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1.
Nat Commun ; 12(1): 5263, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489457

RESUMO

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeo Hidrolases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Evolução Molecular , Células HEK293 , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Lenalidomida/química , Lenalidomida/farmacologia , Camundongos , Organoides , Peptídeo Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Nat Commun ; 12(1): 5178, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34462441

RESUMO

Animals maintain metabolic homeostasis by modulating the activity of specialized organs that adjust internal metabolism to external conditions. However, the hormonal signals coordinating these functions are incompletely characterized. Here we show that six neurosecretory cells in the Drosophila central nervous system respond to circulating nutrient levels by releasing Capa hormones, homologs of mammalian neuromedin U, which activate the Capa receptor (CapaR) in peripheral tissues to control energy homeostasis. Loss of Capa/CapaR signaling causes intestinal hypomotility and impaired nutrient absorption, which gradually deplete internal nutrient stores and reduce organismal lifespan. Conversely, increased Capa/CapaR activity increases fluid and waste excretion. Furthermore, Capa/CapaR inhibits the release of glucagon-like adipokinetic hormone from the corpora cardiaca, which restricts energy mobilization from adipose tissue to avoid harmful hyperglycemia. Our results suggest that the Capa/CapaR circuit occupies a central node in a homeostatic program that facilitates the digestion and absorption of nutrients and regulates systemic energy balance.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neuropeptídeos/metabolismo , Nutrientes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Metabolismo Energético , Feminino , Homeostase , Hormônios de Inseto/metabolismo , Longevidade , Masculino , Neuropeptídeos/genética , Oligopeptídeos/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Ácido Pirrolidonocarboxílico/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
3.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361042

RESUMO

Various neurodegenerative disorders are associated with human NTE/PNPLA6 dysfunction. Mechanisms of neuropathogenesis in these diseases are far from clearly elucidated. Hereditary spastic paraplegia belongs to a type of neurodegeneration associated with NTE/PNLPLA6 and is implicated in neuron death. In this study, we used Drosophila melanogaster to investigate the consequences of neuronal knockdown of swiss cheese (sws)-the evolutionarily conserved ortholog of human NTE/PNPLA6-in vivo. Adult flies with the knockdown show longevity decline, locomotor and memory deficits, severe neurodegeneration progression in the brain, reactive oxygen species level acceleration, mitochondria abnormalities and lipid droplet accumulation. Our results suggest that SWS/NTE/PNPLA6 dysfunction in neurons induces oxidative stress and lipid metabolism alterations, involving mitochondria dynamics and lipid droplet turnover in neurodegeneration pathogenesis. We propose that there is a complex mechanism in neurological diseases such as hereditary spastic paraplegia, which includes a stress reaction, engaging mitochondria, lipid droplets and endoplasmic reticulum interplay.


Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Encéfalo/citologia , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster , Metabolismo dos Lipídeos , Mitocôndrias/ultraestrutura , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Estresse Oxidativo
4.
Nat Commun ; 12(1): 4173, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234137

RESUMO

The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we show that cellular protein O-GlcNAcylation exhibits robust 24-hour rhythm and represents a key post-translational mechanism that regulates circadian physiology. We observe strong correlation between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms are severely dampened when we subject flies to time-restricted feeding at unnatural feeding time. This suggests the presence of clock-regulated buffering mechanisms that prevent excessive O-GlcNAcylation at non-optimal times of the day-night cycle. We show that this buffering mechanism is mediated by the expression and activity of GFAT, OGT, and OGA, which are regulated through integration of circadian and metabolic signals. Finally, we generate a mathematical model to describe the key factors that regulate daily O-GlcNAcylation rhythm.


Assuntos
Ritmo Circadiano/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/biossíntese , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Acetilglucosamina/metabolismo , Animais , Animais Geneticamente Modificados , Vias Biossintéticas/genética , Relógios Circadianos/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Comportamento Alimentar/fisiologia , Feminino , Perfilação da Expressão Gênica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Masculino , Modelos Animais , N-Acetilglucosaminiltransferases/genética , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo
5.
Cell Death Dis ; 12(7): 671, 2021 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-34218254

