Transcription factors of the bHLH-PAS family play vital roles in animal development, physiology, and disease. Two members of the family require binding of low-molecular weight ligands for their activity: the vertebrate aryl hydrocarbon receptor (AHR) and the insect juvenile hormone receptor (JHR). In the fly Drosophila melanogaster, the paralogous proteins GCE and MET constitute the ligand-binding component of JHR complexes. Whilst GCE/MET and AHR are phylogenetically heterologous, their mode of action is similar. JHR is targeted by several synthetic agonists that serve as insecticides disrupting the insect endocrine system. AHR is an important regulator of human endocrine homeostasis, and it responds to environmental pollutants and endocrine disruptors. Whether AHR signaling is affected by compounds that can activate JHR has not been reported. To address this question, we screened a chemical library of 50,000 compounds to identify 93 novel JHR agonists in a reporter system based on Drosophila cells. Of these compounds, 26% modulated AHR signaling in an analogous reporter assay in a human cell line, indicating a significant overlap in the agonist repertoires of the two receptors. To explore the structural features of agonist-dependent activation of JHR and AHR, we compared the ligand-binding cavities and their interactions with selective and common ligands of AHR and GCE. Molecular dynamics modeling revealed ligand-specific as well as conserved side chains within the respective cavities. Significance of predicted interactions was supported through site-directed mutagenesis. The results have indicated that synthetic insect juvenile hormone agonists might interfere with AHR signaling in human cells.
- Klíčová slova
- aryl hydrocarbon receptor, endocrine disruptors, high-throughput screening, juvenile hormone receptor, ligand binding domain,
- MeSH
- buněčné linie MeSH
- Drosophila melanogaster * metabolismus genetika účinky léků MeSH
- juvenilní hormony metabolismus farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- proteiny Drosophily * metabolismus genetika chemie MeSH
- receptory aromatických uhlovodíků * agonisté metabolismus genetika MeSH
- signální transdukce účinky léků MeSH
- transkripční faktory bHLH * metabolismus genetika agonisté MeSH
- transkripční faktory metabolismus genetika MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- AHR protein, human MeSH Prohlížeč
- gce protein, Drosophila MeSH Prohlížeč
- juvenilní hormony MeSH
- ligandy MeSH
- MET protein, Drosophila MeSH Prohlížeč
- proteiny Drosophily * MeSH
- receptory aromatických uhlovodíků * MeSH
- transkripční faktory bHLH * MeSH
- transkripční faktory MeSH
Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3' untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3' UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.
- Klíčová slova
- Dosage compensation, Hrp48, RNA binding protein, RNA recognition motif, Translation regulation,
- MeSH
- 3' nepřekládaná oblast * MeSH
- DNA vazebné proteiny MeSH
- Drosophila melanogaster * metabolismus genetika MeSH
- heterogenní jaderné ribonukleoproteiny MeSH
- messenger RNA metabolismus genetika chemie MeSH
- proteiny Drosophily * metabolismus chemie genetika MeSH
- proteiny vázající RNA * metabolismus chemie genetika MeSH
- proteosyntéza MeSH
- simulace molekulární dynamiky MeSH
- transkripční faktory metabolismus chemie genetika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 3' nepřekládaná oblast * MeSH
- DNA vazebné proteiny MeSH
- heterogenní jaderné ribonukleoproteiny MeSH
- Hrb27C protein, Drosophila MeSH Prohlížeč
- messenger RNA MeSH
- msl-2 protein, Drosophila MeSH Prohlížeč
- proteiny Drosophily * MeSH
- proteiny vázající RNA * MeSH
- transkripční faktory MeSH
The minute wasp Habrobracon hebetor venom (HH venom) is a potent cocktail of toxins that paralyzes the victim's muscles and suppresses humoral and cellular immunity. This study examined the effect of HH venom on specific biochemical, physiological, and ultrastructural characteristics of the thoracic and nervous (CNS) tissues of Drosophila melanogaster under in vitro conditions. Venom treatment modulated the activities of superoxide dismutase (SOD) and catalase (CAT), endogenous Drome-AKH level, and affected the relative viability of the cells. Additionally, it reduced the expression of genes related to the immune system in the CNS, including Keap1, Relish, Nox, Eiger, Gadd45, and Domeless, as well as in the thoracic muscles, except for Nox. Besides, venom treatment led to deteriorative changes in the ultrastructure of muscle cells, particularly affecting the mitochondria. When venom and Drosophila melanogaster-adipokinetic hormone (Drome-AKH) were applied together, the effects of the venom alone were often modulated. The harmful effect of the venom on SOD activity was relatively reduced and the activity returned to a level similar to that of the control. In the CNS, the simultaneous application of venom and hormones abolished the suppression of previously reported immune-related genes (except for Gadd45), whereas in the muscles, this was only true for Eiger. Additionally, Drome-AKH restored cell structure to a level comparable to that of the control and lessened the harmful effects of HH venom on muscle mitochondria. These findings suggest a general body response of D. melanogaster to HH venom and a partial defensive role of Drome-AKH in this process.
