Additive and Specific Effects of Elicitor Treatments on the Metabolic Profile of Arabidopsis thaliana.

Several elicitors of plant defense have been identified and numerous efforts to use them in the field have been made. Exogenous elicitor treatments mimic the in planta activation of pattern-triggered immunity (PTI), which relies on the perception of pathogen-associated molecular patterns (PAMPs) such as bacterial flg22 or fungal chitins. Early transcriptional responses to distinct PAMPs are mostly overlapping, regardless of the elicitor being used. However, it remains poorly known if the same patterns are observed for metabolites and proteins produced later during PTI. In addition, little is known about the impact of a combination of elicitors on PTI and the level of induced resistance to pathogens. Here, we monitored Arabidopsis thaliana resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) following application of flg22 and chitosan elicitors, used individually or in combination. A slight, but not statistically significant increase in induced resistance was observed when the elicitors were applied together when compared with individual treatments. We investigated the effect of these treatments on the metabolome by using an untargeted analysis. We found that the combination of flg22 and chitosan impacted a higher number of metabolites and deregulated specific metabolic pathways compared with the elicitors individually. These results contribute to a better understanding of plant responses to elicitors, which might help better rationalize their use in the field. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

The 26RFa (QRFP)/GPR103 Neuropeptidergic System: A Key Regulator of Energy and Glucose Metabolism.

BACKGROUND: Obesity and type 2 diabetes are strongly associated pathologies, currently considered as a worldwide epidemic problem. Understanding the mechanisms that drive the development of these diseases would enable to develop new therapeutic strategies for their prevention and treatment. Particularly, the role of the brain in energy and glucose homeostasis has been studied for 2 decades. In specific, the hypothalamus contains well-identified neural networks that regulate appetite and potentially also glucose homeostasis. A new concept has thus emerged, suggesting that obesity and diabetes could be due to a dysfunction of the same, still poorly understood, neural networks. SUMMARY: The neuropeptide 26RFa (also termed QRFP) belongs to the family of RFamide regulatory peptides and has been identified as the endogenous ligand of the human G protein-coupled receptor GPR103 (QRFPR). The primary structure of 26RFa is strongly conserved during vertebrate evolution, suggesting its crucial roles in the control of vital functions. Indeed, the 26RFa/GPR103 peptidergic system is reported to be involved in the control of various neuroendocrine functions, notably the control of energy metabolism in which it plays an important role, both centrally and peripherally, since 26RFa regulates feeding behavior, thermogenesis and lipogenesis. Moreover, 26RFa is reported to control glucose homeostasis both peripherally, where it acts as an incretin, and centrally, where the 26RFa/GPR103 system relays insulin signaling in the brain to control glucose metabolism. KEY MESSAGES: This review gives a comprehensive overview of the role of the 26RFa/GPR103 system as a key player in the control of energy and glucose metabolism. In a pathophysiological context, this neuropeptidergic system represents a prime therapeutic target whose mechanisms are highly relevant to decipher.

Selenoprotein T, a potential treatment of attention-deficit/hyperactivity disorder and comorbid pain in neonatal 6-OHDA lesioned mice.

pathological pain and Attention-deficit/hyperactivity disorder (ADHD) are two complex multifactorial syndromes. The comorbidity of ADHD and altered pain perception is well documented in children, adolescents, and adults. According to pathophysiological investigations, the dopaminergic system's dysfunction provides a common basis for ADHD and comorbid pain. Growing evidence suggests that oxidative stress may be crucial in both pathologies. Recent studies revealed that a small peptide encompassing the redox-active site of selenoprotein T (PSELT), protects dopaminergic neurons and fibers as well as lesioned nerves in animal models. The current study aims to examine the effects of PSELT treatment on ADHD-like symptoms and pain sensitivity, as well as the role of catecholaminergic systems in these effects. Our results demonstrated that intranasal administration of PSELT reduced the hyperactivity in the open field, decreased the impulsivity displayed by 6-OHDA-lesioned male mice in the 5-choice serial reaction time task test and improved attentional performance. In addition, PSELT treatment significantly increased the nociception threshold in both normal and inflammatory conditions. Furthermore, anti-hyperalgesic activity was antagonized with sulpiride pre-treatment, but not by phentolamine, or propranolol pre-treatments. The present study suggests that PSELT reduces the severity of ADHD symptoms in mice and possesses potent antinociceptive effects which could be related to the involvement of D2/D3 dopaminergic receptors.