RESUMO

The balanced functionality of cellular proteostatic modules is central to both proteome stability and mitochondrial physiology; thus, the age-related decline of proteostasis also triggers mitochondrial dysfunction, which marks multiple degenerative disorders. Non-functional mitochondria are removed by mitophagy, including Parkin/Pink1-mediated mitophagy. A common feature of neuronal or muscle degenerative diseases, is the accumulation of damaged mitochondria due to disrupted mitophagy rates. Here, we exploit Drosophila as a model organism to investigate the functional role of Parkin/Pink1 in regulating mitophagy and proteostatic responses, as well as in suppressing degenerative phenotypes at the whole organism level. We found that Parkin or Pink1 knock down in young flies modulated proteostatic components in a tissue-dependent manner, increased cell oxidative load, and suppressed mitophagy in neuronal and muscle tissues, causing mitochondrial aggregation and neuromuscular degeneration. Concomitant to Parkin or Pink1 knock down cncC/Nrf2 overexpression, induced the proteostasis network, suppressed oxidative stress, restored mitochondrial function, and elevated mitophagy rates in flies' tissues; it also, largely rescued Parkin or Pink1 knock down-mediated neuromuscular degenerative phenotypes. Our in vivo findings highlight the critical role of the Parkin/Pink1 pathway in mitophagy, and support the therapeutic potency of Nrf2 (a druggable pathway) activation in age-related degenerative diseases.


Assuntos
Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Mitocôndrias Musculares/enzimologia , Mitofagia , Músculo Esquelético/enzimologia , Degeneração Neural , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/patologia , Músculo Esquelético/patologia , Neurônios/patologia , Estresse Oxidativo , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteostase , Proteínas Repressoras/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética
6.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34313318

RESUMO

Heterozygosity of ribosomal protein genes causes a variety of developmental abnormalities in humans, which are collectively known as ribosomopathies, yet the underlying mechanisms remain elusive. Here, we analyzed Drosophila Minute (M)/+ mutants, a group of mutants heterozygous for ribosomal protein genes that exhibit a characteristic thin-bristle phenotype. We found that, although M/+ flies develop essentially normal wings, simultaneous deletion of one copy of the Hippo pathway effector yki resulted in severe wing growth defects. These defects were caused by JNK-mediated cell death in the wing pouch via Eiger/TNF signaling. The JNK activation in M/+, yki/+ wing discs required the caspase Dronc, which is normally blocked by DIAP1. Notably, heterozygosity of yki reduced DIAP1 expression in the wing pouch, leading to elevation of Dronc activity. Dronc and JNK formed a positive-feedback loop that amplifies Dronc activation, leading to apoptosis. Our observations suggest a mechanism of robust tissue growth whereby tissues with reduced ribosomal protein prevent ectopic apoptosis via Yki activity.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Animais , Apoptose , Morte Celular , Regulação para Baixo , Drosophila/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Nucleares/genética , Transdução de Sinais , Transativadores/genética , Asas de Animais/anatomia & histologia , Asas de Animais/metabolismo
7.
Mol Cell ; 81(16): 3356-3367.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34297910

RESUMO

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.


Assuntos
Proteínas de Drosophila/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , RNA Polimerase II/genética , Transcrição Genética , Animais , Drosophila melanogaster/genética , Metiltransferases/genética , Regiões Promotoras Genéticas/genética
8.
Am J Hum Genet ; 108(9): 1669-1691, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34314705

RESUMO

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.


Assuntos
Deficiências do Desenvolvimento/genética , Proteínas de Drosophila/genética , Oftalmopatias Hereditárias/genética , Deficiência Intelectual/genética , Carioferinas/genética , Anormalidades Musculoesqueléticas/genética , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genética , Alelos , Sequência de Aminoácidos , Animais , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo , Masculino , Anormalidades Musculoesqueléticas/metabolismo , Anormalidades Musculoesqueléticas/patologia , Mutação , Neurônios/metabolismo , Neurônios/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sequenciamento Completo do Genoma , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
9.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34297127

RESUMO

Meiosis in female oocytes lacks centrosomes, the microtubule-organizing centers. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, we examined the role of protein phosphatase 2A (PP2A) in Drosophila oocyte meiosis. We found that both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which has a B subunit encoded by two partially redundant paralogs, wdb and wrd, is also required for maintenance of sister chromatid cohesion, establishment of end-on microtubule attachments, and metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332 and kinetochore protein SPC105R. Although BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. We propose at least three populations of PP2A-B56 regulate meiosis, two of which depend on SPC105R and a third that is associated with the spindle.


