Serine metabolism in the brain regulates starvation-induced sleep suppression in Drosophila melanogaster.

Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. Here, we identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. We propose that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs.

A homeostatic sleep-stabilizing pathway in Drosophila composed of the sex peptide receptor and its ligand, the myoinhibitory peptide.

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.

The novel gene twenty-four defines a critical translational step in the Drosophila clock.

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms. While attention has been focused on transcriptional and post-translational mechanisms, other post-transcriptional modes have been less clearly delineated. Here we report mutants of a novel Drosophila gene twenty-four (tyf) that show weak behavioural rhythms. Weak rhythms are accompanied by marked reductions in the levels of the clock protein Period (PER) as well as more modest effects on Timeless (TIM). Nonetheless, PER induction in pacemaker neurons can rescue tyf mutant rhythms. TYF associates with a 5'-cap-binding complex, poly(A)-binding protein (PABP), as well as per and tim transcripts. Furthermore, TYF activates reporter expression when tethered to reporter messenger RNA even in vitro. Taken together, these data indicate that TYF potently activates PER translation in pacemaker neurons to sustain robust rhythms, revealing a new and important role for translational control in the Drosophila circadian clock.

vIRF3 encoded by Kaposi's sarcoma-associated herpesvirus inhibits T-cell factor-dependent transcription via a CREB-binding protein-interaction motif.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Like other herpesviruses, KSHV has two distinct life cycles: latent and lytic. Among KSHV latent genes, viral interferon regulatory factor 3 (vIRF3), which shares homology with cellular IRFs, is a multifunctional protein. To identify unknown functions of vIRF3, we performed luciferase-reporter assays in the presence of vIRF3. These analyses revealed that overexpression of vIRF3 inhibited T-cell factor (TCF)-dependent transcriptional activity. This TCF-dependent transcription was associated with the Wnt signaling pathway, which normally regulates embryonic development, but contributes to oncogenesis under dysregulated conditions. Using a mutagenesis analysis, we identified a CREB-binding protein-interaction motif (LXXLL) in vIRF3 as an important region for its inhibitory activity. Collectively, our findings provide insight into the dysregulation of host signaling pathways in KSHV-infected cells.

Kibra and aPKC regulate starvation-induced autophagy in Drosophila.

Autophagy is a bulk degradation system that functions in response to cellular stresses such as metabolic stress, endoplasmic reticulum stress, oxidative stress, and developmental processes. During autophagy, cytoplasmic components are captured in double-membrane vesicles called autophagosomes. The autophagosome fuses with the lysosome, producing a vacuole known as an autolysosome. The cellular components are degraded by lysosomal proteases and recycled. Autophagy is important for maintaining cellular homeostasis, and the process is evolutionarily conserved. Kibra is an upstream regulator of the hippo signaling pathway, which controls organ size by affecting cell growth, proliferation, and apoptosis. Kibra is mainly localized in the apical membrane domain of epithelial cells and acts as a scaffold protein. We found that Kibra is required for autophagy to function properly. The absence of Kibra caused defects in the formation of autophagic vesicles and autophagic degradation. We also found that the well-known cell polarity protein aPKC interacts with Kibra, and its activity affects autophagy upstream of Kibra. Constitutively active aPKC decreased autophagic vesicle formation and autophagic degradation. We confirmed the interaction between aPKC and Kibra in S2 cells and Drosophila larva. Taken together, our data suggest that Kibra and aPKC are essential for regulating starvation-induced autophagy.

Dopamine signalling in mushroom bodies regulates temperature-preference behaviour in Drosophila.

The ability to respond to environmental temperature variation is essential for survival in animals. Flies show robust temperature-preference behaviour (TPB) to find optimal temperatures. Recently, we have shown that Drosophila mushroom body (MB) functions as a center controlling TPB. However, neuromodulators that control the TPB in MB remain unknown. To identify the functions of dopamine in TPB, we have conducted various genetic studies in Drosophila. Inhibition of dopamine biosynthesis by genetic mutations or treatment with chemical inhibitors caused flies to prefer temperatures colder than normal. We also found that dopaminergic neurons are involved in TPB regulation, as the targeted inactivation of dopaminergic neurons by expression of a potassium channel (Kir2.1) induced flies with the loss of cold avoidance. Consistently, the mutant flies for dopamine receptor gene (DopR) also showed a cold temperature preference, which was rescued by MB-specific expression of DopR. Based on these results, we concluded that dopamine in MB is a key component in the homeostatic temperature control of Drosophila. The current findings will provide important bases to understand the logic of thermosensation and temperature preference decision in Drosophila.

Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila.

Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription-factor heterodimer, CLOCK/CYCLE (CLK/CYC), transcriptionally activates the clock components period (per), timeless (tim), Par domain protein 1 (Pdp1), and vrille (vri), which feed back and regulate distinct features of CLK/CYC function. Microarray studies have identified numerous rhythmically expressed transcripts, some of which are potential direct CLK targets. Here we demonstrate a circadian function for one such target, a bHLH-Orange repressor, CG17100/CLOCKWORK ORANGE (CWO). cwo is rhythmically expressed, and levels are reduced in Clk mutants, suggesting that cwo is CLK activated in vivo. cwo mutants display reduced-amplitude molecular and behavioral rhythms with lengthened periods. Molecular analysis suggests that CWO acts, in part, by repressing CLK target genes. We propose that CWO acts as a transcriptional and behavioral rhythm amplifier.

DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication.

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

Functional role of CREB-binding protein in the circadian clock system of Drosophila melanogaster.

Rhythmic histone acetylation underlies the oscillating expression of clock genes in the mammalian circadian clock system. Cellular factors that contain histone acetyltransferase and histone deacetylase activity have been implicated in these processes by direct interactions with clock genes, but their functional relevance remains to be assessed by use of appropriate animal models. Here, using transgenic fly models, we show that CREB-binding protein (CBP) participates in the transcriptional regulation of the Drosophila CLOCK/CYCLE (dCLK/CYC) heterodimer. CBP knockdown in pigment dispersing factor-expressing cells lengthens the period of adult locomotor rhythm with the prolonged expression of period and timeless genes, while CBP overexpression in timeless-expressing cells causes arrhythmic circadian behaviors with the impaired expression of these dCLK/CYC-induced clock genes. In contrast to the mammalian circadian clock system, CBP overexpression attenuates the transcriptional activity of the dCLK/CYC heterodimer in cultured cells, possibly by targeting the PER-ARNT-SIM domain of dCLK. Our data suggest that the Drosophila circadian clock system has evolved a distinct mechanism to tightly regulate the robust transcriptional potency of the dCLK/CYC heterodimer.

march5 Governs the Convergence and Extension Movement for Organization of the Telencephalon and Diencephalon in Zebrafish Embryos.

MARCH5 is a RING finger E3 ligase involved in mitochondrial integrity, cellular protein homeostasis, and the regulation of mitochondrial fusion and fission. To determine the function of MARCH5 during development, we assessed transcript expression in zebrafish embryos. We found that march5 transcripts were of maternal origin and evenly distributed at the 1-cell stage, except for the mid-blastula transition, with expression predominantly in the developing central nervous system at later stages of embryogenesis. Overexpression of march5 impaired convergent extension movement during gastrulation, resulting in reduced patterning along the dorsoventral axis and alterations in the ventral cell types. Overexpression and knockdown of march5 disrupted the organization of the developing telencephalon and diencephalon. Lastly, we found that the transcription of march5 was tightly regulated by the transcriptional regulators CHOP, C/EBPα, Staf, Znf143a, and Znf76. These results demonstrate the essential role of March5 in the development of zebrafish embryos.

The DOUBLETIME protein kinase regulates phosphorylation of the Drosophila PDP1epsilon.

Reversible phosphorylation of clock proteins plays an important role in circadian timekeeping as it is a key post-translational mechanism that regulates the activity, stability and subcellular localization of core clock proteins. The kinase DOUBLETIME (DBT), a Drosophila ortholog of mammalian casein kinase Iepsilon, regulates circadian phosphorylation of two essential clock proteins, PERIOD and dCLOCK. We present evidence that Par Domain Protein 1epsilon (PDP1epsilon), a transcription factor and mediator of clock output in Drosophila, is phosphorylated in vivo and in cultured cells by DBT activity. We also demonstrate that DBT interacts with PDP1epsilon and promotes its degradation by the ubiquitin-proteasome pathway in cultured cells. In addition, PDP1epsilon nuclear localization is decreased by dbt RNA interference in S2 cell system. These results suggest that DBT regulates phosphorylation, stability and localization of PDP1epsilon, and that it has multiple targets in the Drosophila circadian system.

