Search Results (47)

Leptin, an adipocyte-derived hormone, plays a central role in the regulation of energy homeostasis by acting on distinct hypothalamic nuclei. This review explores recent advances in our understanding of leptin’s region-specific actions within the arcuate nucleus, ventromedial hypothalamus, dorsomedial hypothalamus, and lateral hypothalamus, highlighting their contributions to appetite regulation, energy expenditure, and neuroendocrine function. In the hypothalamic arcuate nucleus, leptin’s differential regulation of pro-opiomelanocortin and agouti-related peptide/neuropeptide Y neurons is now complemented by the identification of novel leptin-responsive neuronal populations—such as those expressing prepronociceptin, basonuclin 2, and Pirt—as well as a growing array of cellular and molecular modulators, including secreted factors like angiopoietin-like growth factor, zinc-α2-glycoprotein, and spexin, intracellular regulators such as Rap1, growth factor receptor-bound protein 10, and spliced X-box binding protein 1. In the ventromedial hypothalamus, leptin integrates with both peripheral (e.g., cholecystokinin) and central (e.g., pituitary adenylate cyclase-activating polypeptide) signals, while epigenetic mechanisms, such as those mediated by Jumonji domain-containing protein D3, regulate leptin receptor expression and sensitivity. The dorsomedial hypothalamus is increasingly recognized for coordinating leptin’s effects on metabolism, circadian rhythms, and respiration through distinct neuronal populations, including a subset of neurons co-expressing GLP-1 receptors that mediate leptin’s metabolic effects. In the lateral hypothalamus, leptin modulates reward-driven feeding via GABAergic neuronal populations—circuits that are particularly susceptible to disruption following early life trauma. Together, these insights reveal a sophisticated neurobiological framework through which leptin orchestrates systemic physiology. Understanding the heterogeneity of leptin signaling opens new avenues for restoring leptin sensitivity and developing personalized therapeutic strategies to combat obesity and related metabolic disorders. Full article

This study explored the biosynthetic mechanisms and structural diversity of pyoverdines (PVDs) produced by Pseudomonas fulva. Genomic analysis using antiSMASH identified the PVD biosynthetic gene cluster, although the C-terminal peptide sequence could not be predicted. Subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed the full peptide structure, including modified residues, such as N-acetylhydroxyornithine and cyclohydroxyornithine, and confirmed the presence of several PVD isoforms with different chromophore side chains. Comparative LC-MS analysis across Pseudomonas species demonstrated that P. fulva produces unique PVD molecular mass patterns. The bioinformatic and structural modeling of non-ribosomal peptide synthetase PvdL open reading frame 3 revealed that the A2 and A3 adenylation domains are lysine selective. Although their sequences differ from known lysine-specific signatures, AlphaFold3-based structural prediction revealed conserved substrate-binding configurations, suggesting that similar substrate-binding features may have arisen independently. Notably, Thr²⁹⁷, a unique residue in the non-ribosomal code, likely plays a key role in lysine recognition. The high degree of sequence similarity between the A2 and A3 domains may reflect domain duplication and could be involved in the diversification of the PVD structure. Further functional and ecological studies are required to assess the physiological significance of P. fulva PVDs in microbial iron acquisition. Full article

Peptaibols are linear fungal peptides featuring α,α-dialkylated amino acids (e.g., α-aminoisobutyric acid (Aib), isovaline (Iva)) and characteristic C-terminal alcohol groups. Despite their promising antibacterial and antiplasmodial activities, detailed biosynthetic studies remain limited. A genome-guided study of the fungus Trichodema sp. SK1-7, isolated from decaying wood, revealed the production of previously described trichorozin IV (1), along with novel SF4-type peptaibol 2 (trichorozin V). The structures of these compounds were elucidated through MS analysis, NMR study and advanced Marfey’s method. The genome of Trichoderma sp. SK1-7 harbors two PKS-NRPS hybrid gene clusters containing 14 and 18 adenylation domains. Analysis of the modular architecture suggested that trichorozins are synthesized by a 14-module protein via a module skipping mechanism. Genome mining revealed several types of short peptaibol synthase architectures (10–14 adenylation domains) across various Trichoderma species, accompanied by similar long peptaibol synthases. Furthermore, putative Aib/Iva biosynthesis machinery in Trichoderma was identified, showing specific architectures potentially involved in regulating peptaibol biosynthesis. Feeding experiments demonstrated that peptaibol production depends on the ratio of Iva/Aib. The isolated compounds exhibited moderate antibacterial and cytotoxic activities along with a synergistic effect when combined with membrane-targeting antibiotics. Our findings suggest that genome-guided approaches hold promise for further development of peptabiotics with a wide range of applications, including antibiotic adjuvants. Full article

