Pheromone Signaling Pathway Research Articles

Environmental concentrations of acetaminophen and its metabolites promote the spread of antibiotic resistance genes through pheromone signaling pathway

The massive use of acetaminophen (APAP), an antipyretic and analgesic drug, especially during the COVID-19 pandemic, has caused the environmental residue and pollution of acetaminophen and its metabolites. However, the environmental ecological and health issues they may cause are still unclear. In this study, we found that environmental concentrations of APAP and its metabolites could promote the conjugative transfer of antibiotic resistance genes mediated by plasmid pCF10 through pheromone-like effect rather than the commonly mechanism found in current studies. Conjugative transfer studies showed that APAP and its metabolites increased the frequency of conjugative transfer of ARGs by up to 3.3 times, reaching 3.2 × 10-2. We explored the underling mechanisms using qPCR, western blot, and SEM images. This study demonstrates that APAP stimulates pheromone secretion, subsequently triggering conjugation and accelerating downstream gene expression. Furthermore, APAP functions as a pheromone, initially facilitating plasmid cleavage and peptidoglycan hydrolysis, followed by the promotion of adhesion protein PrgB expression and T4SS scaffold construction, ultimately enhancing DNA strand transfer via T4CP. This study provides a theoretical basis for understanding the environmental hazards of APAP and the environmental sustainability.

Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease.

We previously demonstrated that we could hijack the fungal pheromone signaling pathway to provide a living yeast biosensor where peptide biomarkers were recognized by G-protein-coupled receptors and engineered to transcribe a readout. Here, we demonstrated that the protease could be reintroduced to the biosensor to provide a simple mechanism for distinguishing single-amino-acid changes in peptide ligands that, otherwise, would likely be difficult to detect using binding-based assays. We characterized the dose-response curves for five fungal pheromone G-protein-coupled receptors, peptides, and proteases-Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Schizosaccharomyces octosporus, and Schizosaccharomyces japonicus. Alanine scanning was carried out for the most selective of these-S. cerevisiae and C. albicans-with and without the protease. Two peptide variants were discovered, which showed diminished cleavage by the protease (CaPep2A and CaPep2A13A). Those peptides were then distinguished by utilizing the biosensor strains with and without the protease, which selectively cleaved and altered the apparent concentration of peptide required for half-maximal activation for 2 peptides-CaPep and CaPep13A, respectively-by more than one order of magnitude. These results support the hypothesis that the living yeast biosensor with a sequence-specific protease can translate single-amino-acid changes into more than one order of magnitude apparent shift in the concentration of peptide required for half-maximal activation. With further engineering by computational modeling and directed evolution, the biosensor could likely distinguish a wide variety of peptide sequences beyond the alanine scanning carried out here. In the future, we envision incorporating proteases into our living yeast biosensor for use as a point of care diagnostic, a scalable communication language, and other applications.

Sex Pheromone Receptors of Lepidopteran Insects

The sex pheromone receptors (SPRs) of Lepidopteran insects play important roles in chemical communication. In the sex pheromone detection processes, sex pheromone molecule (SPM), SPR, co-receptor (Orco), pheromone binding protein (PBP), sensory neuron membrane protein (SNMP), and pheromone degradation enzyme (PDE) play individual and cooperative roles. Commonly known as butterfly and moth, the Lepidopteran insects are widely distributed throughout the world, most of which are pests. Comprehensive knowledge of the SPRs of Lepidopteran insects would help the development of sex lure technology and the sex communication pathway research. In this review, we summarized SPR/Orco information from 10 families of Lepidopteran insects from corresponding studies. According to the research progress in the literature, we speculated the evolution of SPRs/Orcos and phylogenetically analyzed the Lepidopteran SPRs and Orcos with the neighbor-joining tree and further concluded the relationship between the cluster of SPRs and their ligands; we analyzed the predicted structural features of SPRs and gave our prediction results of SPRs and Orcos with Consensus Constrained TOPology Prediction (CCTOP) and SwissModel; we summarized the functional characterization of Lepidopteran SPRs and SPR-ligand interaction and then described the progress in the sex pheromone signaling pathways and metabotropic ion channel. Further studies are needed to work out the cryo-electron microscopy (EM) structure of SPR and the SPR-ligand docking pattern in a biophysical perspective, which will directly facilitate the understanding of sex pheromone signal transduction pathways and provide guidance in the sex lure technology in field pest control.

Kel1 is a phosphorylation-regulated noise suppressor of the pheromone signaling pathway.

