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1.
Cell ; 133(5): 852-63, 2008 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-18510929

RESUMO

Activity of the big brain (bib) gene influences Notch signaling during Drosophila nervous system development. We demonstrate that Bib, which belongs to the aquaporin family of channel proteins, is required for endosome maturation in Drosophila epithelial cells. In the absence of Bib, early endosomes arrest and form abnormal clusters, and cells exhibit reduced acidification of endocytic trafficking organelles. Bib acts downstream of Hrs in early endosome morphogenesis and regulates biogenesis of endocytic compartments prior to the formation of Rab7-containing late endosomes. Abnormal endosome morphology caused by loss of Bib is accompanied by overaccumulation of Notch, Delta, and other signaling molecules as well as reduced intracellular trafficking of Notch to nuclei. Analysis of several endosomal trafficking mutants reveals a correlation between endosomal acidification and levels of Notch signaling. Our findings reveal an unprecedented role for an aquaporin in endosome maturation, trafficking, and acidification.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Animais , Aquaporinas/metabolismo , Drosophila melanogaster/metabolismo , Presenilinas/metabolismo , Transporte Proteico
2.
Development ; 140(14): 3018-27, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23785054

RESUMO

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of the Notch signaling pathway is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is crucial for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila epithelial tissues, we recovered mutations that disrupt the Catsup gene, which encodes the Drosophila ortholog of the mammalian ZIP7 zinc transporter. Loss of Catsup function causes Notch to accumulate abnormally in the endoplasmic reticulum (ER) and Golgi compartments, resulting in impaired Notch signaling. In addition, Catsup mutant cells exhibit elevated ER stress, suggesting that impaired zinc homeostasis causes increased levels of misfolded proteins within the secretory compartment.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Discos Imaginais/metabolismo , Transporte Proteico , Via Secretória , Zinco/metabolismo , Animais , Apoptose , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Estresse do Retículo Endoplasmático , Mutação , Receptores Notch/metabolismo , Transdução de Sinais , Asas de Animais/embriologia
3.
PLoS Genet ; 9(11): e1003917, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24244188

RESUMO

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, we recovered mutants that disrupt genes encoding serine palmitoyltransferase and acetyl-CoA carboxylase. Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EFGR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. Our analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation.


Assuntos
Diferenciação Celular/genética , Drosophila melanogaster/crescimento & desenvolvimento , Metabolismo dos Lipídeos , Transdução de Sinais/genética , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endocitose/genética , Receptores ErbB/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína Wnt1/genética
4.
Development ; 139(3): 558-67, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22190636

RESUMO

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Resposta a Proteínas não Dobradas , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/biossíntese , Retículo Endoplasmático/metabolismo , Feminino , Fucosiltransferases/biossíntese , Proteínas de Choque Térmico HSC70/metabolismo , Masculino , Neurogênese , Transdução de Sinais
5.
Semin Cell Dev Biol ; 23(4): 465-72, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22309842

RESUMO

Dysregulated Notch signaling has been implicated in numerous human diseases, including a broad spectrum of cancers. Mutations in Notch1 are prevalent in T-cell acute lymphoblastic leukemia, and abnormal expression of different human Notch receptors contributes to B-cell tumors as well as cancers of the breast, lung, pancreas, skin, prostate, colon, brain and other tissues. Several γ-secretase inhibitors, small chemical compounds that were initially developed to inhibit the activity of the γ-secretase aspartyl protease in Alzheimer's disease, are now being explored for their potential chemotherapeutic applications in Notch-associated cancers. An alternative approach involves the development of antibodies to inhibit specific Notch receptors, their activating ligands, or other components of the Notch pathway in tumors. Here we review recent progress and current challenges in the use of these strategies to modulate Notch signaling for cancer therapy.


Assuntos
Neoplasias/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Imunoterapia , Neoplasias/tratamento farmacológico , Receptores Notch/antagonistas & inibidores
6.
J Neurosci ; 30(28): 9510-22, 2010 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-20631179

RESUMO

Alzheimer's disease (AD) is the leading cause of cognitive loss and neurodegeneration in the developed world. Although its genetic and environmental causes are not generally known, familial forms of the disease (FAD) are attributable to mutations in a single copy of the Presenilin (PS) and amyloid precursor protein genes. The dominant inheritance pattern of FAD indicates that it may be attributable to gain or change of function mutations. Studies of FAD-linked forms of presenilin (psn) in model organisms, however, indicate that they are loss of function, leading to the possibility that a reduction in PS activity might contribute to FAD and that proper psn levels are important for maintaining normal cognition throughout life. To explore this issue further, we have tested the effect of reducing psn activity during aging in Drosophila melanogaster males. We have found that flies in which the dosage of psn function is reduced by 50% display age-onset impairments in learning and memory. Treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium during the aging process prevented the onset of these deficits, and treatment of aged flies reversed the age-dependent deficits. Genetic reduction of Drosophila metabotropic glutamate receptor (DmGluRA), the inositol trisphosphate receptor (InsP(3)R), or inositol polyphosphate 1-phosphatase also prevented these age-onset cognitive deficits. These findings suggest that reduced psn activity may contribute to the age-onset cognitive loss observed with FAD. They also indicate that enhanced mGluR signaling and calcium release regulated by InsP(3)R as underlying causes of the age-dependent cognitive phenotypes observed when psn activity is reduced.


