Sleep, a reversible quiescent state found in both invertebrate and vertebrate animals, disconnects animals from their environment and is highly regulated for coordination with wakeful activities, such as reproduction. The fruit fly, Drosophila melanogaster, has proven to be a valuable model for studying the regulation of sleep by circadian clock and homeostatic mechanisms. Here, we demonstrate that the sex peptide receptor (SPR) of Drosophila, known for its role in female reproduction, is also important in stabilizing sleep in both males and females. Mutants lacking either the SPR or its central ligand, myoinhibitory peptide (MIP), fall asleep normally, but have difficulty in maintaining a sleep-like state. Our analyses have mapped the SPR sleep function to pigment dispersing factor (pdf) neurons, an arousal center in the insect brain. MIP downregulates intracellular cAMP levels in pdf neurons through the SPR. MIP is released centrally before and during night-time sleep, when the sleep drive is elevated. Sleep deprivation during the night facilitates MIP secretion from specific brain neurons innervating pdf neurons. Moreover, flies lacking either SPR or MIP cannot recover sleep after the night-time sleep deprivation. These results delineate a central neuropeptide circuit that stabilizes the sleep state by feeding a slow-acting inhibitory input into the arousal system and plays an important role in sleep homeostasis.
SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference- mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.
Histamine and its two receptors, histamine-gated chloride channel subunit 1 (HisCl1) and ora transientless (Ort), are known to control photoreception and temperature sensing in Drosophila. However, histamine signaling in the context of neural circuitry for sleep-wake behaviors has not yet been examined in detail. Here, we obtained mutant flies with compromised or enhanced histamine signaling and tested their baseline sleep. Hypomorphic mutations in histidine decarboxylase (HDC), an enzyme catalyzing the conversion from histidine to histamine, caused an increase in sleep duration. Interestingly, hisCl1 mutants but not ort mutants showed long-sleep phenotypes similar to those in hdc mutants. Increased sleep duration in hisCl1 mutants was rescued by overexpressing hisCl1 in circadian pacemaker neurons expressing a neuropeptide pigment dispersing factor (PDF). Consistently, RNA interference (RNAi)-mediated depletion of hisCl1 in PDF neurons was sufficient to mimic hisCl1 mutant phenotypes, suggesting that PDF neurons are crucial for sleep regulation by the histamine-HisCl1 signaling. Finally, either hisCl1 mutation or genetic ablation of PDF neurons dampened wake-promoting effects of elevated histamine signaling via direct histamine administration. Taken together, these data clearly demonstrate that the histamine-HisCl1 receptor axis can activate and maintain the wake state in Drosophila and that wake-activating signals may travel via the PDF neurons.
Vibrio vulnificus is an opportunistic human pathogen that causes severe infections in susceptible individuals. While the components of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) have been shown to regulate numerous targets, little such information is available for the V. vulnificus PTS. Here we show that enzyme IIA(Glc) of the PTS regulates the peptidase activity of a mammalian insulysin homolog in V. vulnificus. While interaction of IIA(Glc) with the insulysin homolog is independent of the phosphorylation state of IIA(Glc), only unphosphorylated IIA(Glc) activates the insulysin homolog. Taken together, our results suggest that the V. vulnificus insulysin-IIA(Glc) complex plays a role in survival in the host by sensing glucose.
During propagation, yeast prions show a strict sequence preference that confers the specificity of prion assembly. Although propagations of [PSI(+)] and [RNQ(+)] are independent of each other, the appearance of [PSI(+)] is facilitated by the presence of [RNQ(+)]. To explain the [RNQ(+)] effect on the appearance of [PSI(+)], the cross-seeding model was suggested, in which Rnq1 aggregates act as imperfect templates for Sup35 aggregation. If cross-seeding events take place in the cytoplasm of yeast cells, the collision frequency between Rnq1 aggregates and Sup35 will affect the appearance of [PSI(+)]. In this study, to address whether cross-seeding occurs in vivo, a new [PSI(+)] induction method was developed that exploits a protein fusion between the prion domain of Sup35 (NM) and Rnq1. This fusion protein successfully joins preexisting Rnq1 aggregates, which should result in the localization of NM around the Rnq1 aggregates and hence in an increased collision frequency between NM and Rnq1 aggregates. The appearance of [PSI(+)] could be induced very efficiently, even with a low expression level of the fusion protein. This study supports the occurrence of in vivo cross-seeding between Sup35 and Rnq1 and provides a new tool that can be used to dissect the mechanism of the de novo appearance of prions.
Components of the bacterial phosphoenolpyruvate (PEP) : carbohydrate phosphortransferase system (PTS) have multiple regulatory roles in addition to PEP-dependent transport/phosphorylation of numerous carbohydrates. We have recently shown that, in an opportunistic human pathogen, Vibrio vulnificus, enzyme IIA(Glc) (EIIA(Glc)) interacts with a peptidase that has high sequence similarity to mammalian insulin-degrading enzymes, called Vibrio insulin-degrading enzyme (vIDE). Although the vIDE-EIIA(Glc) interaction is independent of the phosphorylation state of EIIA(Glc), vIDE shows no peptidase activity unless complexed with the unphosphorylated form of EIIA(Glc). A deletion mutant of ideV, the gene encoding vIDE, shows remarkably lower degrees of survival and virulence than the wild-type strain in mice, implying that vIDE is a virulence factor. In this study, we investigated regulation of ideV expression at the transcriptional level. Primer extension analysis identified two different transcriptional start sites of ideV: P(L) for the longer transcript and P(S) for the shorter transcript. We performed ligand fishing experiments by using the promoter region of ideV and found that the cAMP receptor protein (CRP) specifically binds to the promoter. DNase I footprinting experiments revealed that CRP binds to a region between the two promoters. In vitro transcription assays showed that CRP activates ideV P(S) transcription in the presence of cAMP whose concentration is regulated by EIIA(Glc). These results suggest that EIIA(Glc) regulates the expression level of vIDE as well as its activity.
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