Mechanisms have evolved that allow cells to detect signals and generate an appropriate response. The accuracy of these responses relies on the ability of cells to discriminate between signal and noise. How cells filter noise in signaling pathways is not well understood. Here, we analyze noise suppression in the yeast pheromone signaling pathway and show that the poorly characterized protein Kel1 serves as a major noise suppressor and prevents cell death. At the molecular level, Kel1 prevents spontaneous activation of the pheromone response by inhibiting membrane recruitment of Ste5 and Far1. Only a hypophosphorylated form of Kel1 suppresses signaling, reduces noise, and prevents pheromone-associated cell death, and our data indicate that the MAPK Fus3 contributes to Kel1 phosphorylation. Taken together, Kel1 serves as a phospho-regulated suppressor of the pheromone pathway to reduce noise, inhibit spontaneous activation of the pathway, regulate mating efficiency, and prevent pheromone-associated cell death.

How to Select a Mate: Kel1 is a Phosphorylation-Regulated Suppressor of the Pheromone Signaling Pathway

Mechanisms have evolved that allow cells to detect signals and generate an appropriate response. The accuracy of these responses relies on the ability of cells to discriminate between signal and noise. How cells filter noise in signaling pathways is not well understood. We have analyzed noise suppression in the yeast pheromone signaling pathway. By combining synthetic genetic array screening, mass spectrometry and single-cell time-resolved microscopy, we discovered that the poorly characterized protein Kel1 serves as a major noise suppressor of the pathway. At the molecular level, Kel1 suppresses spontaneous activation of the pheromone response by inhibiting membrane recruitment of Ste5 and Far1. Kel1 is regulated by phosphorylation, and only the hypophosphorylated form of Kel1 suppresses signaling, reduces noise and prevents pheromone-associated cell death. Our data indicate that in response to pheromone the MAPKs Fus3 and Kss1 phosphorylate Kel1 to relieve inhibition of the pheromone pathway. Taken together, Kel1 serves as a phospho-regulated suppressor of the pheromone pathway to reduce noise, inhibit spontaneous activation of the pathway, regulate mating efficiency and to prevent pheromone-associated cell death.

Prey sensing and response in a nematode-trapping fungus is governed by the MAPK pheromone response pathway

Detection of surrounding organisms in the environment plays a major role in the evolution of interspecies interactions, such as predator-prey relationships. Nematode-trapping fungi (NTF) are predators that develop specialized trap structures to capture, kill, and consume nematodes when food sources are limited. Despite the identification of various factors that induce trap morphogenesis, the mechanisms underlying the differentiation process have remained largely unclear. Here, we demonstrate that the highly conserved pheromone-response MAPK pathway is essential for sensing ascarosides, a conserved molecular signature of nemaotdes, and is required for the predatory lifestyle switch in the NTF Arthrobotrys oligospora. Gene deletion of STE7 (MAPKK) and FUS3 (MAPK) abolished nematode-induced trap morphogenesis and conidiation and impaired the growth of hyphae. The conserved transcription factor Ste12 acting downstream of the pheromone-response pathway also plays a vital role in the predation of A. oligospora. Transcriptional profiling of a ste12 mutant identified a small subset of genes with diverse functions that are Ste12 dependent and could trigger trap differentiation. Our work has revealed that A. oligospora perceives and interprets the ascarosides produced by nematodes via the conserved pheromone signaling pathway in fungi, providing molecular insights into the mechanisms of communication between a fungal predator and its nematode prey.

Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes.

Sexual and asexual development in filamentous ascomycetes is controlled by components of conserved signaling pathways. Here, we investigated the development of mutant strains lacking genes for kinases MAK2, MEK2, and MIK2, as well as the scaffold protein HAM5 of the pheromone response (PR) pathway. All had a defect in fruiting body development and hyphal fusion. Another phenotype was a defect in melanin-dependent ascospore germination. However, this deficiency was observed only in kinase deletion mutants, but not in strains lacking HAM5. Notably, the same developmental phenotypes were previously described for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) mutants, but the germination defect was only seen in NOX2 mutants. These data suggest a molecular link between the pheromone signaling pathway and both NOX complexes. Using data from yeast two-hybrid (Y2H) analysis, we found that the scaffolding protein HAM5 interacts with NOR1, the regulator of NOX1 and NOX2 complexes. This interaction was further confirmed using differently fluorescent-labeled proteins to demonstrate that NOR1 and HAM5 co-localize at cytoplasmic spots and tips of mature hyphae. This observation was supported by phenotypic characterization of single and double mutants. The oxidative stress response and the initiation of fruiting bodies were similar in Δham5Δnor1 and Δham5, but distinctly reduced in Δnor1, indicating that the double deletion leads to a partial suppression of the Δnor1 phenotype. We conclude that the PR and NOX1 complexes are connected by direct interaction between HAM5 and NOR1. In contrast, PR kinases are linked to the NOX2 complex without participation of HAM5.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein βγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gβγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