In non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products—namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), N-propargyloxy-carbonyl-L-lysine (Prop-Lys), or O-propargyl-L-tyrosine (Prop-Tyr). Subsequently, chemo-selective labeling is used to visualize the clickable cyanopeptides using Alexa Fluor 488 (A488). In this study, the time-lapse build up or decline of azide- or alkyne-modified MCs or APs was visualized during maximum growth, specifically MC biosynthesis in Microcystis aeruginosa and AP biosynthesis in Planktothrix agardhii. Throughout the time-lapse build up or decline, the A488 signal occurred with heterogeneous intracellular distribution. There was a fast increase or decrease in the A488 signal for either Prop-Tyr or Prop-Lys, while a delayed or unobservable A488 signal for Phe-Az was related to increased cell size as well as a reduction in growth and autofluorescence. The proportion of clickable MC/AP in peptide extracts as recorded by a chemical–analytical technique correlated positively with A488 labeling intensity quantified via laser-scanning confocal microscopy for individual cells or via flow cytometry at the population level. It is concluded that chemical modification of MC/AP can be used to track intracellular dynamics in biosynthesis using both analytical chemistry and high-resolution imaging. Full article

Recently, the use of click chemistry for localization of chemically modified cyanopeptides has been introduced, i.e., taking advantage of promiscuous adenylation (A) domains in non-ribosomal peptide synthesis (NRPS), allowing for the incorporation of clickable non-natural amino acids (non-AAs) into their peptide products. In this study, time-lapse experiments have been performed using pulsed feeding of three different non-AAs in order to observe the synthesis or decline of azide- or alkyne-modified microcystins (MCs) or anabaenopeptins (APs). The cyanobacteria Microcystis aeruginosa and Planktothrix agardhii were grown under maximum growth rate conditions (r = 0.35–0.6 and 0.2–0.4 (day⁻¹), respectively) in the presence of non-AAs for 12–168 h. The decline of the azide- or alkyne-modified MC or AP was observed via pulse-feeding. In general, the increase in clickable MC/AP in peptide content reached a plateau after 24–48 h and was related to growth rate, i.e., faster-growing cells also produced more clickable MC/AP. Overall, the proportion of clickable MC/AP in the intracellular fraction correlated with the proportion observed in the dissolved fraction. Conversely, the overall linear decrease in clickable MC/AP points to a rather constant decline via dilution by growth instead of a regulated or induced release in the course of the synthesis process. Full article

Increasing numbers of reports have revealed novel catalytically active cryptic guanylate cyclases (GCs) and adenylate cyclases (ACs) operating within complex proteins in prokaryotes and eukaryotes. Here we review the structural and functional aspects of some of these cyclases and provide examples that illustrate their roles in the regulation of the intramolecular functions of complex proteins, such as the phytosulfokine receptor (PSKR), and reassess their contribution to signal generation and tuning. Another multidomain protein, Arabidopsis thaliana K⁺ uptake permease (AtKUP5), also harbors multiple catalytically active sites including an N-terminal AC and C-terminal phosphodiesterase (PDE) with an abscisic acid-binding site. We argue that this architecture may enable the fine-tuning and/or sensing of K⁺ flux and integrate hormone responses to cAMP homeostasis. We also discuss how searches with motifs based on conserved amino acids in catalytic centers led to the discovery of GCs and ACs and propose how this approach can be applied to discover hitherto masked active sites in bacterial, fungal, and animal proteomes. Finally, we show that motif searches are a promising approach to discover ancient biological functions such as hormone or gas binding. Full article

Nonribosomal peptides (NRPs) are biosynthesized by nonribosomal peptide synthetases (NRPSs) and are widely distributed in both terrestrial and marine organisms. Many NRPs and their analogs are biologically active and serve as therapeutic agents. The adenylation (A) domain is a key catalytic domain that primarily controls the sequence of a product during the assembling of NRPs and thus plays a predominant role in the structural diversity of NRPs. Engineering of the A domain to alter substrate specificity is a potential strategy for obtaining novel NRPs for pharmaceutical studies. On the basis of introducing the catalytic mechanism and multiple functions of the A domains, this article systematically describes several representative NRPS engineering strategies targeting the A domain, including mutagenesis of substrate-specificity codes, substitution of condensation-adenylation bidomains, the entire A domain or its subdomains, domain insertion, and whole-module rearrangements. Full article

