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1.
Science ; 367(6483): 1230-1234, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165583

RESUMO

How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and "seed" for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-ß unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.


Assuntos
Amiloide/química , Proteínas de Drosophila/química , Memória de Longo Prazo , Neurônios/metabolismo , Agregados Proteicos , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Animais , Microscopia Crioeletrônica , Drosophila melanogaster , Glutamina/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica
2.
PLoS One ; 15(2): e0228348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032373

RESUMO

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.


Assuntos
Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Transmissão Sináptica , Sinaptotagminas/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Potenciais Pós-Sinápticos Excitadores , Humanos , Larva/metabolismo , Larva/fisiologia , Mutagênese Sítio-Dirigida , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sinaptotagminas/química , Sinaptotagminas/genética
3.
Nat Commun ; 11(1): 858, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051406

RESUMO

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs), and silence transposons in animal gonads. Here, we report the crystal structure of the Drosophila PIWI-clade Argonaute Piwi in complex with endogenous piRNAs, at 2.9 Å resolution. A structural comparison of Piwi with other Argonautes highlights the PIWI-specific structural features, such as the overall domain arrangement and metal-dependent piRNA recognition. Our structural and biochemical data reveal that, unlike other Argonautes including silkworm Siwi, Piwi has a non-canonical DVDK tetrad and lacks the RNA-guided RNA cleaving slicer activity. Furthermore, we find that the Piwi mutant with the canonical DEDH catalytic tetrad exhibits the slicer activity and readily dissociates from less complementary RNA targets after the slicer-mediated cleavage, suggesting that the slicer activity could compromise the Piwi-mediated co-transcriptional silencing. We thus propose that Piwi lost the slicer activity during evolution to serve as an RNA-guided RNA-binding platform, thereby ensuring faithful co-transcriptional silencing of transposons.


Assuntos
Proteínas Argonauta/classificação , Proteínas de Drosophila/química , Drosophila/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Bombyx/metabolismo , Linhagem Celular , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Drosophila/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inativação Gênica , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , RNA Guia/metabolismo , RNA Interferente Pequeno/metabolismo , RNA não Traduzido
4.
Immunity ; 52(2): 374-387.e6, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32075729

RESUMO

Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. Here, we found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. Our findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms.


Assuntos
Drosophila melanogaster/imunologia , Hemolinfa/metabolismo , Metabolismo dos Lipídeos/imunologia , Túbulos de Malpighi/imunologia , Espécies Reativas de Oxigênio/imunologia , Imunidade Adaptativa , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diglicerídeos/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fezes/química , Peroxidação de Lipídeos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Túbulos de Malpighi/metabolismo , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/imunologia
5.
Nucleic Acids Res ; 48(7): 3906-3921, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32025726

RESUMO

RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site - contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.


Assuntos
Proteínas de Drosophila/metabolismo , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Fertilidade/genética , Sequências Repetitivas Dispersas , Masculino , Mutação , Domínios Proteicos , RNA Helicases/metabolismo , Interferência de RNA , Transporte de RNA , RNA Antissenso , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Transcrição Genética
6.
Nat Struct Mol Biol ; 27(1): 78-83, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907454

RESUMO

The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/química , Humanos , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização Proteica
7.
Nat Neurosci ; 23(2): 172-175, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907439

RESUMO

Arc, a neuronal gene that is critical for synaptic plasticity, originated through the domestication of retrotransposon Gag genes and mediates intercellular messenger RNA transfer. We report high-resolution structures of retrovirus-like capsids formed by Drosophila dArc1 and dArc2 that have surface spikes and putative internal RNA-binding domains. These data demonstrate that virus-like capsid-forming properties of Arc are evolutionarily conserved and provide a structural basis for understanding their function in intercellular communication.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/ultraestrutura , Sequência de Aminoácidos , Animais , Capsídeo , Drosophila melanogaster , Conformação Proteica
8.
Biochim Biophys Acta Gene Regul Mech ; 1863(1): 194446, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706027