Glycogen phosphorylase isoenzyme GPbb versus GPmm regulation of ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and counter-regulatory hormone profiles during hypoglycemia: Role of L-lactate and octadecaneuropeptide.

Glucose accesses the brain primarily via the astrocyte cell compartment, where it passes through the glycogen shunt before catabolism to the oxidizable fuel L-lactate. Glycogen phosphorylase (GP) isoenzymes GPbb and GPmm impose distinctive control of ventromedial hypothalamic nucleus (VMN) glucose-regulatory neurotransmission during hypoglycemia, but lactate and/or gliotransmitter involvement in those actions is unknown. Lactate or the octadecaneuropeptide receptor antagonist cyclo(1-8)[DLeu5] OP (LV-1075) did not affect gene product down-regulation caused by GPbb or GPmm siRNA, but suppressed non-targeted GP variant expression in a VMN region-specific manner. Hypoglycemic up-regulation of neuronal nitric oxide synthase was enhanced in rostral and caudal VMN by GPbb knockdown, yet attenuated by GPMM siRNA in the middle VMN; lactate or LV-1075 reversed these silencing effects. Hypoglycemic inhibition of glutamate decarboxylase65/67 was magnified by GPbb (middle and caudal VMN) or GPmm (middle VMN) knockdown, responses that were negated by lactate or LV-1075. GPbb or GPmm siRNA enlarged hypoglycemic VMN glycogen profiles in rostral and middle VMN. Lactate and LV-1075 elicited progressive rostral VMN glycogen augmentation in GPbb knockdown rats, but stepwise-diminution of rostral and middle VMN glycogen after GPmm silencing. GPbb, not GPmm, knockdown caused lactate or LV-1075 - reversible amplification of hypoglycemic hyperglucagonemia and hypercorticosteronemia. Results show that lactate and octadecaneuropeptide exert opposing control of GPbb protein in distinct VMN regions, while the latter stimulates GPmm. During hypoglycemia, GPbb and GPmm may respectively diminish (rostral, caudal VMN) or enhance (middle VMN) nitrergic transmission and each oppose GABAergic signaling (middle VMN) by lactate- and octadecaneuropeptide-dependent mechanisms.

Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

The Heterogeneity of Response of PC12 Cells from Different Laboratories to Nerve Growth Factor and Pituitary Adenylate Cyclase-Activating Polypeptide Questions the Reproducibility of Studies Carried Out with Tumor Cell Lines.

BACKGROUND: PC12 pheochromocytoma tumor cell lines are widely used to decipher the intracellular signaling mechanisms mediating the effects of some growth factors. Nevertheless, the disparity in appearance of some PC12 cell lines used in the different publications questions our ability to compare the results obtained by the numerous laboratories which use them. This led us to analyze the phenotypic aspect and transcriptomic expression of 5 PC12 cell lines from different origins under control conditions and after treatment with nerve growth factor (NGF) or pituitary adenylate cyclase-activating polypeptide (PACAP). METHODS: Characterization of the 5 PC12 cell lines was conducted using imaging techniques and high-throughput real-time PCR combined with bioinformatics analysis. RESULTS: The results show that the 5 cell lines are very variable in terms of shape, proliferation rate, motility, adhesion to the substrate, and gene expression. This high heterogeneity of the cell lines is also found when looking at their response to NGF or PACAP on gene expression or differentiation, with even in some cases opposite effects, as, for example, on cell proliferation. Actually, only 2 of the cell lines tested exhibited some phenotypic similarities with each other, even though the transcriptomic analyses show that they are far from identical. DISCUSSION/CONCLUSION: As this issue of cell heterogenicity is not restricted to PC12 cells, the present results highlight the need to facilitate the supply of cell lines at low cost, the necessity to standardize practices regarding the use of cell lines, and the requirement to define precise markers of established cell lines which should be monitored in every publication. Regarding this latter point, the present data show that transcriptomic analysis by real-time PCR using a panel of genes of interest is easy to implement and provides a reliable method to control the possible drift of the cells over time in culture. Transcriptomic phenotyping combined with bioinformatics analysis can also be a useful approach to predict the response of the cells to treatments in terms of cell signaling activation, which can help to choose among several cell lines the most appropriate one for the investigation of a particular mechanism. Taken together, the results from this study highlight the need to use well-characterized cell lines with standardized protocols to generate reproducible results from 1 laboratory to the other.