Pheromones and Nutritional Signals Regulate the Developmental Reliance on let-7 Family MicroRNAs in C. elegans

Adverse environmental conditions can affect rates ofanimal developmental progression and lead to temporary developmental quiescence (diapause), exemplified by the dauer larva stage of the nematode Caenorhabditis elegans (C.elegans). Remarkably, patterns of cell division and temporal cell-fate progression in C.elegans larvae are not affected by changes in developmental trajectory. However, the underlying physiological and gene regulatory mechanisms that ensure robust developmental patterning despite substantial plasticity in developmental progression are largely unknown. Here, we report thatdiapause-inducing pheromones correct heterochronic developmental cell lineage defects caused by insufficient expression of let-7 family microRNAs in C.elegans. Moreover, two conserved endocrine signaling pathways, DAF-7/TGF-β and DAF-2/Insulin, that confer on the larva diapause and non-diapause alternative developmental trajectories interact with the nuclear hormone receptor, DAF-12, to initiate and regulate a rewiring of the genetic circuitry controlling temporal cell fates. This rewiring includes engagement of certain heterochronic genes, lin-46, lin-4, and nhl-2, that are previously associated with an altered genetic program in post-diapause animals, in combination with a novel ligand-independent DAF-12 activity, to downregulate the critical let-7 family target Hunchback-like-1 (HBL-1). Our results show how pheromone or endocrine signaling pathways can coordinately regulate both developmental progression and cell-fate transitions in C.elegans larvae under stress so that the developmental schedule of cell fates remains unaffected by changes in developmental trajectory.

Fungal Sex: The Ascomycota.

This article provides an overview of sexual reproduction in the ascomycetes, a phylum of fungi that is named after the specialized sacs or "asci" that hold the sexual spores. They have therefore also been referred to as the Sac Fungi due to these characteristic structures that typically contain four to eight ascospores. Ascomycetes are morphologically diverse and include single-celled yeasts, filamentous fungi, and more complex cup fungi. The sexual cycles of many species, including those of the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the filamentous saprobes Neurospora crassa, Aspergillus nidulans, and Podospora anserina, have been examined in depth. In addition, sexual or parasexual cycles have been uncovered in important human pathogens such as Candida albicans and Aspergillus fumigatus, as well as in plant pathogens such as Fusarium graminearum and Cochliobolus heterostrophus. We summarize what is known about sexual fecundity in ascomycetes, examine how structural changes at the mating-type locus dictate sexual behavior, and discuss recent studies that reveal that pheromone signaling pathways can be repurposed to serve cellular roles unrelated to sex.

Evolved differences in larval social behavior mediated by novel pheromones

Pheromones, chemical signals that convey social information, mediate many insect social behaviors, including navigation and aggregation. Several studies have suggested that behavior during the immature larval stages of Drosophila development is influenced by pheromones, but none of these compounds or the pheromone-receptor neurons that sense them have been identified. Here we report a larval pheromone-signaling pathway. We found that larvae produce two novel long-chain fatty acids that are attractive to other larvae. We identified a single larval chemosensory neuron that detects these molecules. Two members of the pickpocket family of DEG/ENaC channel subunits (ppk23 and ppk29) are required to respond to these pheromones. This pheromone system is evolving quickly, since the larval exudates of D. simulans, the sister species of D. melanogaster, are not attractive to other larvae. Our results define a new pheromone signaling system in Drosophila that shares characteristics with pheromone systems in a wide diversity of insects.

The Link between Morphotype Transition and Virulence in Cryptococcus neoformans

Cryptococcus neoformans is a ubiquitous human fungal pathogen. This pathogen can undergo morphotype transition between the yeast and the filamentous form and such morphological transition has been implicated in virulence for decades. Morphotype transition is typically observed during mating, which is governed by pheromone signaling. Paradoxically, components specific to the pheromone signaling pathways play no or minimal direct roles in virulence. Thus, the link between morphotype transition and virulence and the underlying molecular mechanism remain elusive. Here, we demonstrate that filamentation can occur independent of pheromone signaling and mating, and both mating-dependent and mating-independent morphotype transition require the transcription factor Znf2. High expression of Znf2 is necessary and sufficient to initiate and maintain sex-independent filamentous growth under host-relevant conditions in vitro and during infection. Importantly, ZNF2 overexpression abolishes fungal virulence in murine models of cryptococcosis. Thus, Znf2 bridges the sex-independent morphotype transition and fungal pathogenicity. The impacts of Znf2 on morphological switch and pathogenicity are at least partly mediated through its effects on cell adhesion property. Cfl1, a Znf2 downstream factor, regulates morphogenesis, cell adhesion, biofilm formation, and virulence. Cfl1 is the first adhesin discovered in the phylum Basidiomycota of the Kingdom Fungi. Together with previous findings in other eukaryotic pathogens, our findings support a convergent evolution of plasticity in morphology and its impact on cell adhesion as a critical adaptive trait for pathogenesis.