RESUMO

Recently, the concept has arisen that a special class of architectural proteins exists, which are responsible not only for global chromosome architecture but also for the local regulation of enhancer-promoter interactions. Here, we describe a new architectural protein, with a total size of only 375 aa, which contains an N-terminal zinc finger-associated domain (ZAD) and a cluster of five zinc finger C2H2 domains at the C-terminus. This new protein, named ZAD and Architectural Function 1 protein (ZAF1 protein), is weakly and ubiquitously expressed, with the highest expression levels observed in oocytes and embryos. The cluster of C2H2 domains recognizes a specific 15-bp consensus site, located predominantly in promoters, near transcription start sites. The expression of ZAF1 by a tissue-specific promoter led to the complete blocking of the eye enhancer when clusters of ZAF1 binding sites flanked the eye enhancer in transgenic lines, suggesting that the loop formed by the ZAF1 protein leads to insulation. The ZAF1 protein also supported long-range interactions between the yeast GAL4 activator and the white promoter in transgenic Drosophila lines. A mutant protein lacking the ZAD failed to block the eye enhancer or to support distance interactions in transgenic lines. Taken together, these results suggest that ZAF1 is a minimal architectural protein that can be used to create a convenient model for studying the mechanisms of distance interactions.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Elementos Facilitadores Genéticos , Olho/metabolismo , Proteínas do Olho/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Dedos de Zinco
9.
Biochim Biophys Acta Mol Cell Res ; 1867(2): 118607, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31733261

RESUMO

The zinc/iron permease (ZIP/SLC39A) family plays an important role in metal ion transport and is essential for diverse physiological processes. Members of the ZIP family function primarily in the influx of transition metal ions zinc and iron, into cytoplasm from extracellular space or intracellular organelles. The molecular determinants defining metal ion selectivity among ZIP family members remain unclear. Specifically, we reported before that the Drosophila ZIP family member ZIP13 (dZIP13), functions as an iron exporter and was responsible for pumping iron into the secretory pathway. ZIP13 protein is unique in that it differs from the other LIV-1 subfamily members at transmembrane domain IV (TM4), wherein relative positions of the conserved H and D residues in the HNXXD sequence motif are switched, generating a DNXXH motif. In this study, we undertook an in vivo approach to explore the significance of this D/H exchange. Comparative functional analysis of mutants revealed that the relative positions of D and H are critical for the physiological roles of dZIP13 and its close homologue dZIP7. Swapping D/H position of this DNXXH sequence in dZIP13 resulted in loss of iron activity; normal dZIP13 could not complement dZIP7 loss, but swapping the two relative amino acid positions D and H in dZIP13 was sufficient to make it functionally analogous to its close homologue dZIP7. This work provides the first in vivo functional analysis of a structural motif required to differentiate different transporting functions of ZIPs.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ferro/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Humanos , Larva/química , Larva/metabolismo , Espectrometria de Massas , Domínios Proteicos , Alinhamento de Sequência , Zinco/análise , Zinco/metabolismo
10.
PLoS One ; 14(12): e0226061, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856229

RESUMO

Collective migration plays critical roles in animal development, physiological events, and cancer metastasis. However, the molecular mechanisms of collective cell migration are not well understood. Drosophila border cells represent an excellent in vivo genetic model to study collective cell migration and identify novel regulatory genes for cell migration. Using the Mosaic Analysis with a Repressible Cell Marker (MARCM) system, we screened 240 P-element insertion lines to identify essential genes for border cell migration. Two genes were uncovered, including dlg5 (discs large 5) and CG31689. Further analysis showed that Dlg5 regulates the apical-basal polarity and cluster integrity in border cell clusters. Dlg5 is enriched in lateral surfaces between border cells and central polar cells but also shows punctate localization between border cells. We found that the distribution of Dlg5 in border cell clusters is regulated by Armadillo. Structure-function analysis revealed that the N-terminal Coiled-coil domain and the C-terminal PDZ3-PDZ4-SH3-GUK domains but not the PDZ1-PDZ2 domains of Dlg5 are required for BC migration. The Coiled-coil domain and the PDZ4-SH3-GUK domains are critical for Dlg5's cell surface localization in border cell clusters.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Guanilato Quinases/metabolismo , Oogênese , Animais , Proteínas do Domínio Armadillo/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Polaridade Celular , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Genes Reporter , Guanilato Quinases/antagonistas & inibidores , Guanilato Quinases/química , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismo , Domínios Proteicos , Proteína Quinase C/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo
11.
BMC Genomics ; 20(Suppl 13): 932, 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31881842

RESUMO

Proteins play essential roles in almost all life processes. The prediction of protein function is of significance for the understanding of molecular function and evolution. Network alignment provides a fast and effective framework to automatically identify functionally conserved proteins in a systematic way. However, due to the fast growing genomic data, interactions and annotation data, there is an increasing demand for more accurate and efficient tools to deal with multiple PPI networks. Here, we present a novel global alignment algorithm NetCoffee2 based on graph feature vectors to discover functionally conserved proteins and predict function for unknown proteins. To test the algorithm performance, NetCoffee2 and three other notable algorithms were applied on eight real biological datasets. Functional analyses were performed to evaluate the biological quality of these alignments. Results show that NetCoffee2 is superior to existing algorithms IsoRankN, NetCoffee and multiMAGNA++ in terms of both coverage and consistency. The binary and source code are freely available under the GNU GPL v3 license at https://github.com/screamer/NetCoffee2.