Exploring a Structural Data Mining Approach to Design Linkers for Head-to-Tail Peptide Cyclization.

Peptides have recently regained interest as therapeutic candidates, but their development remains confronted with several limitations including low bioavailability. Backbone head-to-tail cyclization, i.e., setting a covalent peptide bond linking the last amino acid with the first one, is one effective strategy of peptide-based drug design to stabilize the conformation of bioactive peptides while preserving peptide properties in terms of low toxicity, binding affinity, target selectivity, and preventing enzymatic degradation. Starting from an active peptide, it usually requires the design of a linker of a few amino acids to make it possible to cyclize the peptide, possibly preserving the conformation of the initial peptide and not affecting its activity. However, very little is known about the sequence-structure relationship requirements of designing linkers for peptide cyclization in a rational manner. Recently, we have shown that large-scale data-mining of available protein structures can lead to the precise identification of protein loop conformations, even from remote structural classes. Here, we transpose this approach to linkers, allowing head-to-tail peptide cyclization. First we show that given a linker sequence and the conformation of the linear peptide, it is possible to accurately predict the cyclized peptide conformation. Second, and more importantly, we show that it seems possible to elaborate on the information inferred from protein structures to propose effective candidate linker sequences constrained by length and amino acid composition, providing the first framework for the rational design of head-to-tail cyclization linkers. Finally, we illustrate this for two peptides using a limited set of amino-acids likely not to interfere with peptide function. For a linear peptide derived from Nrf2, the peptide cyclized starting from the experimental structure showed a 26-fold increase in the binding affinity. For urotensin II, a peptide already cyclized by a disulfide bond that exerts a broad array of biological activities, we were able, starting from models of the structure, to design a head-to-tail cyclized peptide, the first synthesized bicyclic 14-residue long urotensin II analogue, showing a retention of in vitro activity. Although preliminary, our results strongly suggest that such an approach has strong potential for cyclic peptide-based drug design.

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell-line activation.

BACKGROUND: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte-derived macrophages via activation of an unknown G protein-coupled receptor (GPCR). OBJECTIVE: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas-related G protein-coupled receptor X2). METHODS: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In particular, to investigate the immunomodulatory actions, we used ß-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays, immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining. RESULTS: P17 activated MRGPRX2 in a dose-dependent manner in ß-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and ß-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked ß-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-π interaction with the Phe172 of MRGPRX2 and [Ala8]P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17. CONCLUSIONS: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.

The Gliopeptide ODN, a Ligand for the Benzodiazepine Site of GABAA Receptors, Boosts Functional Recovery after Stroke.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

The 26RFa (QRFP)/GPR103 neuropeptidergic system in mice relays insulin signalling into the brain to regulate glucose homeostasis.