Assuntos
Cognição/fisiologia , Aprendizagem/fisiologia , Memória/fisiologia , Presenilinas/genética , Fatores Etários , Análise de Variância , Animais , Animais Geneticamente Modificados , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Cognição/efeitos dos fármacos , Corte , Drosophila melanogaster , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Aprendizagem/efeitos dos fármacos , Lítio/farmacologia , Masculino , Memória/efeitos dos fármacos , Corpos Pedunculados/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Presenilinas/metabolismo , Distribuição Aleatória , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo
7.
Neuron ; 50(3): 359-75, 2006 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-16675392

RESUMO

Genetic analysis of familial Alzheimer's disease has revealed that mutations in the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however, presenilin mutations might also influence tau hyperphosphorylation and neurodegeneration through gamma-secretase-independent mechanisms. To address this possibility and determine whether other components of the gamma-secretase complex possess similar regulatory functions, we analyzed the roles of presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced neurodegeneration. Here, we show that presenilin and nicastrin prevent tau toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these transmembrane proteins differentially regulate the intracellular localization of GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity neither interfered with these kinase pathways nor induced aberrant tau phosphorylation. These results establish new in vivo molecular functions for the three components of the gamma-secretase complex and reveal a different mechanism that might contribute to neuronal degeneration in Alzheimer's disease.


Assuntos
Proteínas de Drosophila/metabolismo , Endopeptidases/metabolismo , Junções Intercelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Secretases da Proteína Precursora do Amiloide , Animais , Animais Geneticamente Modificados , Regulação para Baixo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endopeptidases/efeitos dos fármacos , Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Junções Intercelulares/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Mutação/genética , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Presenilina-1 , Proteína Quinase C/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologia , Proteínas tau/genética , Proteínas tau/toxicidade
8.
Mol Pharmacol ; 77(4): 567-74, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20064975

RESUMO

The gamma-secretase aspartyl protease is responsible for the cleavage of numerous type I integral membrane proteins, including amyloid precursor protein (APP) and Notch. APP cleavage contributes to the generation of toxic amyloid beta peptides in Alzheimer's disease, whereas cleavage of the Notch receptor is required for normal physiological signaling between differentiating cells. Mutagenesis studies as well as in vivo analyses of Notch and APP activity in the presence of pharmacological inhibitors indicate that these substrates can be differentially modulated by inhibition of mammalian gamma-secretase, although some biochemical studies instead show nearly identical dose-response inhibitor effects on Notch and APP cleavages. Here, we examine the dose-response effects of several inhibitors on Notch and APP in Drosophila melanogaster cells, which possess a homogeneous form of gamma-secretase. Four different inhibitors that target different domains of gamma-secretase exhibit similar dose-response effects for both substrates, including rank order of inhibitor potencies and effective concentration ranges. For two inhibitors, modest differences in inhibitor dose responses toward Notch and APP were detected, suggesting that inhibitors might be identified that possess some discrimination in their ability to target alternative gamma-secretase substrates. These findings also indicate that despite an overall conservation in inhibitor potencies toward different gamma-secretase substrates, quantitative differences might exist that could be relevant for the development of therapeutically valuable substrate-specific inhibitors.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Inibidores de Proteases/farmacologia , Receptores Notch/metabolismo , Secretases da Proteína Precursora do Amiloide/fisiologia , Animais , Relação Dose-Resposta a Droga , Drosophila melanogaster
9.
Differentiation ; 77(4): 424-32, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19281789

RESUMO

Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53% of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28% and 30%, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46%, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16%). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.


Assuntos
Predisposição Genética para Doença , Neoplasias Renais/genética , Tumor de Wilms/genética , Alquilantes/farmacologia , Animais , Western Blotting , Cruzamento , Etilnitrosoureia/farmacologia , Feminino , Perfilação da Expressão Gênica , Rim/efeitos dos fármacos , Neoplasias Renais/fisiopatologia , Masculino , Mutação , Ratos , Ratos Endogâmicos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas WT1/genética , Tumor de Wilms/fisiopatologia
11.
Curr Biol ; 16(10): 1026-33, 2006 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-16713961