Optimal ratio of scaffold complex to free Fus3 to maximise the accumulation of phosphorylated Fus3 in yeast pheromone signalling pathway

In this study, the author considers the design rule of the intracellular signalling pathway. In yeast pheromone signalling pathway, scaffold Ste5 tethers the components of signalling pathway, Ste11, Ste7 and Fus3. Even though scaffold complex is independently produced before stimuli, excessively expressed Fus3 as compared with scaffold exists in cytoplasm as free kinase. How the ratio of scaffold complex to the free Fus3 is determined is not clear yet. First, the contribution of free Fus3 to signal transduction is theoretically shown by using a simplified model of pheromone signalling pathway. Next, the optimum expression levels of Ste5, Ste11, Ste7 and Fus3 are systematically explored by using the detailed model and genetic algorithm under the constraint that the total expression level of these four genes is limited. Excessive expression of Fus3 is advantageous for the efficient signalling without stall of the signal transduction. The result suggests that the component of signalling pathway is optimally expressed to maximise the accumulation of phosphorylated Fus3 at a fixed time point under the constraint that the total gene expression is limited. The proposed model provides further insight into the signalling network from the point of view of not only its function but also its optimality.

Computational Mass Spectrometry–Based Proteomics

Proteomics is defined as the system-widecharacterization of all the proteins in anorganism in terms of their sequence,localization, abundance, post-translationalmodifications, and biomolecular interac-tions. Modern proteomic investigationsare increasingly quantitative and compre-hensive [1]. Examples include the relativequantification of over 4,000 proteins inhaploid and diploid yeast, which identifiedthe pheromone signaling pathway asenriched in differential abundance [2];determination of site- and time-specificdynamics of more than 6,000 phosphory-lation sites of HeLa cells stimulated withepidermal growth factor [3]; and charac-terization of 232 multiprotein complexesin Saccharomyces cerevisiae, which proposednew cellular roles for 344 proteins [4].Such investigations are now successfullyutilized in functional biology [5,6], geno-mics [7,8], and biomedical research [9].Challenges of proteomic studies stemfrom the complexity of the proteome andto its broad dynamic range. For example,the human genome contains around20,000 protein coding genes. Their trans-lation, combined with splicing or proteol-ysis, yields an estimated 50,000–500,000proteins, and over 10 million differentprotein forms can be derived by somaticDNA rearrangements and post-transla-tional modifications [10]. The abundanceof protein species in human plasma spansmore than 10 orders of magnitude [11].Unlike oligonucleotides, proteins cannotbe amplified, and therefore the objectivesof proteomics are achieved by sensitiveand scalable technologies identifying andquantifying proteins [12]. The overallmass spectrometry–based proteomic work-flow is summarized in Figure 1.

Discovering pathways by orienting edges in protein interaction networks

Modern experimental technology enables the identification of the sensory proteins that interact with the cells’ environment or various pathogens. Expression and knockdown studies can determine the downstream effects of these interactions. However, when attempting to reconstruct the signaling networks and pathways between these sources and targets, one faces a substantial challenge. Although pathways are directed, high-throughput protein interaction data are undirected. In order to utilize the available data, we need methods that can orient protein interaction edges and discover high-confidence pathways that explain the observed experimental outcomes. We formalize the orientation problem in weighted protein interaction graphs as an optimization problem and present three approximation algorithms based on either weighted Boolean satisfiability solvers or probabilistic assignments. We use these algorithms to identify pathways in yeast. Our approach recovers twice as many known signaling cascades as a recent unoriented signaling pathway prediction technique and over 13 times as many as an existing network orientation algorithm. The discovered paths match several known signaling pathways and suggest new mechanisms that are not currently present in signaling databases. For some pathways, including the pheromone signaling pathway and the high-osmolarity glycerol pathway, our method suggests interesting and novel components that extend current annotations.

The Ustilago maydis Clp1 protein orchestrates pheromone and b-dependent signaling pathways to coordinate the cell cycle and pathogenic development.