AIMS/HYPOTHESIS: 26RFa (pyroglutamilated RFamide peptide [QRFP]) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localised in the hypothalamus. In this study we investigated whether 26RFa neurons are involved in the hypothalamic regulation of glucose homeostasis. METHODS: 26Rfa+/+, 26Rfa-/- and insulin-deficient male C57Bl/6J mice were used in this study. Mice received an acute intracerebroventricular (i.c.v.) injection of 26RFa, insulin or the 26RFa receptor (GPR103) antagonist 25e and were subjected to IPGTTs, insulin tolerance tests, acute glucose-stimulated insulin secretion tests and pyruvate tolerance tests (PTTs). Secretion of 26RFa by hypothalamic explants after incubation with glucose, leptin or insulin was assessed. Expression and quantification of the genes encoding 26RFa, agouti-related protein, the insulin receptor and GPR103 were evaluated by quantitative reverse transcription PCR and RNAscope in situ hybridisation. RESULTS: Our data indicate that i.c.v.-injected 26RFa induces a robust antihyperglycaemic effect associated with an increase in insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26Rfa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26Rfa are mainly localised in the lateral hypothalamic area, that they co-express the gene encoding the insulin receptor and that insulin induces the expression of 26Rfa in these neurons. Concurrently, the central antihyperglycaemic effect of insulin is abolished in the presence of a GPR103 antagonist and in 26RFa-deficient mice. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production. CONCLUSION/INTERPRETATION: We have identified a novel mechanism in the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system, and we provide evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

The SELENOT mimetic PSELT promotes nerve regeneration by increasing axonal myelination in a facial nerve injury model in female rats.

Peripheral nerve injury (PNI) is frequent and many patients suffer lifelong disabilities in severe cases. Although the peripheral nervous system is able to regenerate, its potential is limited. In this study, we tested in a nerve regeneration model in rat the potential beneficial effect of a short mimetic peptide, named PSELT, which derives from SELENOT, an essential thioredoxin-like selenoprotein endowed with neuroprotective and antioxidant activities. For this purpose, the right facial nerve of female Long-Evans rats was axotomized then bridged with a free femoral vein interposition graft. PSELT (1 µM) was injected into the vein immediately and 48 h after the injury, and the effects observed were compared to those found after an end-to-end suture used as a gold standard treatment. Whisking behavior, electrophysiological potential, and histological analyses were performed 3 months after injury to determine the effects of these treatments. These analyses revealed that PSELT-treated animals exhibit a better motor recovery in terms of protraction amplitude and velocity of vibrissae compared to control and end-sutured nerve animal groups. Moreover, administration of PSELT following injury enhanced muscle innervation, axonal elongation, and myelination of newly formed nerve fibers. Altogether, these results indicate that a PSELT-based treatment is sufficient to enhance facial nerve myelination and regeneration and could represent a new therapeutic tool to treat PNI.

Acute but Not Chronic Central Administration of the Neuropeptide 26RFa (QRFP) Improves Glucose Homeostasis in Obese/Diabetic Mice.

INTRODUCTION: The aim of the study is to investigate whether acute or chronic central administration of the hypothalamic neuropeptide 26RFa may ameliorate the glycemic control of obese/diabetic mice. METHODS: Mice were treated for 4 months with a high-fat (HF) diet and received a single i.c.v. injection of 26RFa (3 µg) or a chronic i.c.v. administration of the peptide during 28 days via osmotic minipumps (25 µg/day). i.p. and oral glucose (GLU) tolerance tests, insulin (INS) tolerance test, glucose-stimulated insulin secretion (GSIS), food/water intake, horizontal/vertical activity, energy expenditure, meal pattern, and whole-body composition were monitored. In addition, 26RFa and GPR103 mRNA expressions as well as plasma 26RFa levels were evaluated by RT-QPCR and radioimmunoassay. RESULTS: Acute administration of 26RFa in HF mice induced a robust antihyperglycemic effect by enhancing INS secretion, whereas chronic administration of the neuropeptide is unable to improve glucose homeostasis in these obese/diabetogenic conditions. By contrast, chronic 26RFa treatment induced an increase of the body weight accompanied with an enhanced food intake and a decreased energy expenditure. Finally, we show that the HF diet does not alter the hypothalamic expression of the 26RFa/GPR103 neuropeptidergic system nor the levels of circulating 26RFa. CONCLUSION: Our data indicate that the central beneficial effect of 26RFa on glucose homeostasis, by potentiating GSIS, is preserved in HF mice. However, chronic administration of the neuropeptide is unable to balance glycemia in these pathophysiological conditions, suggesting that the hypothalamic 26RFa/GPR103 neuropeptidergic system mainly affects short-term regulation of glucose metabolism.