RESUMO

To assess the potential of Drosophila to analyze clinically graded aspects of human disease, we developed a transgenic fly model to characterize Presenilin (PS) gene mutations that cause early-onset familial Alzheimer's disease (FAD). FAD exhibits a wide range in severity defined by ages of onset from 24 to 65 years . PS FAD mutants have been analyzed in mammalian cell culture, but conflicting data emerged concerning correlations between age of onset and PS biochemical activity . Choosing from over 130 FAD mutations in Presenilin-1, we introduced 14 corresponding mutations at conserved residues in Drosophila Presenilin (Psn) and assessed their biological activity in transgenic flies by using genetic, molecular, and statistical methods. Psn FAD mutant activities were tightly linked to their age-of-onset values, providing evidence that disease severity in humans primarily reflects differences in PS mutant lesions rather than contributions from unlinked genetic or environmental modifiers. Our study establishes a precedent for using transgenic Drosophila to study clinical heterogeneity in human disease.


Assuntos
Doença de Alzheimer/genética , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Proteínas de Membrana/fisiologia , Adulto , Idade de Início , Idoso , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila/genética , Proteínas de Drosophila/genética , Heterogeneidade Genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação , Presenilina-1 , Presenilinas
12.
Dev Cell ; 2(1): 69-78, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11782315

RESUMO

Nicastrin is genetically linked to Notch/lin-12 signaling in C. elegans and is part of a large multiprotein complex along with Presenilin. Here we describe the isolation and characterization of Drosophila Nicastrin (Nic) mutants. Nic mutants and tissue clones display characteristic Notch-like phenotypes. Genetic and inhibitor studies indicate a function for Nicastrin in the gamma-secretase step of Notch processing, similar to Presenilin. Further, Nicastrin is genetically required for signaling from membrane-anchored activated Notch. In the absence of Nicastrin, Presenilin is destabilized and mature C-terminal subunits are absent. Nicastrin might recruit gamma-secretase substrates into the proteolytic complex as a prerequisite for Presenilin maturation and active complex assembly.


Assuntos
Drosophila/genética , Endopeptidases/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Proteínas de Drosophila , Mutação/fisiologia , Presenilina-1 , Receptores Notch , Transdução de Sinais/fisiologia
13.
J Cell Biol ; 161(4): 685-90, 2003 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-12771124

RESUMO

The gamma-secretase complex is required for intramembrane cleavage of several integral membrane proteins, including the Notch receptor, where it generates an active signaling fragment. Four putative gamma-secretase components have been identified-presenilin (Psn), nicastrin (Nct), Aph-1, and Pen-2. Here, we use a stepwise coexpression approach to investigate the role of each new component in gamma-secretase assembly and activation. Coexpression of all four proteins leads to high level accumulation of mature Psn and increased proteolysis of Notch. Aph-1 and Nct may form a subcomplex that stabilizes the Psn holoprotein at an early step in gamma-secretase assembly. Subcomplex levels of Aph-1 are down-regulated by stepwise addition of Psn, suggesting that Aph-1 might not enter the mature complex. In contrast, Pen-2 accumulates proportionally with Psn, and is associated with Psn endoproteolysis during gamma-secretase assembly. These results demonstrate that Aph-1 and Pen-2 are essential cofactors for Psn, but that they play different roles in gamma-secretase assembly and activation.


Assuntos
Proteínas de Caenorhabditis elegans , Drosophila melanogaster/metabolismo , Endopeptidases/metabolismo , Proteínas de Homeodomínio/metabolismo , Glicoproteínas de Membrana/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Mutação , Presenilina-1 , Receptores Notch , Transdução de Sinais
14.
FASEB J ; 21(10): 2368-78, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17428965

RESUMO

It has been shown that presenilin is involved in maintaining Ca2+ homeostasis in neurons, including regulating endoplasmic reticulum (ER) Ca2+ storage. From studies of primary cultures and cell lines, however, its role in stress-induced responses is still controversial. In the present study we analyzed the effects of presenilin mutations on membrane currents and synaptic functions in response to stress using an in vivo preparation. We examined voltage-gated K+ and Ca2+ currents at the Drosophila larval neuromuscular junction (NMJ) with voltage-clamp recordings. Our data showed that both currents were generally unaffected by loss-of-function or Alzheimer's disease (AD) -associated presenilin mutations under normal or stress conditions induced by heat shock (HS) or ER stress. In larvae expressing the mutant presenilins, prolonged Ca2+ tail current, reflecting slower deactivation kinetics of Ca2+ channels, was observed 1 day after stress treatments were terminated. It was further demonstrated that the L-type Ca2+ channel was specifically affected under these conditions. Moreover, synaptic plasticity at the NMJ was reduced in larvae expressing the mutant presenilins. At the behavioral level, memory in adult flies was impaired in the presenilin mutants 1 day after HS. The results show that presenilin function is important during the poststress period and its impairment contributes to memory dysfunction observed during adaptation to normal conditions after stress. Our findings suggest a new stress-related mechanism by which presenilin may be implicated in the neuropathology of AD.