Assuntos
Algoritmos , Proteínas/metabolismo , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Entropia , Humanos , Camundongos , Mapas de Interação de Proteínas , Proteínas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Elife ; 82019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31718774

RESUMO

Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They play important roles in neurons, as suggested by inhibition of axon growth and regeneration through the ARF activator Efa6 in C. elegans, and by neurodevelopmental disorders linked to the mammalian kinesin Kif21A. How cortical collapse factors influence axon growth is little understood. Here we studied them, focussing on the function of Drosophila Efa6 in experimentally and genetically amenable fly neurons. First, we show that Drosophila Efa6 can inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane-attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of explorative MTs: in growth cones they enhance axon growth, in axon shafts they cause excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over-expression causes the opposite phenotypes. Taken together, our work conceptually links molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised fashion when fusing it to specifically localising protein domains.


Assuntos
Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Microtúbulos/metabolismo , Polimerização , Motivos de Aminoácidos , Animais , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Drosophila/química , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Cones de Crescimento/metabolismo , Camundongos , Células NIH 3T3 , Peptídeos/metabolismo , Domínios Proteicos , Pseudópodes/metabolismo
13.
Nature ; 575(7781): 203-209, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31666698

RESUMO

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 Testes
14.
BMC Cancer ; 19(1): 1009, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660888

RESUMO

BACKGROUND: The high expression of BLM (Bloom syndrome) helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to screen potential antiproliferative small molecules from 12 small molecules (the derivatives of bisbenzylisoquinoline alkaloids tetrandrine and fangchinoline) by targeting BLM642-1290 helicase. Then we explore the inhibitory mechanism of those small molecules on proliferation of MDA-MB-435 breast cancer cells. METHODS: Fluorescence polarization technique was used to screen small molecules which inhibited the DNA binding and unwinding of BLM642-1290 helicase. The effects of positive small molecules on the ATPase and conformation of BLM642-1290 helicase were studied by the malachite green-phosphate ammonium molybdate colorimetry and ultraviolet spectral scanning, respectively. The effects of positive small molecules on growth of MDA-MB-435 cells were studied by MTT method, colony formation and cell counting method. The mRNA and protein levels of BLM helicase in the MDA-MB-435 cells after positive small molecule treatments were examined by RT-PCR and ELISA, respectively. RESULTS: The compound HJNO (a tetrandrine derivative) was screened out which inhibited the DNA binding, unwinding and ATPase of BLM642-1290 helicase. That HJNO could bind BLM642-1290helicase to change its conformationcontribute to inhibiting the DNA binding, ATPase and DNA unwinding of BLM642-1290 helicase. In addition, HJNO showed its inhibiting the growth of MDA-MB-435 cells. The values of IC50 after drug treatments for 24 h, 48 h and 72 h were 19.9 µmol/L, 4.1 µmol/L and 10.9 µmol/L, respectively. The mRNA and protein levels of BLM helicase in MDA-MB-435 cells increased after HJNO treatment. Those showed a significant difference (P < 0.05) compared with negative control when the concentrations of HJNO were 5 µmol/L and 10 µmol/L, which might contribute to HJNO inhibiting the DNA binding, ATPase and DNA unwinding of BLM helicase. CONCLUSION: The small molecule HJNO was screened out by targeting BLM642-1290 helicase. And it showed an inhibition on MDA-MB-435 breast cancer cells expansion.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzilisoquinolinas/farmacologia , Neoplasias da Mama/tratamento farmacológico , RecQ Helicases/genética , RecQ Helicases/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Benzilisoquinolinas/química , Benzilisoquinolinas/metabolismo , Benzilisoquinolinas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/química , DNA/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/enzimologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração Inibidora 50 , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos
15.
Nat Cell Biol ; 21(10): 1261-1272, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570835

RESUMO

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/ultraestrutura , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oócitos/metabolismo , Oócitos/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
Biomater Sci ; 7(11): 4708-4719, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31565713

RESUMO

Glucan particles derived from yeast have been recently proposed as potential drug delivery carriers. Here, we demonstrate the potential of glucan particles for protein delivery in vivo, using the insect Drosophila melanogaster as a model organism. By employing genetic tools, we demonstrate the capacity of yeast glucan particles to spread efficiently through the Drosophila body, to enter macrophages and to deliver an active transcription factor protein successfully. Moreover, the glucan particles were nontoxic and induced only minimal immune response. The injection of glucan particles did not impair the ability of Drosophila to fight and survive infection by pathogenic bacteria. From this study, Drosophila emerges as an excellent model to test and develop drug delivery systems based on glucan particles, specifically aimed to regulate macrophages.


