Biologically active peptides are synthesized as larger inactive proprotein peptide precursors which are processed by the concerted action of a cascade of enzymes. Among the proprotein convertases, PC2 is widely expressed in neuroendocrine tissues and has been proposed to be the major convertase involved in the biosynthesis of neuropeptides. In this study, we have examined the role of the Caenorhabditis elegans orthologue PC2/EGL-3 in the processing of proprotein peptide precursors. We recently isolated and identified 60 endogenous peptides in the nematode C. elegans by twodimensional nanoscale liquid chromatography -quadrupole time-of-flight tandem mass spectrometry. In the present study, we compare the peptide profile of different C. elegans strains, including PC2/EGL-3 mutants. For this purpose, we used an offline approach in which HPLC fractions are analysed by a matrix-assisted laser desorption ionisation -time of flight mass spectrometer. This differential peptidomic approach unambiguously provides evidence for the role of PC2/EGL-3 in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors in nematodes. Keywords: EGL-3, FMRFamide-like peptide gene, mass spectrometry, neuropeptide, neuropeptide-like protein gene, proprotein convertase. J. Neurochem. (2006Neurochem. ( ) 98, 1999Neurochem. ( -2012 Neuropeptides are derived from large proprotein peptide precursor genes, which encode for a single or multiple peptides. Several highly regulated and specific post-translational events are required for the formation of biologically active peptides from their larger inactive precursor proteins. A typical proprotein peptide precursor contains an amino terminal signal peptide, which is cleaved off upon entrance into the secretory pathway by a signal peptidase. Subsequently, proprotein convertases (PCs) cleave the remaining part of the precursor at specific cleavage sites. KR and RR constitute a large majority of all cleavage places, while RK and KK are found with much lower frequency (Veenstra 2000;Fricker 2005). In addition to these sites, some cleavages occur at sites containing pairs of basic amino acids separated by two, four, six or eight other residues. These sites were earlier described as 'monobasic' (Veenstra 2000). After cleavage from the proprotein peptide precursor, the peptide is further processed by a specific carboxypeptidase that removes the carboxyterminal basic amino acids. Next, the carboxyterminal glycine residue is transformed into an amide by a-amidating enzymes. This post-translational modification (conversion of a glycine into an amide) is a common feature of secretory peptides.In mammals, seven members of the kex2/subtilisin-like PCs have been described in detail (Rouille et al. 1995;Beinfeld 1998;Canaff et al. 1999). These are designated furin, PC1/3, PC2, PC4, PC5/6, PC7/8/LPC and PACE4 and they all share a characteristic overall similarity to the prototype member of the family, Kex2p, that was initially identified in yeast (Julius et a...

Completion of the Caenorhabditis elegans genome sequencing project in 1998 has provided more insight into the complexity of nematode neuropeptide signaling. Several C. elegans neuropeptide precursor genes, coding for approximately 250 peptides, have been predicted from the genomic database. One can, however, not deduce whether all these peptides are actually expressed, nor is it possible to predict all post-translational modifications. Using two dimensional nanoscale liquid chromatography combined with tandem mass spectrometry and database mining, we analyzed a mixed stage C. elegans extract. This peptidomic setup yielded 21 peptides derived from formerly predicted neuropeptide-like protein (NLP) precursors and 28 predicted FMRFamide-related peptides. In addition, we were able to sequence 11 entirely novel peptides derived from nine peptide precursors that were not predicted or identified in any way previously. Some of the identified peptides display profound sequence similarities with neuropeptides from other invertebrates, indicating that these peptides have a long evolutionary history.

Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of a-helical, cationic peptides. For the first time, a comparison of the primary structures of a-helical pore-forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40-50 amino acids contain a typical scorpion conserved sequence S(x) 3 KxWxS(x) 5 L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a poreforming peptide previously isolated from the venom of the South-African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell-selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram-negative bacteria (1.3-25 lM), while melittin and mastoparan, two well-known cytolytic peptides, were more effective against Gram-positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.

The pars intercerebralis±corpora cardiaca system (PI±CC) of insects is the endocrinological equivalent of the hypothalamus±pituitary system of vertebrates. Peptide profiles of the pars intercerebralis and the corpora cardiaca were characterized using simple sampling protocols in combination with MALDI-TOF and electrospray ionization double quadrupole time of flight (ESI-Qq-TOF) mass spectrometric technologies. The results were compared with earlier results of conventional sequencing methods and immunocytochemical methods. In addition to many known peptides, several m/z signals corresponding to putative novel peptides were observed in the corpora cardiaca and/or pars intercerebralis. Furthermore, for a number of peptides evidence was provided about their localization and MALDI-TOF analysis of the released material from the corpora cardiaca yielded information on the hormonal status of particular brain peptides.

Epilepsy is a common neurological disorder characterized by recurrent seizures. These seizures are due to abnormal excessive and synchronous neuronal activity in the brain caused by a disruption of the delicate balance between excitation and inhibition. Neuropeptides can contribute to such misbalance by modulating the effect of classical excitatory and inhibitory neurotransmitters. In this review, we discuss 21 different neuropeptides that have been linked to seizure disorders. These neuropeptides show an aberrant expression and/or release in animal seizure models and/or epilepsy patients. Many of these endogenous peptides, like adrenocorticotropic hormone, angiotensin, cholecystokinin, cortistatin, dynorphin, galanin, ghrelin, neuropeptide Y, neurotensin, somatostatin, and thyrotropin-releasing hormone, are able to suppress seizures in the brain. Other neuropeptides, such as arginine-vasopressine peptide, corticotropin-releasing hormone, enkephalin, β-endorphin, pituitary adenylate cyclase-activating polypeptide, and tachykinins have proconvulsive properties. For oxytocin and melanin-concentrating hormone both pro- and anticonvulsive effects have been reported, and this seems to be dose or time dependent. All these neuropeptides and their receptors are interesting targets for the development of new antiepileptic drugs. Other neuropeptides such as nesfatin-1 and vasoactive intestinal peptide have been less studied in this field; however, as nesfatin-1 levels change over the course of epilepsy, this can be considered as an interesting marker to diagnose patients who have suffered a recent epileptic seizure.