Aplysia allatotropin-related peptide and its newly identified d-amino acid-containing epimer both activate a receptor and a neuronal target.
ABSTRACT: l- to d-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the d-residue is critical for the biological function of the resulting d-amino acid-containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-l-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a d-amino acid-containing peptide (d2-ATRP) and identified a specific G protein-coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-l-residue analogs, both l-ATRP and d2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d2-ATRP was much more stable than its all-l-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the l- to d-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that l- to d-residue isomerization can occur even in an all-l-residue peptide with a known biological activity and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.
Project description:Neuropeptides in several animals undergo an unusual post-translational modification, the isomerization of an amino acid residue from the l-stereoisomer to the d-stereoisomer. The resulting d-amino acid-containing peptide (DAACP) often displays biological activity higher than that of its all-l-residue analogue, with the d-residue being critical for function in many cases. However, little is known about the full physiological roles played by DAACPs, and few studies have examined the interaction of DAACPs with their cognate receptors. Here, we characterized the signaling of several DAACPs derived from a single neuropeptide prohormone, the Aplysia californica achatin-like neuropeptide precursor (apALNP), at their putative receptor, the achatin-like neuropeptide receptor (apALNR). We first used quantitative polymerase chain reaction and in situ hybridization experiments to demonstrate receptor ( apALNR) expression throughout the central nervous system; on the basis of the expression pattern, we identified novel physiological functions that may be mediated by apALNR. To gain insight into ligand signaling through apALNR, we created a library of native and non-native neuropeptide analogues derived from apALNP (the neuropeptide prohormone) and evaluated them for activity in cells co-transfected with apALNR and the promiscuous G? subunit G?-16. Several of these neuropeptide analogues were also evaluated for their ability to induce circuit activity in a well-defined neural network associated with feeding behavior in intact ganglia from Aplysia. Our results reveal the specificity of apALNR and provide strong evidence that this receptor mediates diverse physiological functions throughout the central nervous system. Finally, we show that some native apALNP-derived DAACPs exhibit enhanced stability toward endogenous proteases, suggesting that the d-residues in these DAACPs may increase the peptide lifetime, in addition to influencing receptor specificity, in the nervous system. Ultimately, these studies provide insight into signaling at one of the few known DAACP-specific receptors and advance our understanding of the roles that l- to d-residue isomerization play in neuropeptide signaling.
Project description:RNA-based protein synthesis produces L-amino acid-containing proteins and peptides. D-amino acid-containing peptides (DAACPs) can be generated from L-amino acid peptides via post-translational modification. In the nervous system, the conformational change of a single L-amino acid in a peptide to its D-form results in altered bioactivity, with some DAACPs having orders-of-magnitude enhanced efficacy. However, this modification is often overlooked when characterizing endogenous peptides. Here, with the use of matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry, neuropeptides that have the second residue isomerized to the D-isoform are distinguished from their L-epimers via differences in the relative amounts of specific fragment ions during tandem MS. With the appropriate fragment ions chosen, and in some cases with the use of metal adducts, epimer discrimination is optimized. Specifically, the cardioexcitatory peptide Asn-(D)Trp-Phe-amide (NdWFa) was assayed directly from neurons isolated from the sea slug Aplysia californica; the fraction of the peptide with the second residue (W) in the D- versus L-form was 90 ± 10%. We demonstrate that this approach is well suited for confirming DAACPs directly from cells and tissue, advancing our understanding of the l to d modification and the role it plays in cell-to-cell signaling.