Intracellular small interfering RNA delivery using genetically engineered double-stranded RNA binding protein domain.

BACKGROUND: A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double-stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery. METHODS: A double-stranded RNA binding domain (dsRBD) of human double-stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines. RESULTS: The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down-regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation. CONCLUSIONS: The results suggest that dsRBD-based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity.

Peli1b governs the brain patterning via ERK signaling pathways in zebrafish embryos.

Pellino proteins are associated with immune and stress responses through their effects on NF-κB signaling and B-cell development, and through their role as a scaffold in TLR/IL-1R signaling pathways. However, their function during embryonic development is unclear. Here, we report the developmental expression patterns and functions of peli1b, which encodes a zebrafish ortholog of human Pellino1. Maternal peli1b transcripts were present in zebrafish embryos at the 1-cell stage and zygotic transcripts appeared in the shield area at 6 hours post fertilization (hpf), particularly in the neural plate of the dorsal region. peli1b transcripts were concentrated in the somites, lens, myogenic cells, lateral plate mesoderm, and presomitic mesoderm at 12 hpf, but expression shifted to the telencephalon, diencephalon, hindbrain, and rhombomeres (r1-7) at 24 hpf. Distribution of peli1b transcripts was further restricted to the telencephalon, diencephalon, hindbrain, eyes, and pectoral fins at 48 hpf. Knock-down of peli1b with a peli1b antisense morpholino resulted in significant developmental defects and a reduction in size of the telencephalon, diencephalon, rhombomeres (r1-7), and spinal cord at 24 hpf. When peli1b-knock-down embryos were analyzed for zic3, a marker associated with the central nervous system, we found lower levels of zic3 transcripts in the shield area at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, and hindbrain at 14 hpf. Finally, the ERK3/4 inhibitor SB203580 also induced a significant reduction in the level of zic3 transcripts in the neural plate at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, hindbrain, segmental plate, dorsal spinal cord, and dorsal posterior neural plate at 14 hpf. It is thus likely that the association between Peli1b and brain development in zebrafish embryos occurs via ERK3/4 pathways.

Drosophila CrebB is a Substrate of the Nonsense-Mediated mRNA Decay Pathway that Sustains Circadian Behaviors.

Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.

Sumoylation of the novel protein hRIP{beta} is involved in replication protein A deposition in PML nuclear bodies.

Replication protein A (RPA) is a single-stranded-DNA-binding protein composed of three subunits with molecular masses of 70, 32, and 14 kDa. The protein is involved in multiple processes of eukaryotic DNA metabolism, including DNA replication, repair, and recombination. In Xenopus, Xenopus RPA-interacting protein alpha has been identified as a carrier molecule of RPA into the nucleus. In this study, human RPA-interacting protein alpha (hRIPalpha) and five novel splice isoforms (named hRIPalpha, hRIPbeta, hRIPgamma, hRIPdelta1, hRIPdelta2, and hRIPdelta3 according to the lengths of their encoding peptides) were cloned. Among hRIP isoforms, hRIPalpha and hRIPbeta were found to be the major splice isoforms and to show different subcellular localizations. While hRIPalpha localized to the cytoplasm, hRIPbeta was found in the PML nuclear body. Modification of hRIPbeta by sumoylation was found to be required for localization to the PML nuclear body. The results of the present work demonstrate that hRIPbeta transports RPA into the PML nuclear body and releases RPA upon UV irradiation. hRIPbeta thus plays an important role in RPA deposition in PML nuclear bodies and thereby supplements RPA for DNA metabolism.

High-Amplitude Circadian Rhythms in Drosophila Driven by Calcineurin-Mediated Post-translational Control of sarah.