Dopamine receptors (DARs) are important transmembrane receptors responsible for receiving extracellular signals in the DAR-mediated signaling pathway, and are involved in a variety of physiological functions. Herein, the D1 DAR gene from Marsupenaeus japonicus (MjDAD1) was identified and characterized. The protein encoded by MjDAD1 has the typical structure and functional domains of the G-protein coupled receptor family. MjDAD1 expression was significantly upregulated in the gills and hepatopancreas after low temperature stress. Moreover, double-stranded RNA-mediated silencing of MjDAD1 significantly changed the levels of protein kinases (PKA and PKC), second messengers (cyclic AMP (cAMP), cyclic cGMP, calmodulin, and diacyl glycerol), and G-protein effectors (adenylate cyclase and phospholipase C). Furthermore, MjDAD1 silencing increased the apoptosis rate of gill and hepatopancreas cells. Thus, following binding to their specific receptors, G-protein effectors are activated by MjDAD1, leading to DAD1-cAMP/PKA pathway-mediated regulation of caspase-dependent mitochondrial apoptosis. We suggest that MjDAD1 is indispensable for the environmental adaptation of M. japonicus. Full article

Nonribosomal peptide synthetases (NRPSs) are a class of cytosolic enzymes that synthesize a range of bio-active secondary metabolites including antibiotics and siderophores. They are widespread among both prokaryotes and eukaryotes but are considered rare among animals. Recently, several novel NRPS genes have been described in nematodes, schistosomes, and arthropods, which led us to investigate how prevalent NRPS genes are in the animal kingdom. We screened 1059 sequenced animal genomes and showed that NRPSs were present in 7 out of the 19 phyla analyzed. A phylogenetic analysis showed that the identified NRPSs form clades distinct from other adenylate-forming enzymes that contain similar domains such as fatty acid synthases. NRPSs show a remarkably scattered distribution over the animal kingdom. They are especially abundant in rotifers and nematodes. In rotifers, we found a large variety of domain architectures and predicted substrates. In the nematode Plectus sambesii, we identified the beta-lactam biosynthesis genes L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase that catalyze the formation of beta-lactam antibiotics in fungi and bacteria. These genes are also present in several species of Collembola, but not in other hexapods analyzed so far. In conclusion, our survey showed that NRPS genes are more abundant and widespread in animals than previously known. Full article

4-Coumarate: coenzyme A ligase (4CL; EC 6.2.1.12) is an important enzyme in the phenylpropanoid metabolic pathway that controls the biosynthesis of lignin and flavonoids. In this study, to identify the function of the Ag4CL3 gene of celery, the Ag4CL3 gene was cloned from celery cv. “Nanxuan Liuhe Ziqin”. Sequence analysis results showed that the Ag4CL3 gene contained an open reading frame (ORF) with a length of 1688 bp, and 555 amino acids were encoded. The Ag4CL3 protein was highly conserved among different plant species. Phylogenetic analysis demonstrated that the 4CL proteins from celery and carrot belonged to the same clade. The Ag4CL3 protein was mainly composed of 31.89% α-helixes, 18.02% extended strands, 6.67% β-turns, and 43.42% random coils, and the signal peptide was unfound. A total of 62 phosphorylation sites and a class-I superfamily of adenylate-forming domains were found. As the growth time increased, the plant height and stem thickness also increased, and the petiole lignin content increased and became lignified gradually. The relative expression levels of the Ag4CL3 gene in “Nanxuan Liuhe Ziqin” petioles were higher than those in other tissues, with the highest level occurring 70 d after sowing. The lignin contents in the transgenic Arabidopsis thaliana lines hosting the Ag4CL3 gene were higher than those in the WT. In this study, the overexpression of Ag4CL3 led to the significant upregulation of lignin biosynthesis gene expression in transgenic A. thaliana plants, except for AtPAL, AtCCR, and AtLAC. This study speculates that Ag4CL3 genes are related to lignin synthesis in A. graveolens. Full article

Dithiolopyrrolone antibiotics are well known for their outstanding biological activities, and their biosynthesis has been studied vigorously. However, the biosynthesis mechanism of the characteristic bicyclic scaffold is still unknown after years of research. To uncover this mechanism, a multi-domain non-ribosomal peptide synthase DtpB from the biosynthetic gene cluster of thiolutin was selected as an object to study. We discovered that its adenylation domain not only recognized and adenylated cysteine, but also played an essential role in the formation of the peptide bond. Notably, an eight-membered ring compound was also discovered as an intermediate during the formation of the bicyclic structure. Based on these findings, we propose a new mechanism for the biosynthesis of the bicyclic scaffold of dithiolopyrrolones, and unveil additional functions of the adenylation domain. Full article