Structural and Functional Characterization of Orcokinin B-like Neuropeptides in the Cuttlefish (Sepia officinalis).

The cuttlefish (Sepia officinalis) is a Cephalopod mollusk that lives in the English Channel and breeds in coastal spawning grounds in spring. A previous work showed that the control of egg-laying is monitored by different types of regulators, among which neuropeptides play a major role. They are involved in the integration of environmental cues, and participate in the transport of oocytes in the genital tract and in the secretion of capsular products. This study addresses a family of neuropeptides recently identified and suspected to be involved in the control of the reproduction processes. Detected by mass spectrometry and immunocytochemistry in the nerve endings of the accessory sex glands of the females and ovary, these neuropeptides are also identified in the hemolymph of egg-laying females demonstrating that they also have a hormone-like role. Released in the hemolymph by the sub-esophageal mass, a region that innervates the genital tract and the neurohemal area of the vena cava, in in vitro conditions these neuropeptides modulated oocyte transport and capsular secretion. Finally, in silico analyses indicated that these neuropeptides, initially called FLGamide, had extensive structural homology with orcokinin B, which motivated their name change.

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes.

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four Cα-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by Cα H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,ß-dehydro-amino acid intermediate during Cα-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

Glial endozepines and energy balance: Old peptides with new tricks.

The contribution of neuroglial interactions to the regulation of energy balance has gained increasing acceptance in recent years. In this context, endozepines, endogenous analogs of benzodiazepine derived from diazepam-binding inhibitor, are now emerging as major players. Produced by glial cells (astrocytes and tanycytes), endozepines have been known for two decades to exert potent anorexigenic effects by acting at the hypothalamic level. However, it is only recently that their modes of action, including the mechanisms by which they modulate energy metabolism, have begun to be elucidated. The data available today are abundant, significant, and sometimes contradictory, revealing a much more complex regulation than initially expected. Several mechanisms of action of endozepines seem to coexist at the central level, particularly in the hypothalamus. The brainstem has also recently emerged as a potential site of action for endozepines. In addition to their central anorexigenic effects, endozepines may also display peripheral effects promoting orexigenic actions, adding to their complexity and raising yet more questions. In this review, we attempt to provide an overview of our current knowledge in this rapidly evolving field and to pinpoint questions that remain unanswered.

Neuroprotective effects of PACAP against paraquat-induced oxidative stress in the Drosophila central nervous system.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that can arise after long-term exposure to environmental oxidative stressors, such as the herbicide paraquat (PQ). Here we investigated the potential neuroprotective action of vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) against PQ in Drosophila. We found that pre-treatment with this neuropeptide applied to the ventral nerve cord (VNC) at low doses markedly extended the survival of wild-type decapitated flies exposed to neurotoxic levels of PQ or dopamine (DA). In contrast and interestingly, application of a PACAP receptor antagonist, PACAP-6-38, had opposite effects, significantly decreasing the resistance of flies to PQ. PACAP also reduced PQ-induced caspase activation and reactive oxygen species (ROS) accumulation in the VNC. We then searched for the endogenous neuropeptide receptor potentially involved in PACAP-mediated neuroprotection in Drosophila. Knocking down the gene encoding the receptor Han/PDFR of the neuropeptide pigment-dispersing factor (PDF) in all neurons conferred to flies higher resistance to PQ, whereas PDFR downregulation restricted to PDF or DA neurons did not increase PQ resistance, but remarkably suppressed the neuroprotective action of PACAP. Further experiments performed with Pdf and Pdfr-deficient mutant strains confirmed that PDF and its receptor are required for PACAP-mediated neuroprotection in flies. We also provide evidence using split-green fluorescent protein (split-GFP) reconstitution that PDF neurons make synaptic contacts onto DA neurons in the abdominal VNC. Our results therefore suggest that the protective action of PACAP against PQ-induced defects in the Drosophila nervous system involves the modulation of PDFR signaling in a small number of interconnected neurons.

A putative new melatonin binding site in sheep brain, MTx: preliminary observations and characteristics.