- Klíčová slova
- Adipokinetic hormone, Habrobracon venom, Immune responsive genes, Muscle structure, Oxidative stress,
- MeSH
- centrální nervový systém účinky léků MeSH
- Drosophila melanogaster * účinky léků MeSH
- hmyzí hormony * metabolismus MeSH
- katalasa metabolismus genetika MeSH
- kyselina pyrrolidonkarboxylová * analogy a deriváty MeSH
- oligopeptidy * farmakologie toxicita MeSH
- proteiny Drosophily * metabolismus genetika MeSH
- sršňovití * účinky léků MeSH
- superoxiddismutasa metabolismus MeSH
- vosí jedy * toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adipokinetic hormone MeSH Prohlížeč
- hmyzí hormony * MeSH
- katalasa MeSH
- kyselina pyrrolidonkarboxylová * MeSH
- oligopeptidy * MeSH
- proteiny Drosophily * MeSH
- superoxiddismutasa MeSH
- vosí jedy * MeSH
In multicellular organisms, the indole melatonin synthesized by aralkylamine N-acetyltransferase (AANATI) serves as an antioxidant. To test this, sex-mixed 3-day-old mated fly adults bw1 and AANAT1 homozygous recessive loss-of-function mutant (bw AANAT1lo) of Drosophila melanogaster were fed by a standard diet or by one containing paraquat (PQ, 1,1'-dimethyl-4,4'-bipyridilium dichloride hydrate) at a final concentration of 15.5 mM. Experiment lasted 8 h and began at 11 a.m. In bw1 flies the paraquat treatment resulted in a significant (evaluated by Student's t-tests) decrease of the superoxide dismutase (SOD) activity and an increase the catalase (CAT) and glutathione S-transferase (GST) activities. Meanwhile, in these flies, total Antioxidative capacity (TAC) was significantly curbed by the paraquat presence. Importantly, these changes were not observed in the AANAT1-mutants. Thus, melatonin seems to play an important defence role against the oxidative stress elicited by paraquat.
- Klíčová slova
- arylalkylamine‐N‐acetyltransferase, drosophila, mutant, oxidative stress, paraquat, stress response,
- MeSH
- acetyltransferasy metabolismus genetika MeSH
- antioxidancia * metabolismus MeSH
- Drosophila melanogaster * účinky léků enzymologie MeSH
- glutathiontransferasa * metabolismus genetika MeSH
- katalasa metabolismus genetika MeSH
- melatonin farmakologie metabolismus MeSH
- oxidační stres * účinky léků MeSH
- paraquat * farmakologie MeSH
- proteiny Drosophily metabolismus genetika MeSH
- superoxiddismutasa metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetyltransferasy MeSH
- antioxidancia * MeSH
- glutathiontransferasa * MeSH
- katalasa MeSH
- melatonin MeSH
- paraquat * MeSH
- proteiny Drosophily MeSH
- superoxiddismutasa MeSH
Insect prophenoloxidases (PPOs) are important immunity proteins for defending against the invading pathogens and parasites. As a Type-Ⅲ copper-containing proteins, unlike Homo sapiens tyrosinases, the insect PPOs and most bacterial tyrosinases contain no signal peptides for unknown reason, however they can still be released. To this end, we fused different signal peptides to Drosophila melanogaster PPOs for in vitro and in vivo expression, respectively. We demonstrate that an artificial signal peptide can help PPO secretion in vitro. The secreted PPO appeared larger than wild-type PPO on molecular weight sizes due to glycosylation when expressed in S2 cells. Two asparagine residues for potential glycosylation in PPO1 were identified when a signal peptide was fused. After purification, the glycosylated PPO1 lost zymogen activity. When PPO1 containing a signal peptide was over-expressed in Drosophila larvae, the glycosylation and secretion of PPO1 was detected in vivo. Unlike insect PPO, human tyrosinase needs a signal peptide for protein expression and maintaining enzyme activity. An artificial signal peptide fused to bacterial tyrosinase had no influence on the protein expression and enzyme activity. These Type-Ⅲ copper-containing proteins from different organisms may evolve to perform their specific functions. Intriguingly, our study revealed that the addition of calcium inhibits PPO secretion from the transiently cultured larval hindguts in vitro, indicating that the calcium concentration may regulate PPO secretion. Taken together, insect PPOs can maintain enzyme activities without any signal peptide.