Assuntos
Drosophila/fisiologia , Junção Neuromuscular/fisiologia , Presenilinas/fisiologia , Doença de Alzheimer/fisiopatologia , Animais , Sinalização do Cálcio , Clonagem Molecular , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Heterozigoto , Homozigoto , Humanos , Larva/fisiologia , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Presenilinas/genética , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição
15.
Curr Biol ; 14(24): R1043-5, 2004 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-15620635

RESUMO

Notch signaling is regulated by ubiquitination of the receptor and its extracellular ligands. New studies reveal distinct ubiquitination-dependent endosomal sorting pathways in which ligand-bound Notch is activated while unliganded Notch is recycled or degraded, facilitating signaling while preventing inappropriate activation of unstimulated receptors.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Drosophila , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Ligantes , Receptores Notch
18.
J Alzheimers Dis ; 46(4): 913-28, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26402626

RESUMO

The amyloid-ß protein precursor (AßPP) is subjected to sequential intramembrane proteolysis by α-, ß-, andγ-secretases, producing secreted amyloid-ß (Aß) peptides and a cytoplasmically released AßPP Intracellular Domain (AICD). AICD complexes with transcription factors in the nucleus, suggesting that this AßPP fragment serves as an active signaling effector that regulates downstream genes, although its nuclear targets are poorly defined. To further understand this potential signaling mechanism mediated by AßPP, we performed a transcriptomic identification of the Drosophila genome that is regulated by the fly AßPP orthologue in fly mushroom body neurons, which control learning- and memory-based behaviors. We find significant changes in expression of 245 genes, representing approximately 1.6% of the Drosophila genome, with the changes ranging from +6 fold to -40 fold. The largest class of responsive targets corresponds to non-protein coding genes and includes microRNAs that have been previously implicated in Alzheimer's disease pathophysiology. Several genes were identified in our Drosophila microarray analyses that have also emerged as putative AßPP targets in similar mammalian transcriptomic studies. Our results also indicate a role for AßPP in cellular pathways involving the regulation of Drosophila Casein Kinase II, mitochondrial oxidative phosphorylation, RNA processing, and innate immunity. Our findings provide insights into the intracellular events that are regulated by AßPP activity in healthy neurons and that might become dysregulated as a result of abnormal AßPP proteolysis in AD.


Assuntos
Precursor de Proteína beta-Amiloide/deficiência , Regulação da Expressão Gênica/genética , Corpos Pedunculados/citologia , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Análise em Microsséries , RNA Mensageiro , Transcriptoma
19.
Mol Cell Biol ; 30(13): 3165-75, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20421416

RESUMO

The intramembrane aspartyl protease gamma-secretase plays a fundamental role in several signaling pathways involved in cellular differentiation and has been linked with a variety of human diseases, including Alzheimer's disease. Here, we describe a transgenic Drosophila model for in vivo-reconstituted gamma-secretase, based on expression of epitope-tagged versions of the four core gamma-secretase components, Presenilin, Nicastrin, Aph-1, and Pen-2. In agreement with previous cell culture and yeast studies, coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase. We demonstrate that in vivo-reconstituted gamma-secretase has biochemical properties and a subcellular distribution resembling those of endogenous gamma-secretase. However, analysis of the cleavage of alternative substrates in transgenic-fly assays revealed unexpected functional differences in the activity of reconstituted gamma-secretase toward different substrates, including markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor. These findings indicate that in vivo under physiological conditions, additional factors differentially modulate the activity of gamma-secretase toward its substrates. Thus, our approach for the first time demonstrates the overall functionality of reconstituted gamma-secretase in a multicellular organism and the requirement for substrate-specific factors for efficient in vivo cleavage of certain substrates.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Drosophila melanogaster/enzimologia , Secretases da Proteína Precursora do Amiloide/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fenótipo , Presenilinas/genética , Presenilinas/metabolismo , Transdução de Sinais/fisiologia , Especificidade por Substrato/genética
20.
Dev Cell ; 16(5): 633-47, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19460341

RESUMO

Notch signaling controls numerous cell-fate specification events in multicellular organisms, and dysregulated Notch signaling causes several diseases with underlying developmental defects. A key step in Notch receptor activation is its intramembrane proteolysis, which releases an intracellular fragment that participates directly in transcriptional regulation of nuclear target genes. Despite the apparent simplicity of this mechanism, a host of posttranslational processes regulate Notch activity during its synthesis and secretion, ligand-dependent activation at the surface, endocytic trafficking, and degradation. This review describes the core developmental logic of Notch signaling and how regulatory mechanisms tailor Notch pathway outputs to specific developmental scenarios.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Vesículas Citoplasmáticas/metabolismo , Endocitose , Processamento de Proteína Pós-Traducional
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