Assuntos
Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Sistemas de Liberação de Medicamentos , Glucanos/metabolismo , Leveduras/química , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Glucanos/química , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
17.
PLoS One ; 14(10): e0219878, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31658274

RESUMO

The extraordinarily strong non-covalent interaction between biotin and avidin (kD = 10-14-10-16) has permitted this interaction to be used in a wide variety of experimental contexts. The Biotin Acceptor Peptide (BAP), a 15 amino acid motif that can be biotinylated by the E. coli BirA protein, has been fused to proteins-of-interest, making them substrates for in vivo biotinylation. Here we report on the construction and characterization of a modified BirA bearing signals for secretion and endoplasmic reticulum (ER) retention, for use in experimental contexts requiring biotinylation of secreted proteins. When expressed in the Drosophila female germline or ovarian follicle cells under Gal4-mediated transcriptional control, the modified BirA protein could be detected and shown to be enzymatically active in ovaries and progeny embryos. Surprisingly, however, it was not efficiently retained in the ER, and instead appeared to be secreted. To determine whether this secreted protein, now designated secBirA, could biotinylate secreted proteins, we generated BAP-tagged versions of two secreted Drosophila proteins, Torsolike (Tsl) and Gastrulation Defective (GD), which are normally expressed maternally and participate in embryonic pattern formation. Both Tsl-BAP and GD-BAP were shown to exhibit normal patterning activity. Co-expression of Tsl-BAP together with secBirA in ovarian follicle cells resulted in its biotinylation, which permitted its isolation from both ovaries and progeny embryos using Avidin-coupled affinity matrix. In contrast, co-expression with secBirA in the female germline did not result in detectable biotinylation of GD-BAP, possibly because the C-terminal location of the BAP tag made it inaccessible to BirA in vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretion in vivo, providing access to powerful experimental approaches for secreted proteins-of-interest. However, efficient biotinylation of target proteins may vary depending upon the location of the BAP tag or other structural features of the protein.


Assuntos
Carbono-Nitrogênio Ligases/química , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Embrião não Mamífero/química , Proteínas de Escherichia coli/química , Ovário/química , Proteínas Repressoras/química , Animais , Biotinilação , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrião não Mamífero/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Ovário/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
18.
Nat Struct Mol Biol ; 26(11): 1013-1022, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659330

RESUMO

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.


Assuntos
Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Guanosina Trifosfato/química , Transposases/química , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Forma A/química , DNA de Forma B/química , Drosophila melanogaster/genética , Modelos Moleculares , Conformação Proteica
19.
Proc Natl Acad Sci U S A ; 116(44): 22205-22211, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31615886

RESUMO

The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. Here we use the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, we show that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.


Assuntos
Proteínas de Drosophila/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Morfogênese , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/genética , Trombocitopenia/congênito , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Domínios Proteicos , Trombocitopenia/genética
20.
EMBO Rep ; 20(12): e48296, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31576653

RESUMO

Eggless/SETDB1 (Egg), the only essential histone methyltransferase (HMT) in Drosophila, plays a role in gene repression, including piRNA-mediated transposon silencing in the ovaries. Previous studies suggested that Egg is post-translationally modified and showed that Windei (Wde) regulates Egg nuclear localization through protein-protein interaction. Monoubiquitination of mammalian SETDB1 is necessary for the HMT activity. Here, using cultured ovarian somatic cells, we show that Egg is monoubiquitinated and phosphorylated but that only monoubiquitination is required for piRNA-mediated transposon repression. Egg monoubiquitination occurs in the nucleus. Egg has its own nuclear localization signal, and the nuclear import of Egg is Wde-independent. Wde recruits Egg to the chromatin at target gene silencing loci, but their interaction is monoubiquitin-independent. The abundance of nuclear Egg is governed by that of nuclear Wde. These results illuminate essential roles of nuclear monoubiquitination of Egg and the role of Wde in piRNA-mediated transposon repression.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Núcleo Celular/genética , Núcleo Celular/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Feminino , Inativação Gênica , Histona-Lisina N-Metiltransferase/química , Técnicas In Vitro , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Ovário/citologia , Ovário/metabolismo , Domínios Proteicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ubiquitinação
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