Assuntos
Proteínas de Drosophila , Cinetocoros , Proteína Fosfatase 2 , Fuso Acromático , Animais , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Drosophila , Proteínas de Drosophila/genética , Feminino , Meiose , Microtúbulos , Oócitos , Proteína Fosfatase 2/genética
10.
Nat Commun ; 12(1): 4170, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234130

RESUMO

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Genoma de Inseto , Elementos Isolantes/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , RNA-Seq , Proteínas Repressoras/genética , Fatores de Transcrição/genética
11.
Science ; 373(6550)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210853

RESUMO

The mechanisms by which macrophages regulate energy storage remain poorly understood. We identify in a genetic screen a platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF)-family ortholog, Pvf3, that is produced by macrophages and is required for lipid storage in fat-body cells of Drosophila larvae. Genetic and pharmacological experiments indicate that the mouse Pvf3 ortholog PDGFcc, produced by adipose tissue-resident macrophages, controls lipid storage in adipocytes in a leptin receptor- and C-C chemokine receptor type 2-independent manner. PDGFcc production is regulated by diet and acts in a paracrine manner to control lipid storage in adipose tissues of newborn and adult mice. At the organismal level upon PDGFcc blockade, excess lipids are redirected toward thermogenesis in brown fat. These data identify a macrophage-dependent mechanism, conducive to the design of pharmacological interventions, that controls energy storage in metazoans.


Assuntos
Adipócitos/imunologia , Dieta Hiperlipídica , Proteínas de Drosophila/metabolismo , Metabolismo Energético , Linfocinas/metabolismo , Macrófagos/imunologia , Obesidade/imunologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Termogênese , Tecido Adiposo Marrom/imunologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Hemócitos/imunologia , Fígado/imunologia , Linfocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Derivado de Plaquetas/genética , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201604

RESUMO

The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3'UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G0 flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G0 flies produced G1 mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii.


Assuntos
Sistemas CRISPR-Cas , Drosophila/genética , Marcação de Genes/métodos , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Drosophila/embriologia , Proteínas de Drosophila/genética , Embrião não Mamífero , Feminino , Proteínas de Fluorescência Verde/genética , Resposta ao Choque Térmico/genética , Masculino , Mutagênese , Pigmentação/genética , Temperatura , Transgenes
13.
Nat Commun ; 12(1): 4061, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210982

RESUMO

PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Proteínas de Transporte/química , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Masculino , Metilação , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Domínios Proteicos , Proteína-Arginina N-Metiltransferases , RNA Interferente Pequeno/química
14.
Nat Commun ; 12(1): 4268, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257295

RESUMO

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct-processive or distributive-modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2's cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.


Assuntos
Proteínas de Drosophila/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/genética , Ribonuclease III/metabolismo , Imagem Individual de Molécula/métodos , Animais , Drosophila , Proteínas de Drosophila/genética , Modelos Teóricos , RNA Helicases/genética , Interferência de RNA/fisiologia , Ribonuclease III/genética
15.
Yi Chuan ; 43(6): 615-622, 2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-34284991

RESUMO

Lipid is one of the important components of living organisms. The precise regulation and homeostasis maintenance of lipid metabolism are essential to human health. The ubiquitination pathway regulates lipid metabolism by degrading lipid-related proteins. Ppa encodes an F-box protein, which is a member of the SCF ubiquitination complex. Previous studies reported that Ppa regulated the body segmentation and the correct localization of centromere histones, while its function in lipid metabolism has not been reported. In this study, Drosophila melanogaster was used to explore the function of Ppa in lipid storage. The subcellular localization of PPA was detected by fusion with green fluorescent protein. The deletion mutant of Ppa was constructed via CRISPR/Cas9 technology. The morphological changes of lipid droplets in deletion mutants and Ppa overexpression flies were analyzed by BODIPY 493/503 or Nile red staining. Further, Ppa was overexpressed in the deletion mutant to verify its function. The results showed that PPA-GFP fusion protein were localized in the nuclei of salivary gland and fat body. Compared with the control flies, the lipid droplets in Ppa deletion mutants became smaller, and overexpression of Ppa exhibited larger lipid droplets. Overexpression of Ppa in the deletion mutant could restore the lipid droplets to normal state. In summary, this study demonstrated that Ppa could promote lipid storage in Drosophila.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Metabolismo dos Lipídeos/genética , Lipídeos
16.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201772

