RESUMO
Zinc is an essential trace element and participates in a variety of biological processes. ZnT (SLC30) family members are generally responsible for zinc efflux across the membrane regulating zinc homeostasis. In mammals, the only predominantly plasma membrane resident ZnT has been reported to be ZnT1, and ZnT1-/ZnT1- mice die at the embryonic stage. In Drosophila, knock down of ZnT1 homologue (dZnT1//ZnT63C/CG17723) results in growth arrest under zinc-limiting conditions. To investigate the essentiality of dZnT1 for zinc homeostasis, as well as its role in dietary zinc uptake especially under normal physiological conditions, we generated dZnT1 mutants by the CRISPER/Cas9 method. Homozygous mutant dZnT1 is lethal, with substantial zinc accumulation in the iron cell region, posterior midgut as well as gastric caeca. Expression of human ZnT1 (hZnT1), in the whole body or in the entire midgut, fully rescued the dZnT1 mutant lethality, whereas tissue-specific expression of hZnT1 in the iron cell region and posterior midgut partially rescued the developmental defect of the dZnT1 mutant. Supplementation of zinc together with clioquinol or hinokitiol conferred a limited but observable rescue upon dZnT1 loss. Our work demonstrated the absolute requirement of dZnT1 in Drosophila survival and indicated that the most essential role of dZnT1 is in the gut.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Zinco/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Dieta , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Técnicas de Silenciamento de Genes , Genes de Insetos , Humanos , Absorção Intestinal/genética , Absorção Intestinal/fisiologia , Masculino , Mutação , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oligoelementos/administração & dosagem , Oligoelementos/metabolismo , Oligoelementos/farmacocinética , Zinco/administração & dosagem , Zinco/farmacocinéticaRESUMO
Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Proteína Fosfatase 2/fisiologia , Proteínas Quinases S6 Ribossômicas/fisiologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Ceramidas/farmacologia , Modelos Animais de Doenças , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Mutação com Ganho de Função , Técnicas de Silenciamento de Genes , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Mutação de Sentido Incorreto , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.
Assuntos
Alelos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteína Huntingtina/antagonistas & inibidores , Proteína Huntingtina/genética , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Mutação/genética , Animais , Ataxina-3/genética , Autofagossomos/metabolismo , Autofagia , Modelos Animais de Doenças , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/efeitos dos fármacos , Neurônios/citologia , Peptídeos/genética , Fenótipo , Reprodutibilidade dos TestesRESUMO
Alpha-ketoglutarate (AKG) is a key metabolite of the tricarboxylic acid (TCA) cycle, an essential process influencing the mitochondrial oxidative respiration rate. Recent studies have shown that dietary AKG reduces mTOR pathway activation by inhibiting ATP synthase, thereby extending the lifespan of nematodes. Although AKG also extends lifespan in fruit flies, the antiaging mechanisms of AKG in these organisms remain unclear. In the present study, we explored changes in gene expression associated with the extension of Drosophila lifespan mediated by dietary AKG. Supplementation of the flies' diets with 5 µM AKG extended their lifespan but reduced their reproductive performance. Dietary AKG also enhanced vertical climbing ability, but did not protect against oxidative stress or increase tolerance to starvation. AKG-reared flies were resistant to heat stress and demonstrated higher expression of heat shock protein genes (Hsp22 and Hsp70) than control flies. In addition, AKG significantly upregulated mRNA expression of cry, FoxO, HNF4, p300, Sirt1 and AMPKα, and downregulated expression of HDAC4, PI3K, TORC, PGC, and SREBP. The metabolic effects of AKG supplementation included a reduction in the ATP/ADP ratio and increased autophagy. Collectively, these observations indicate that AKG extends Drosophila lifespan by activating AMPK signaling and inhibiting the mTOR pathway.
Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/fisiologia , Ácidos Cetoglutáricos/farmacologia , Longevidade/efeitos dos fármacos , Proteínas Associadas à Matriz Nuclear/antagonistas & inibidores , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Comportamento Alimentar , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases/genética , Estresse FisiológicoRESUMO
Harsh climate induces physiological stress thus compromising organismal survival. Our previous studies demonstrated that curcumin (CUR) supplementation increased survival of turtle under heat stress (HS). Here, we span this work to investigate the survival and lifespan of HS Drosophila fed a diet supplemented with CUR. For this purpose, female and male flies were fed basal diet (N) and CUR diet (0.2 mg/g), and exposed to three conditions: 25°C and 29°C continuously, and 34 °C for 2 h at days 1, 4, and 7, then kept at 25 °C. Lifespan analysis showed that, compared to N-25 °C flies, the mean lifespans of N-29 °C and N-34 °C flies were decreased significantly by 8.5-15.7% in males, and 3.7-7.9% in females. Conversely, in the CUR-supplemented diet, mean lifespans of C-29 °C and C-34 °C flies were significantly extended by 8.7-16.4% in males, and by 8.9-12.8% in females, compared to that of temperature-matched flies fed basal diets. The MDA levels of C-34 °C flies were significantly lower than those of N-34 °C flies, indicating CUR reduced oxidative stress caused by HS. Furthermore, CUR palliated the increased oxidative stress caused by HS, by increasing the expression of SOD1, CAT, and PHGPx and decreasing the expression of Hsp70 and Hsp83. Our results indicated that CUR supplementation increases the survival rate of Drosophila by enhancing thermal tolerance. © 2018 BioFactors, 44(6):577-587, 2018.
