The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

Cyclotides are plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology that confers them with remarkable structural stability and resistance to proteolytic degradation. Recently, cyclotides have emerged as promising scaffold molecules for designing peptide-based therapeutics. Here, we provide examples of how engineering cyclotides using molecular grafting may lead to the development of novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. Integrating bioactive epitopes into stable cyclotide scaffolds can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. We also discuss and highlight the importance of engineered cyclotides as novel tools to study GPCR signaling.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are co-expressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R-G279S7.44) was identified in an Iranian family: two affected off-springs suffered from early onset L-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A1-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding, but changed the susceptibility to heat denaturation: the thermodynamic stability of A1R-G279S7.44was enhanced by about 2 and 8 K when compared to wildtype A1-receptorand A1R-Y288A7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced indicating a lower energy barrier for conformational transitions in A1R-G279S7.44(73 {plus minus} 23 kJ/mol) than in wildtype A1R(135 {plus minus} 4 kJ/mol) or in A1R-Y288A7.53 (184 {plus minus} 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44was more effective in promoting guanine nucleotide-exchange than wildtype A1R. We detected similar levels of complexes formed between D1-receptors and wildtype A1R or A1R-G279S7.44by co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells co-expressing D1-receptor and A1R-G279S7.44than in those co-expressing wildtype A1R. These observations predict enhanced inhibition of dopa-minergic signaling by A1R-G279S7.44 in vivo consistent with a pathogenic role in Parkinson's disease. Significance Statement Parkinson's diseaseis caused by a loss of dopaminergic input from the substantia nigrato the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes respon-ses elicited by dopamine D1-receptors. We show that this activity is more pronounced in a mutant version of the adenosine A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early onset Parkinson's disease.

The neuropeptides oxytocin (OT) and vasopressin (VP) and their G protein-coupled receptors OTR, V1aR, V1bR, and V2R form an important and widely-distributed neuroendocrine signaling system. In mammals, this signaling system regulates water homeostasis, blood pressure, reproduction, as well as social behaviors such as pair bonding, trust and aggression. There exists high demand for ligands with differing pharmacological profiles to study the physiological and pathological functions of the individual receptor subtypes. Here, we present the pharmacological characterization of an arthropod (Metaseiulus occidentalis) OT/VP-like nonapeptide across the human OT/VP receptors. I8-arachnotocin is a full agonist with respect to second messenger signaling at human V2R (EC50 34 nM) and V1bR (EC50 1.2 µM), a partial agonist at OTR (EC50 790 nM), and a competitive antagonist at V1aR [pA2 6.25 (558 nM)]. Intriguingly, I8-arachnotocin activated the Gαs pathway of V2R without recruiting either β-arrestin-1 or β-arrestin-2. I8-arachnotocin might thus be a novel pharmacological tool to study the (patho)physiological relevance of β-arrestin-1 or -2 recruitment to the V2R. These findings furthermore highlight arthropods as a novel, vast and untapped source for the discovery of novel pharmacological probes and potential drug leads targeting neurohormone receptors.

Ants are emerging model systems to study cellular signaling because distinct castes possess different physiologic phenotypes within the same colony. Here we studied the functionality of inotocin signaling, an insect ortholog of mammalian oxytocin (OT), which was recently discovered in ants. In Lasius ants, we determined that specialization within the colony, seasonal factors, and physiologic conditions down‐regulated the expression of the OT‐like signaling system. Given this natural variation, we interrogated its function using RNAi knockdowns. Next‐generation RNA sequencing of OT‐like precursor knock‐down ants highlighted its role in the regulation of genes involved in metabolism. Knock‐down ants exhibited higher walking activity and increased self‐grooming in the brood chamber. We propose that OT‐like signaling in ants is important for regulating metabolic processes and locomotion.—Liutkevičiūtė, Z., Gil‐Mansilla, E., Eder, T., Casillas‐Pérez, B., Di Giglio, M. G., Muratspahić, E., Grebien, F., Rattei, T., Muttenthaler, M., Cremer, S., Gruber, C. W. Oxytocin‐like signaling in ants influences metabolic gene expression and locomotor activity. FASEB J. 32, 6808–6821 (2018). www.fasebj.org