Project description:Proteins and peptides in nature are almost exclusively made from l-amino acids, and this is even more absolute in the metazoan. With the advent of modern bioanalytical techniques, however, previously unappreciated roles for d-amino acids in biological processes have been revealed. Over 30 d-amino acid containing peptides (DAACPs) have been discovered in animals where at least one l-residue has been isomerized to the d-form via an enzyme-catalyzed process. In Aplysia californica, GdFFD and GdYFD (the lower-case letter "d" indicates a d-amino acid residue) modulate the feeding behavior by activating the Aplysia achatin-like neuropeptide receptor (apALNR). However, little is known about how the three-dimensional conformation of DAACPs influences activity at the receptor, and the role that d-residues play in these peptide conformations. Here, we use a combination of computational modeling, drift-tube ion-mobility mass spectrometry, and receptor activation assays to create a simple model that predicts bioactivities for a series of GdFFD analogs. Our results suggest that the active conformations of GdFFD and GdYFD are similar to their lowest energy conformations in solution. Our model helps connect the predicted structures of GdFFD analogs to their activities, and highlights a steric effect on peptide activity at position 1 on the GdFFD receptor apALNR. Overall, these methods allow us to understand ligand-receptor interactions in the absence of high-resolution structural data.
Project description:A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.
Project description:Compensatory mechanisms are often used to achieve stability by reducing variance, which can be accomplished via negative feedback during homeostatic regulation. In principle, compensation can also be implemented through feedforward mechanisms where a regulator acts to offset the anticipated output variation; however, few such neural mechanisms have been demonstrated. We provide evidence that an Aplysia neuropeptide, identified using an enhanced representational difference analysis procedure, implements feedforward compensation within the feeding network. We named the novel peptide "allatotropin-related peptide" (ATRP) because of its similarity to insect allatotropin. Mass spectrometry confirmed the peptide's identity, and in situ hybridization and immunostaining mapped its distribution in the Aplysia CNS. ATRP is present in the higher-order cerebral-buccal interneuron (CBI) CBI-4, but not in CBI-2. Previous work showed that CBI-4-elicited motor programs have a shorter protraction duration than those elicited by CBI-2. Here we show that ATRP shortens protraction duration of CBI-2-elicited ingestive programs, suggesting a contribution of ATRP to the parametric differences between CBI-4-evoked and CBI-2-evoked programs. Importantly, because Aplysia muscle contractions are a graded function of motoneuronal activity, one consequence of the shortening of protraction is that it can weaken protraction movements. However, this potential weakening is offset by feedforward compensatory actions exerted by ATRP. Centrally, ATRP increases the activity of protraction motoneurons. Moreover, ATRP is present in peripheral varicosities of protraction motoneurons and enhances peripheral motoneuron-elicited protraction muscle contractions. Therefore, feedforward compensatory mechanisms mediated by ATRP make it possible to generate a faster movement with an amplitude that is not greatly reduced, thereby producing stability.
Project description:Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.
Project description:Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues.
Project description:Characterization of endogenous neuropeptides produced from post-translational proteolytic processing of precursor proteins is a demanding task. A variety of complex prohormone processing steps generate molecular diversity from neuropeptide prohormones, making in silico neuropeptide discovery difficult. In addition, the wide range of endogenous peptide concentrations as well as significant peptide complexity further challenge the structural characterization of neuropeptides. Liquid chromatography-mass spectrometry (MS), performed in conjunction with bioinformatics, allows for high-throughput characterization of peptides. Mass analyzers and molecular dissociation techniques render specific characteristics to the acquired data and thus, influence the analysis of the MS data using bioinformatic algorithms for follow-up peptide identification. Here we evaluated the efficacy of several distinct peptidomic workflows using two mass spectrometers, the Thermo Orbitrap Fusion Tribrid and Bruker Impact HD UHR-QqTOF, for confident peptide discovery and characterization. We compared the results in several categories, including the numbers of identified peptides, full-length mature neuropeptides among all identifications, and precursor proteins mapped by the identified peptides. We also characterized the peptide false discovery rate (FDR) based on the occurrence of amidation, a known post-translational modification (PTM) that has been shown to require the presence of a C-terminal glycine. Thus, amidation events without a preceding glycine were considered false-positive amidation assignments. We compared the FDR calculated by the search engine used here to the minimum FDR estimated via false amidation assignments. The search engine severely underestimated the rate of false PTM assignments among the identified peptides, regardless of the specific MS platform used.
Project description:Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. Graphical Abstract ?.
Project description:NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca(2+) binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+) channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+) and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d) of ?14.3 µM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.