Assuntos
Antioxidantes/farmacologia , Curcumina/farmacologia , Suplementos Nutricionais , Drosophila melanogaster/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Termotolerância/efeitos dos fármacos , Animais , Catalase/genética , Catalase/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Longevidade/fisiologia , Masculino , Malondialdeído/antagonistas & inibidores , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Termotolerância/genéticaRESUMO
Insect growth regulators (IGRs) are chemicals that adversely affect the physiological processes associated with insect development and cause abnormalities that impair insect survival. Ecdysone, an insect steroid hormone originally identified as a molting hormone, plays an essential role in developmental transition, such as during molting and metamorphosis. Recently, a member of the epsilon class of glutathione S-transferases (GST), GSTe14, also called Noppera-bo (Nobo), has been identified as essential for regulating the biosynthesis of ecdysone. Knockout or knockdown of the nobo gene causes ecdysone deficiency, leading to either death or arrested phenotype development at the larval stage. It is therefore considered that Nobo is potentially well suited as a target for novel IGRs. In this review, we focus on the development of a high-throughput screening strategy for Nobo inhibitors using a GST fluorogenic substrate.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Descoberta de Drogas , Ecdisteroides/biossíntese , Glutationa Transferase/genética , Glutationa Transferase/fisiologia , Insetos/crescimento & desenvolvimento , Insetos/genética , Hormônios Juvenis/genética , Hormônios Juvenis/fisiologia , Animais , Proteínas de Drosophila/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Ecdisteroides/deficiência , Ecdisteroides/fisiologia , Técnicas de Silenciamento de Genes , Glutationa Transferase/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Larva/genética , Larva/crescimento & desenvolvimento , Metamorfose Biológica/genética , Muda/genéticaRESUMO
We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.
Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/enzimologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/metabolismo , Glutationa Transferase/antagonistas & inibidores , Animais , Proteínas de Drosophila/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaios Enzimáticos , Corantes Fluorescentes/química , Glutationa/química , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Current understanding of amyloid-ß (Aß) metabolism and toxicity provides an extensive list of potential targets for developing drugs for treating Alzheimer's disease. We took two independent approaches, including synaptic-plasticity-based analysis and behavioral screening of synthetic compounds, for identifying single compounds that are capable of rescuing the Aß-induced memory loss in both transgenic fruit fly and transgenic mouse models. Two clinically available drugs and three synthetic compounds not only showed positive effects in behavioral tests but also antagonized the Aß oligomers-induced activation of the epidermal growth factor receptor (EGFR). Such surprising converging outcomes from two parallel approaches lead us to conclude that EGFR is a preferred target for treating Aß-induced memory loss.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Receptores ErbB/antagonistas & inibidores , Transtornos da Memória/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Peptídeos beta-Amiloides/genética , Animais , Animais Geneticamente Modificados , Western Blotting , Células COS , Chlorocebus aethiops , Drosophila , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Gefitinibe , Humanos , Aprendizagem em Labirinto/efeitos dos fármacos , Memantina/farmacologia , Memória/efeitos dos fármacos , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Quinazolinas/farmacologiaRESUMO
The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the 'anti-target' Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.