In mammals, MT1 and MT2 melatonin receptors are high affinity G protein-coupled receptors and are thought to be involved in the integration of the melatonin signaling throughout the brain and periphery. In the present study, we describe a new melatonin binding site, named MTx, with a peculiar pharmacological profile. This site had a low affinity for 2-[125I]-melatonin in saturation assays in hypothalamus and retina (pKD = 9.13 {plus minus} 0.05, Bmax = 1.12 {plus minus} 0.11 fmol/mg protein and pKD = 8.81 {plus minus} 0.50, Bmax = 7.65 {plus minus} 2.64 fmol/mg protein, respectively) and a very high affinity, in competition assays, for melatonin (pKi = 13.08 {plus minus} 0.18), and other endogenous compounds. Using autoradiography, we showed a preferential localization of the MTx in periventricular areas of the sheep brain, with a density 3 to 8 times higher than those observed for ovine MT1 In addition, using a set of well-characterized ligands, we showed that this site did not correspond to any of the following receptors: MT1, MT2, MT3 , D1, D2, noradrenergic, nor 5-HT2 Based on its affinity for melatonin, MTx did not seem to be implicated in the integration of cerebral melatonin concentration variations since they were saturating for MTx. Nevertheless, it remained of prime importance because of its periventricular distribution, in close contact with the CSF, and its peculiar pharmacological profile responding to both melatoninergic and serotoninergic compounds. Significance Statement Herein a putative new melatonin binding site is described in sheep brain parts in close contact with the 3rd ventricle. The characteristics of the pharmacological profile of this site is different from anything previously reported in the literature. The present work forms the basis of future full pharmacological characterization.

Autoimmune encephalitis mediated by B-cell response against N-methyl-d-aspartate receptor.

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a neuropsychiatric disease characterized by an antibody-mediated autoimmune response against NMDAR. Recent studies have shown that anti-NMDAR antibodies are involved in the pathophysiology of the disease. However, the upstream immune and inflammatory processes responsible for this pathogenic response are still poorly understood. Here, we immunized mice against the region of NMDA receptor containing the N368/G369 amino acids, previously implicated in a pathogenic response. This paradigm induced encephalopathy characterized by blood-brain barrier opening, periventricular T2-MRI hyperintensities and IgG deposits into the brain parenchyma. Two weeks after immunization, mice developed clinical symptoms reminiscent of encephalitis: anxiety- and depressive-like behaviours, spatial memory impairment (without motor disorders) and increased sensitivity to seizures. This response occurred independently of overt T-cell recruitment. However, it was associated with B220+ (B cell) infiltration towards the ventricles, where they differentiated into CD138+ cells (plasmocytes). Interestingly, these B cells originated from peripheral lymphoid organs (spleen and cervical lymphoid nodes). Finally, blocking the B-cell response using a depleting cocktail of antibodies reduced the severity of symptoms in encephalitis mice. This study demonstrates that the B-cell response can lead to an autoimmune reaction against NMDAR that drives encephalitis-like behavioural impairments. It also provides a relevant platform for dissecting encephalitogenic mechanisms in an animal model, and enables the testing of therapeutic strategies targeting the immune system in anti-NMDAR encephalitis.

Point-Substitution of Phenylalanine Residues of 26RFa Neuropeptide: A Structure-Activity Relationship Study.

26RFa is a neuropeptide that activates the rhodopsin-like G protein-coupled receptor QRFPR/GPR103. This peptidergic system is involved in the regulation of a wide array of physiological processes including feeding behavior and glucose homeostasis. Herein, the pharmacological profile of a homogenous library of QRFPR-targeting peptide derivatives was investigated in vitro on human QRFPR-transfected cells with the aim to provide possible insights into the structural determinants of the Phe residues to govern receptor activation. Our work advocates to include in next generations of 26RFa(20-26)-based QRFPR agonists effective substitutions for each Phe unit, i.e., replacement of the Phe22 residue by a constrained 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid moiety, and substitution of both Phe24 and Phe26 by their para-chloro counterpart. Taken as a whole, this study emphasizes that optimized modifications in the C-terminal part of 26RFa are mandatory to design selective and potent peptide agonists for human QRFPR.