Regulation of the cell cycle and morphogenetic switching during pathogenic and sexual development in Ustilago maydis is orchestrated by a concerted action of the a and b mating-type loci. Activation of either mating-type locus triggers the G2 cell cycle arrest that is a prerequisite for the formation of the infectious dikaryon; this cell cycle arrest is released only after penetration of the host plant. Here, we show that bW, one of the two homeodomain transcription factors encoded by the b mating-type locus, and the zinc-finger transcription factor Rbf1, a master regulator for pathogenic development, interact with Clp1 (clampless 1), a protein required for the distribution of nuclei during cell division of the dikaryon. In addition, we identify Cib1, a previously undiscovered bZIP transcription factor required for pathogenic development, as a Clp1-interacting protein. Clp1 interaction with bW blocks b-dependent functions, such as the b-dependent G2 cell cycle arrest and dimorphic switching. The interaction of Clp1 with Rbf1 results in the repression of the a-dependent pheromone pathway, conjugation tube formation, and the a-induced G2 cell cycle arrest. The concerted interaction of Clp1 with Rbf1 and bW coordinates a- and b-dependent cell cycle control and ensures cell cycle release and progression at the onset of biotrophic development.

Importance of asparagine residues at positions 13 and 26 on the amino-terminal domain of human somatostatin receptor subtype-5 in signalling

N-linked oligosaccharides or asparagine residues are often involved in G protein-coupled receptor functions. Focusing on Asn13 and Asn26 positioned on N-linked glycosylation motifs in the amino-terminal domain of human somatostatin receptor subtype-5 (hSSTR5), we performed site-directed mutagenesis and evaluated the mutants by using yeast cells as the host strain. This is because analysing the complicated signalling in mammalian cell lines is simplified by the utilization of the monopolistic pheromone signalling pathway in yeast. Western blot analysis and confocal laser scanning microscope observation showed that Asn13 and/or Asn26 mutations had no effects on cell-surface expression of hSSTR5 in yeast. By using an engineered yeast strain of Saccharomyces cerevisiae, which induces the expression of the green fluorescent protein (GFP) reporter gene in response to the agonist-specific signal transduction, it was demonstrated that a single mutation of two asparagine residues attenuated the somatostatin-specific signalling levels, and the double mutant significantly lost the signalling ability. These results clearly show the importance of these asparagine residues in the agonist-specific signalling of hSSTR5, although it was not enough to identify the consequence of oligosaccharides.

Cellular and transcriptional responses of yeast to the cleavage of cytosolic tRNAs induced by colicin D

Colicin D is a plasmid-encoded antibacterial protein that specifically cleaves the anticodon loops of four Escherichia coli tRNA(Arg) species. Here, we report that the catalytic domain of colicin D, which is expressed in Saccharomyces cerevisiae, impairs cell growth by cleaving specific tRNAs. DNA microarray analysis revealed that mating-related genes were upregulated, while genes involved in a range of metabolic processes were downregulated, thereby impairing cell growth. The pheromone-signalling pathway was activated only in alpha cells by tRNA cleavage, which was not observed in 'a' cells or diploid cells. On the basis of these results and on the recent identification of two killer toxins that cleave specific tRNAs, the relationship between tRNA depletion and the resultant cellular response is discussed.

Modeling of G-protein-coupled Receptor Signaling Pathways

Modeling of G-protein-coupled Receptor Signaling Pathways

Oscillatory Phosphorylation of Yeast Fus3 MAP Kinase Controls Periodic Gene Expression and Morphogenesis

Signal-transduction networks can display complex dynamic behavior such as oscillations in the activity of key components [1-6], but it is often unclear whether such dynamic complexity is actually important for the network's regulatory functions [7, 8]. Here, we found that the mitogen-activated protein kinase (MAPK) Fus3, a key regulator of the yeast mating-pheromone response, undergoes sustained oscillations in its phosphorylation and activation state during continuous pheromone exposure. These MAPK activity oscillations led to corresponding oscillations in mating-gene expression. Oscillations in MAPK activity and gene expression required the negative regulator of G protein signaling Sst2 and partially required the MAPK phosphatase Msg5. Peaks in Fus3 activation correlated with periodic rounds of cell morphogenesis, with each peak preceding the formation of an additional mating projection. Preventing projection formation did not eliminate MAPK oscillation, but preventing MAPK oscillation blocked the formation of additional projections. A mathematical model was developed that reproduced several features of the observed oscillatory dynamics. These observations demonstrate a role for MAPK activity oscillation in driving a periodic downstream response and explain how the pheromone signaling pathway, previously thought to desensitize after 1-3 hr, controls morphology changes that continue for a much longer time.