterium involved in a mutualistic relationship with Steinernema nematodes. Xenorhabdus produces a multitude of specialized metabolites by non-ribosomal peptide synthetase (NRPS) pathways to mediate bacterium-nematode-insect interactions. PAX cyclolipopeptides are a family of NRP-type molecules whose ecological role remains poorly understood. In this study, the pleiotropic role of PAX peptides in the life cycle of Xenorhabdus nematophila has been investigated. By mass spectrometry analysis, we first demonstrated that PAX peptides were detected from the pathogenic stage up to the necrotrophic stage. We discovered that the bromothymol blue adsorption phenotype historically used to discriminate Xenorhabdus variants was associated with the presence of PAX peptides. We found that PAX peptides were positively involved in biofilm formation and negatively involved in swimming motility. PAX peptides were also shown to promote in vivo the production of infective Steinernema juveniles, suggesting their involvement in the mutualistic relationship between Xenorhabdus and its nematode partner. Finally, we showed that the paxTABC cluster, as well as PAX peptide production, was conserved across the whole Xenorhabdus genus except in Xenorhabdus poinarii and Xenorhabdus ishibashii . This work has revealed multiple new ecological roles for NRP-type peptides. IMPORTANCE: Xenorhabdus bacteria are models of particular interest for their mutualistic relationship with Steinernema nematodes and their ability to produce a wide range of natural NRP-type bioactive metabolites. These compounds are mostly studied for their medical or industrial applications, but their ecological role is poorly understood. This study provides a dynamic characterization of PAX cyclolipopeptide presence during the Xenorhabdus nematophila life cycle, as well as confirmation of their production by seven different strains within the Xenorhabdus genus. We revealed new multiple functions for PAX peptides in biofilm formation, swimming motility, and juvenile nematode production. A deeper understanding of how PAX peptides interact with the nematode host would provide a better insight into the role of these cyclolipopeptides in bacterial-nematode mutualism.