Trichoderma is recognized as a prolific producer of nonribosomal peptides (NRPs) known as peptaibols, which have remarkable biological properties, such as antimicrobial and anticancer activities, as well as the ability to promote systemic resistance in plants against pathogens. In this study, the sequencing of 11-, 14- and 15-res peptaibols produced by a marine strain of Trichoderma isolated from the ascidian Botrylloides giganteus was performed via liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Identification, based on multilocus phylogeny, revealed that our isolate belongs to the species T. endophyticum, which has never been reported in marine environments. Through genome sequencing and genome mining, 53 biosynthetic gene clusters (BGCs) were identified as being related to bioactive natural products, including two NRP-synthetases: one responsible for the biosynthesis of 11- and 14-res peptaibols, and another for the biosynthesis of 15-res. Substrate prediction, based on phylogeny of the adenylation domains in combination with molecular networking, permitted extensive annotation of the mass spectra related to two new series of 15-res peptaibols, which are referred to herein as “endophytins”. The analyses of synteny revealed that the origin of the 15-module peptaibol synthetase is related to 18, 19 and 20-module peptaibol synthetases, and suggests that the loss of modules may be a mechanism used by Trichoderma species for peptaibol diversification. This study demonstrates the importance of combining genome mining techniques, mass spectrometry analysis and molecular networks for the discovery of new natural products. Full article

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts effective neuroprotective activity through its specific receptor, PAC1-R. We accidentally discovered that as a positive allosteric modulator (PAM) of PAC1-R, the small-molecule PAM (SPAM1) has a hydrazide-like structure, but different binding characteristics, from hydrazide for the N-terminal extracellular domain of PAC1-R (PAC1-R-EC1). SPAM1 had a significant neuroprotective effect against oxidative stress, both in a cell model treated with hydrogen peroxide (H₂O₂) and an aging mouse model induced by D-galactose (D-gal). SPAM1 was found to block the decrease in PACAP levels in brain tissues induced by D-gal and significantly induced the nuclear translocation of PAC1-R in PAC1R-CHO cells and mouse retinal ganglion cells. Nuclear PAC1-R was subjected to fragmentation and the nuclear 35 kDa, but not the 15 kDa fragments, of PAC1-R interacted with SP1 to upregulate the expression of Huntingtin (Htt), which then exerted a neuroprotective effect by attenuating the binding availability of the neuron-restrictive silencer factor (NRSF) to the neuron-restrictive silencer element (NRSE). This resulted in an upregulation of the expression of NRSF-related neuropeptides, including PACAP, the brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), and synapsin-1 (SYN1). The novel mechanism reported in this study indicates that SPAM1 has potential use as a drug, as it exerts a neuroprotective effect by regulating NRSF. Full article

The mitogen-activated protein kinase (MAPK) signaling pathways regulate diverse cellular processes and have been partially characterized in the rice false smut fungus Ustilaginoidea virens. UvSte50 has been identified as a homolog to Saccharomyces cerevisiae Ste50, which is known to be an adaptor protein for MAPK cascades. ΔUvste50 was found to be defective in conidiation, sensitive to hyperosmotic and oxidative stresses, and non-pathogenic. The mycelial expansion of ΔUvste50 inside spikelets of rice terminated at stamen filaments, eventually resulting in a lack of formation of false smut balls on spikelets. We determined that UvSte50 directly interacts with both UvSte7 (MAPK kinase; MEK) and UvSte11 (MAPK kinase kinase; MEKK), where the Ras-association (RA) domain of UvSte50 is indispensable for its interaction with UvSte7. UvSte50 also interacts with UvHog1, a MAP kinase of the Hog1-MAPK pathway, which is known to have important roles in hyphal growth and stress responses in U. virens. In addition, affinity capture–mass spectrometry analysis and yeast two-hybrid assay were conducted, through which we identified the interactions of UvSte50 with UvRas2, UvAc1 (adenylate cyclase), and UvCap1 (cyclase-associated protein), key components of the Ras/cAMP signaling pathway in U. virens. Together, UvSte50 functions as an adaptor protein interacting with multiple components of the MAPK and Ras/cAMP signaling pathways, thus playing critical role in plant infection by U. virens. Full article

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins. Full article