- Klíčová slova
- Enzyme activity, Glycosylation, Insect, Prophenoloxidase, Signal peptide,
- MeSH
- buněčné linie MeSH
- Drosophila melanogaster * imunologie metabolismus MeSH
- glykosylace MeSH
- hmyzí proteiny metabolismus genetika MeSH
- katecholoxidasa * metabolismus MeSH
- larva metabolismus MeSH
- lidé MeSH
- prekurzory enzymů * metabolismus MeSH
- proteinové prekurzory metabolismus MeSH
- proteiny - lokalizační signály * MeSH
- proteiny Drosophily metabolismus genetika MeSH
- tyrosinasa metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hmyzí proteiny MeSH
- katecholoxidasa * MeSH
- prekurzory enzymů * MeSH
- pro-phenoloxidase MeSH Prohlížeč
- proteinové prekurzory MeSH
- proteiny - lokalizační signály * MeSH
- proteiny Drosophily MeSH
- tyrosinasa MeSH
- vápník MeSH
Dietary restriction (DR) slows aging in many animals, while in some cases, the sensory signals from diet alone are sufficient to retard or accelerate lifespan. The digestive tract is a candidate location to sense nutrients, where neuropeptides secreted by enteroendocrine cells (EEC) produce systemic signals in response to food. Here, we measure how Drosophila neuropeptide F (NPF) is secreted into adult circulation by EEC and find that specific EEC differentially respond to dietary sugar and yeast. Female lifespan is increased when gut NPF is genetically depleted, and this manipulation is sufficient to blunt the longevity benefit conferred by DR. Depletion of NPF receptors at insulin-producing neurons of the brain also increases female lifespan, consistent with observations where loss of gut NPF decreases neuronal insulin secretion. The longevity conferred by repressing gut NPF and brain NPF receptors is reversed by treating adults with a juvenile hormone (JH) analog. JH is produced by the adult corpora allata, and inhibition of the insulin receptor at this tissue decreases JH titer and extends lifespan in both males and females, while this longevity is restored to wild type by treating adults with a JH analog. Overall, EEC of the gut modulate Drosophila aging through interorgan communication mediated by a gut-brain-corpora allata axis, and insulin produced in the brain impacts lifespan through its control of JH titer. These data suggest that we consider how human incretins and their analogs, which are used to treat obesity and diabetes, may impact aging.
- Klíčová slova
- aging, incretin, insulin, interorgan communication, juvenile hormone,
- MeSH
- dlouhověkost fyziologie MeSH
- Drosophila melanogaster metabolismus MeSH
- enteroendokrinní buňky metabolismus MeSH
- inzulin * metabolismus MeSH
- juvenilní hormony * metabolismus MeSH
- mozek metabolismus MeSH
- neurony metabolismus MeSH
- neuropeptidy * metabolismus MeSH
- osa mozek-střevo * fyziologie MeSH
- proteiny Drosophily * metabolismus genetika MeSH
- stárnutí metabolismus fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inzulin * MeSH
- juvenilní hormony * MeSH
- neuropeptide F, Drosophila MeSH Prohlížeč
- neuropeptidy * MeSH
- proteiny Drosophily * MeSH
One of the major functions of programmed cell death (apoptosis) is the removal of cells that suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes that, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS promoters at both ends, which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, 7 genes were identified, which suppressed the p53-induced apoptosis. In 4 mutants, the suppression effect resulted from single genes activated by 1 UAS promoter (Pka-R2, Rga, crol, and Spt5). In the other 3 (Orct2, Polr2M, and stg), deleting either UAS promoter eliminated the suppression effect. In qPCR experiments, we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eukaryotic genomes, there are coexpressed gene clusters. Three of the DEP insertion mutants are included, and 2 are in close vicinity of separate coexpressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.
- Klíčová slova
- Drosophila, activating insertional mutagenesis, apoptosis, p53, suppression,
- MeSH
- apoptóza * MeSH
- dominantní geny MeSH
- Drosophila melanogaster * genetika MeSH
- fenotyp MeSH
- inzerční mutageneze * metody MeSH
- nádorový supresorový protein p53 * genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny Drosophily * genetika metabolismus MeSH
- supresorové geny MeSH
- transpozibilní elementy DNA MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- nádorový supresorový protein p53 * MeSH
- p53 protein, Drosophila MeSH Prohlížeč
- proteiny Drosophily * MeSH
- transpozibilní elementy DNA MeSH
The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.