RESUMO

Nucleolar stress occurs when ribosome production or function declines. Nucleolar stress in stem cells or progenitor cells often leads to disease states called ribosomopathies. Drosophila offers a robust system to explore how nucleolar stress causes cell cycle arrest, apoptosis, or autophagy depending on the cell type. We provide an overview of nucleolar stress in Drosophila by depleting nucleolar phosphoprotein of 140 kDa (Nopp140), a ribosome biogenesis factor (RBF) in nucleoli and Cajal bodies (CBs). The depletion of Nopp140 in eye imaginal disc cells generates eye deformities reminiscent of craniofacial deformities associated with the Treacher Collins syndrome (TCS), a human ribosomopathy. We show the activation of c-Jun N-terminal Kinase (JNK) in Drosophila larvae homozygous for a Nopp140 gene deletion. JNK is known to induce the expression of the pro-apoptotic Hid protein and autophagy factors Atg1, Atg18.1, and Atg8a; thus, JNK is a central regulator in Drosophila nucleolar stress. Ribosome abundance declines upon Nopp140 loss, but unusual cytoplasmic granules accumulate that resemble Processing (P) bodies based on marker proteins, Decapping Protein 1 (DCP1) and Maternal expression at 31B (Me31B). Wild type brain neuroblasts (NBs) express copious amounts of endogenous coilin, but coilin levels decline upon nucleolar stress in most NB types relative to the Mushroom body (MB) NBs. MB NBs exhibit resilience against nucleolar stress as they maintain normal coilin, Deadpan, and EdU labeling levels.


Assuntos
Nucléolo Celular/genética , Corpos Enovelados/patologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/genética , Estresse Fisiológico , Animais , Sistemas CRISPR-Cas , Corpos Enovelados/genética , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/fisiologia , Larva/genética , Larva/crescimento & desenvolvimento , Fosfoproteínas , Proteínas de Ligação a RNA/antagonistas & inibidores , Ribossomos/genética , Ribossomos/metabolismo
17.
Nat Commun ; 12(1): 4399, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285221

RESUMO

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. Here, we have investigated Drosophila motor neuron synaptic terminals during ageing. We observed cumulative fragmentation of presynaptic structures accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, we find that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. Our results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections.


Assuntos
Envelhecimento/fisiologia , Neurônios Motores/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Potencial Evocado Motor/fisiologia , Masculino , Microscopia Eletrônica , Modelos Animais , Neurônios Motores/ultraestrutura , Músculos/inervação , Músculos/fisiologia , Músculos/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Fatores de Tempo
18.
Aging (Albany NY) ; 13(11): 14709-14728, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074800

RESUMO

One of the genes which has been linked to the onset of juvenile/early onset Parkinson's disease (PD) is PINK1. There is evidence that supports the therapeutic potential of exercise in the alleviation of PD symptoms. It is possible that exercise may enhance synaptic plasticity, protect against neuro-inflammation and modulate L-Dopa regulated signalling pathways. We explored the effects of exercise on Pink1 deficient Drosophila melanogaster which undergo neurodegeneration and muscle degeneration. We used a 'power-tower' type exercise platform to deliver exercise activity to Pink1- and age matched wild-type Drosophila. Mitochondrial proteomic profiles responding to exercise were obtained. Of the 516 proteins identified, 105 proteins had different levels between Pink1- and wild-type non-exercised Drosophila. Gene ontology enrichment analysis and STRING network analysis highlighted proteins and pathways with altered expression within the mitochondrial proteome. Comparison of the Pink1- exercised proteome to wild-type proteomes showed that exercising the Pink1- Drosophila caused their proteomic profile to return towards wild-type levels.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Mitocondriais/metabolismo , Condicionamento Físico Animal , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eletroforese em Gel Bidimensional , Metabolismo Energético , Regulação da Expressão Gênica , Ontologia Genética , Espectrometria de Massas , Mitocôndrias/metabolismo , Anotação de Sequência Molecular , Mutação/genética , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteômica
19.
Nat Commun ; 12(1): 3778, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145251

RESUMO

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.


Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética
20.
Nucleic Acids Res ; 49(13): 7644-7664, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181727

RESUMO

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.


Assuntos
Proteínas do Complexo SMN/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Dimerização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Humanos , Locomoção , Modelos Moleculares , Mutação , Mutação Puntual , Domínios Proteicos , Multimerização Proteica , Proteínas do Complexo SMN/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
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