Post-translational control is a crucial mechanism for circadian timekeeping. Evolutionarily conserved kinases and phosphatases have been implicated in circadian phosphorylation and the degradation of clock-relevant proteins, which sustain high-amplitude rhythms with 24-hr periodicity in animal behaviors and physiology. Here, we report a novel clock function of the heterodimeric Ca2+/calmodulin-dependent phosphatase calcineurin and its regulator sarah (sra) in Drosophila Genomic deletion of the sra locus dampened circadian locomotor activity rhythms in free-running constant dark after entrainment in light-dark cycles. Poor rhythms in sra mutant behaviors were accompanied by lower expression of two oscillating clock proteins, PERIOD (PER) and TIMELESS (TIM), at the post-transcriptional level. RNA interference-mediated sra depletion in circadian pacemaker neurons was sufficient to phenocopy loss-of-function mutation in sra On the other hand, a constitutively active form of the catalytic calcineurin subunit, Pp2B-14DACT, shortened circadian periodicity in locomotor behaviors and phase-advanced PER and TIM rhythms when overexpressed in clock neurons. Heterozygous sra deletion induced behavioral arrhythmicity in Pp2B-14DACT flies, whereas sra overexpression rescued short periods in these animals. Finally, pharmacological inhibition of calcineurin in either wild-type flies or clock-less S2 cells decreased the levels of PER and TIM, likely by facilitating their proteasomal degradation. Taken together, these data suggest that sra negatively regulates calcineurin by cell-autonomously titrating calcineurin-dependent stabilization of PER and TIM proteins, thereby sustaining high-amplitude behavioral rhythms in Drosophila.

Conessine treatment reduces dexamethasone-induced muscle atrophy by regulating MuRF1 and atrogin-1 expression.

Conessine, a steroidal alkaloid, is a potent histamine H3 antagonist with anti-malarial activity. We recently reported that conessine treatment interferes with H2O2-induced cell death by regulating autophagy. However, the cellular signaling pathways involved in conessine treatment are not fully understood. Here, we report that conessine reduces muscle atrophy by interfering with the expression of atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Promoter reporter assay revealed that conessine treatment inhibits FoxO3a-dependent transcription, NF-kappaB-dependent transcription and p53-dependent transcription. We also showed that conessine treatment reduced dexamethasone-induced expression of MuRF1 and atrogin-1 by the quantitative RT-PCR and Western blot. Finally, we demonstrated that conessine treatment reduced dexamethasone-induced muscle atrophy using differentiated C2C12 cells. These results collectively suggest that conessine is potentially useful in the treatment of muscle atrophy.

Conessine Treatment Reduces Dexamethasone-Induced Muscle Atrophy by Regulating MuRF1 and Atrogin-1 Expression.

Conessine, a steroidal alkaloid, is a potent histamine H3 antagonist with antimalarial activity. We recently reported that conessine treatment interferes with H2O2-induced cell death by regulating autophagy. However, the cellular signaling pathways involved in conessine treatment are not fully understood. Here, we report that conessine reduces muscle atrophy by interfering with the expression of atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Promoter reporter assay revealed that conessine treatment inhibits FoxO3a-dependent transcription, NF-κB-dependent transcription, and p53-dependent transcription. We also showed by quantitative RT-PCR and western blot assays that conessine treatment reduced dexamethasone-induced expression of MuRF1 and atrogin-1. Finally, we demonstrated that conessine treatment reduced dexamethasone-induced muscle atrophy using differentiated C2C12 cells. These results collectively suggest that conessine is potentially useful in the treatment of muscle atrophy.

Dot1 regulates nucleosome dynamics by its inherent histone chaperone activity in yeast.

Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2-Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.

Targeted inhibition of Pdp1epsilon abolishes the circadian behavior of Drosophila melanogaster.

vrille and Par domain protein 1 (Pdp1) epsilon constitute the second transcriptional feedback loop in Drosophila circadian clock system. Their rhythmic expression is controlled by Drosophila Clock (dClk) gene, and they feed back to negatively and positively, respectively, regulate the oscillating transcription from dClk gene. In this study, we characterized the functional domains of PDP1epsilonin vitro using a panel of deletion mutants and showed that PDP1epsilon basic leucine zipper domain can act as a dominant-negative (DN) mutant of wild-type PDP1epsilon. In transgenic flies, the inhibition of PDP1epsilon activity by PDP1(DN) expression or PDP1 knock-down resulted in arrhythmic circadian behavior with altered dorsal projections from small ventral lateral neurons. We propose that one of PDP1-target genes may be involved in the formation of neural connection between the pacemaker cells and their targets for maintaining the rhythmicity of adult locomotor activity under free-running condition.