- Klíčová slova
- Sema2b, axon guidance, glycosaminoglycans, semaphorin, semaphorin bridge model,
- MeSH
- axony metabolismus MeSH
- čichové buňky metabolismus MeSH
- Drosophila melanogaster metabolismus MeSH
- glykosaminoglykany metabolismus MeSH
- navádění axonů * MeSH
- proteiny Drosophily * metabolismus genetika MeSH
- proteoglykany * metabolismus MeSH
- semaforiny * metabolismus genetika MeSH
- signální transdukce * MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykosaminoglykany MeSH
- proteiny Drosophily * MeSH
- proteoglykany * MeSH
- semaforiny * MeSH
Methylation of histone H3 at lysine 36 (H3K36me3) marks active chromatin. The mark is interpreted by epigenetic readers that assist transcription and safeguard the integrity of the chromatin fiber. The chromodomain protein MSL3 binds H3K36me3 to target X-chromosomal genes in male Drosophila for dosage compensation. The PWWP-domain protein JASPer recruits the JIL1 kinase to active chromatin on all chromosomes. Unexpectedly, depletion of K36me3 had variable, locus-specific effects on the interactions of those readers. This observation motivated a systematic and comprehensive study of K36 methylation in a defined cellular model. Contrasting prevailing models, we found that K36me1, K36me2 and K36me3 each contribute to distinct chromatin states. A gene-centric view of the changing K36 methylation landscape upon depletion of the three methyltransferases Set2, NSD and Ash1 revealed local, context-specific methylation signatures. Set2 catalyzes K36me3 predominantly at transcriptionally active euchromatin. NSD places K36me2/3 at defined loci within pericentric heterochromatin and on weakly transcribed euchromatic genes. Ash1 deposits K36me1 at regions with enhancer signatures. The genome-wide mapping of MSL3 and JASPer suggested that they bind K36me2 in addition to K36me3, which was confirmed by direct affinity measurement. This dual specificity attracts the readers to a broader range of chromosomal locations and increases the robustness of their actions.
- MeSH
- chromatin * metabolismus MeSH
- DNA vazebné proteiny metabolismus genetika MeSH
- Drosophila melanogaster genetika metabolismus MeSH
- heterochromatin metabolismus genetika MeSH
- histonlysin-N-methyltransferasa * metabolismus genetika MeSH
- histony * metabolismus MeSH
- lysin metabolismus MeSH
- methyltransferasy metabolismus genetika MeSH
- metylace MeSH
- protein-serin-threoninkinasy MeSH
- proteiny Drosophily * metabolismus genetika MeSH
- transkripční faktory metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ASH1 protein, Drosophila MeSH Prohlížeč
- chromatin * MeSH
- DNA vazebné proteiny MeSH
- heterochromatin MeSH
- histonlysin-N-methyltransferasa * MeSH
- histony * MeSH
- JIL-1 protein, Drosophila MeSH Prohlížeč
- lysin MeSH
- methyltransferasy MeSH
- protein-serin-threoninkinasy MeSH
- proteiny Drosophily * MeSH
- transkripční faktory MeSH
Genes encoding the KDM5 family of transcriptional regulators are disrupted in individuals with intellectual disability (ID). To understand the link between KDM5 and ID, we characterized five Drosophila strains harboring missense alleles analogous to those observed in patients. These alleles disrupted neuroanatomical development, cognition and other behaviors, and displayed a transcriptional signature characterized by the downregulation of many ribosomal protein genes. A similar transcriptional profile was observed in KDM5C knockout iPSC-induced human glutamatergic neurons, suggesting an evolutionarily conserved role for KDM5 proteins in regulating this class of gene. In Drosophila, reducing KDM5 changed neuronal ribosome composition, lowered the translation efficiency of mRNAs required for mitochondrial function, and altered mitochondrial metabolism. These data highlight the cellular consequences of altered KDM5-regulated transcriptional programs that could contribute to cognitive and behavioral phenotypes. Moreover, they suggest that KDM5 may be part of a broader network of proteins that influence cognition by regulating protein synthesis.
- MeSH
- aktivace transkripce MeSH
- Drosophila melanogaster genetika metabolismus MeSH
- Drosophila genetika metabolismus MeSH
- histondemethylasy metabolismus genetika MeSH
- lidé MeSH
- mentální retardace genetika metabolismus MeSH
- mitochondrie metabolismus genetika MeSH
- neurony * metabolismus MeSH
- proteiny Drosophily * genetika metabolismus MeSH
- proteosyntéza MeSH
- ribozomální proteiny * genetika metabolismus MeSH
- ribozomy metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- histondemethylasy MeSH
- KDM5C protein, human MeSH Prohlížeč
- Lid protein, Drosophila MeSH Prohlížeč
- proteiny Drosophily * MeSH
- ribozomální proteiny * MeSH
