Regulation of the pharynx of Caenorhabditis elegans by 5‐HT,octopamine, and FMRFamide‐like neuropeptides

More than fifty FMRFamide‐like neuropeptides have been identified in nematodes. We addressed the role of a subset of these in the control of nematode feeding by electrophysiological recording of the activity of C. elegans pharynx. AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), AF8 (KSAYMRFamide), and GAKFIRFamide (encoded by the C. elegans genes flp‐8, flp‐14, flp‐6, and flp‐5, respectively) increased pharyngeal action potential frequency, in a manner similar to 5‐HT. In contrast, SDPNFLRFamide, SADPNFLRFamide, SAEPFGTMRFamide, KPSVRFamide, APEASPFIRFamide, and AQTVRFamide (encoded by the C. elegans genes flp‐1; flp‐1; flp‐3; flp‐9; flp‐13, and flp‐16, respectively) inhibited the pharynx in a manner similar to octopamine. Only three of the neuropeptides had potent effects at low nanomolar concentrations, consistent with a physiological role in pharyngeal regulation. Therefore, we assessed whether these three peptides mediated their actions either directly on the pharynx or indirectly via the neural circuit controlling its activity by comparing actions between wild‐type and mutants with deficits in synaptic signaling. Our data support the conclusion that AF1 and SAEPFGTMRFamide regulate the activity of the pharynx indirectly, whereas APEASPFIRFamide exerts its action directly. These results are in agreement with the expression pattern for the genes encoding the neuropeptides (Kim and Li, 1999) as both flp‐8 and flp‐3 are expressed in extrapharyngeal neurons, whereas flp‐13 is expressed in I5, a neuron with synaptic output to the pharyngeal muscle. These results provide the first, direct, functional information on the action of neuropeptides in C. elegans. Furthermore, we provide evidence for a putative inhibitory peptidergic synapse, which is likely to have a role in the control of feeding. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 235–244, 2001     

Role of a FMRFamide‐like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide‐like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP‐13A, APEASPFIRFamide; excitatory, FLP‐17A, KSAFVRFamide; excitatory, FLP‐17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP‐8 (excitatory AF1, KNEFIRFamide,) and FLP‐11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp‐3, flp‐13, flp‐14, flp‐17, and flp‐18). For all but one gene (flp‐14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Role of a FMRFamide-like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide-like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP-13A, APEASPFIRFamide; excitatory, FLP-17A, KSAFVRFamide; excitatory, FLP-17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP-8 (excitatory AF1, KNEFIRFamide,) and FLP-11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp-3, flp-13, flp-14, flp-17, and flp-18). For all but one gene (flp-14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans.     

Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering

The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C^γ ring pucker preference and the cis– trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple‐stranded antiparallel β‐sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C^γ ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C^γ‐endo ring pucker in the native structure. Pro37 was replaced with C^γ‐exo biased pucker derivatives: (2S,4R)‐4‐hydroxyproline (4R‐Hyp), (2S,4R)‐4‐fluoroproline (4R‐Flp), (2S,4R)‐4‐methoxyproline (4R‐Mop), and C^γ‐endo biased pucker derivatives: (2S,4S)‐4‐hydroxyproline (4S‐hyp), (2S,4S)‐4‐fluoroproline (4S‐flp), (2S,4S)‐4‐methoxyproline (4S‐mop) to examine how a preorganized pucker affects the folding stability and ligand‐binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S‐flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C? F group. Analysis of ligand‐binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S‐flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4‐substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical. Proteins 2014; 82:67–76. © 2013 Wiley Periodicals, Inc.