Assuntos
Terapia de Alvo Molecular , Neoplasia Endócrina Múltipla Tipo 2b/tratamento farmacológico , Neoplasia Endócrina Múltipla Tipo 2b/genética , Polimedicação , Animais , Benzenossulfonatos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/efeitos adversos , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Masculino , Neoplasia Endócrina Múltipla Tipo 2b/enzimologia , Niacinamida/análogos & derivados , Compostos de Fenilureia , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Piridinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons and the aggression of alpha Synuclein (αS) in the brain. Drosophila mutants and transgenes have provided a platform to understand the mechanistic insight associated with the degenerative diseases. A number of polyphenols have been reported to inhibit the αS aggregation resulting in the possible prevention of PD. The involvement of free radicals in mediating the neuronal death in PD has also been implicated. In the present study, the effect of Nordihydroguaiaretic acid (NDGA) was studied on the climbing ability of the PD model Drosophila expressing normal human alpha synuclein (h-αS) in the neurons. These flies exhibit locomotor dysfunction as the age progresses. NDGA at final concentration of 0.01, 0.1, 0.5, and 1µl/ml was supplemented with the diet and the flies were allowed to feed for the 24 days. NDGA at 0.01 µl/ml did not showed any significant delay in the loss of climbing ability of PD model flies. However, NDGA doses at 0.1, 0.5, and 1.0 µl/ml showed a dose dependent significant (p < .05) delay in the loss of climbing ability of PD model flies as compared to the untreated PD flies. The results suggest that the NDGA is potent in delaying the climbing disability of PD model flies and also supports the utility of this model in studying PD symptoms.
Assuntos
Suplementos Nutricionais , Locomoção/efeitos dos fármacos , Masoprocol/uso terapêutico , Doença de Parkinson/prevenção & controle , Fitoterapia , Extratos Vegetais/uso terapêutico , alfa-Sinucleína/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster , Radicais Livres/metabolismo , Humanos , Masoprocol/farmacologia , Mutação , Doença de Parkinson/metabolismo , Extratos Vegetais/farmacologiaRESUMO
Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila, living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce. Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce -/- background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites.
Assuntos
Apoptose/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos , Animais , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Mutação , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Recent progress in our understanding of genetic mechanisms of aging and longevity provides an opportunity to select some enzymes as targets for pharmacological correction. The phosphoinositide 3-kinase (PI3K) and TOR-kinase cascades are affected in some long-lived mutants of different animals, such as nematodes and mice. The purpose of this study was to investigate the geroprotector efficiency of the inhibitors of enzymes that are known to be affected in long-lived mutants. Experimental animals were exposed to low dozes of LY-294002 (5 microM), wortmannin (0.5 microM), and rapamycin (0.5 microM) separately during their lifetimes. We have shown that the specific PI3K inhibitors (LY-294002 and wortmannin) and the TOR-kinase inhibitor rapamycin slightly increase the median and maximal lifespan of the fruit fly, Drosophila melanogaster.
Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Androstadienos/farmacologia , Animais , Cromonas/farmacologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Longevidade/efeitos dos fármacos , Longevidade/fisiologia , Masculino , Morfolinas/farmacologia , Proteínas Quinases , Sirolimo/farmacologia , Sobrevida , Serina-Treonina Quinases TOR , WortmaninaRESUMO
Signal-mediated translocation of transient receptor potential (TRP) channels is a novel mechanism to fine tune a variety of signaling pathways including neuronal path finding and Drosophila photoreception. In Drosophila phototransduction the cation channels TRP and TRP-like (TRPL) are the targets of a prototypical G protein-coupled signaling pathway. We have recently found that the TRPL channel translocates between the rhabdomere and the cell body in a light-dependent manner. This translocation modifies the ion channel composition of the signaling membrane and induces long-term adaptation. However, the molecular mechanism underlying TRPL translocation remains unclear. Here we report that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, whereas TRPL-eGFP translocation could be directly visualized in intact eyes. TRPL-eGFP failed to translocate to the cell body in flies carrying severe mutations in essential phototransduction proteins, including rhodopsin, Galphaq, phospholipase Cbeta and the TRP ion channel, or in proteins required for TRP function. Our data, furthermore, show that the activation of a small fraction of rhodopsin and of residual amounts of the Gq protein is sufficient to trigger TRPL-eGFP internalization. In addition, we found that endocytosis of TRPL-eGFP occurs independently of dynamin, whereas a mutation of the unconventional myosin III, NINAC, hinders complete translocation of TRPL-eGFP to the cell body. Altogether, this study revealed that activation of the phototransduction cascade is mandatory for TRPL internalization, suggesting a critical role for the light induced conductance increase and the ensuing Ca2+ -influx in the translocation process. The critical role of Ca2+ influx was directly demonstrated when the light-induced TRPL-eGFP translocation was blocked by removing extracellular Ca2+.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Luz , Células Fotorreceptoras de Invertebrados/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Visão Ocular/fisiologia , Animais , Arrestinas/biossíntese , Arrestinas/fisiologia , Cálcio/metabolismo , Cálcio/efeitos da radiação , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/efeitos da radiação , Drosophila melanogaster/efeitos da radiação , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/efeitos da radiação , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Transporte Proteico/fisiologia , Transporte Proteico/efeitos da radiação , Rodopsina/fisiologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/efeitos da radiação , Visão Ocular/efeitos da radiaçãoRESUMO
The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance-voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.