Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
ACS Chem Biol ; 15(12): 3149-3158, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33206504

RESUMO

There is a growing interest in using targeted protein degradation as a therapeutic modality in view of its potential to expand the druggable proteome. One avenue to using this modality is via molecular glue based Cereblon E3 Ligase Modulating Drug compounds. Here, we report the identification of the transcription factor ZBTB16 as a Cereblon neosubstrate. We also report two new Cereblon modulators, CC-3060 and CC-647, that promote ZBTB16 degradation. Unexpectedly, CC-3060 and CC-647 target ZBTB16 for degradation by primarily engaging distinct structural degrons on different zinc finger domains. The reciprocal fusion proteins, ZBTB16-RARα and RARα-ZBTB16, which cause a rare acute promyelocytic leukemia, contain these same structural degrons and can be targeted for proteasomal degradation with Cereblon modulator treatment. Thus, a targeted protein degradation approach via Cereblon modulators may represent a novel therapeutic strategy in acute promyelocytic leukemia where ZBTB16/RARA rearrangements are critical disease drivers.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Leucemia Promielocítica Aguda/metabolismo , Proteólise , Receptor alfa de Ácido Retinoico/metabolismo , Especificidade por Substrato
2.
Mol Neurodegener ; 12(1): 51, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28668092

RESUMO

BACKGROUND: Given multiple studies of brain microRNA (miRNA) in relation to Alzheimer's disease (AD) with few consistent results and the heterogeneity of this disease, the objective of this study was to explore their mechanism by evaluating their relation to different elements of Alzheimer's disease pathology, confounding factors and mRNA expression data from the same subjects in the same brain region. METHODS: We report analyses of expression profiling of miRNA (n = 700 subjects) and lincRNA (n = 540 subjects) from the dorsolateral prefrontal cortex of individuals participating in two longitudinal cohort studies of aging. RESULTS: We confirm the association of two well-established miRNA (miR-132, miR-129) with pathologic AD in our dataset and then further characterize this association in terms of its component neuritic ß-amyloid plaques and neurofibrillary tangle pathologies. Additionally, we identify one new miRNA (miR-99) and four lincRNA that are associated with these traits. Many other previously reported associations of microRNA with AD are associated with the confounders quantified in our longitudinal cohort. Finally, by performing analyses integrating both miRNA and RNA sequence data from the same individuals (525 samples), we characterize the impact of AD associated miRNA on human brain expression: we show that the effects of miR-132 and miR-129-5b converge on certain genes such as EP300 and find a role for miR200 and its target genes in AD using an integrated miRNA/mRNA analysis. CONCLUSIONS: Overall, miRNAs play a modest role in human AD, but we observe robust evidence that a small number of miRNAs are responsible for specific alterations in the cortical transcriptome that are associated with AD.


Assuntos
Doença de Alzheimer/metabolismo , Emaranhados Neurofibrilares/metabolismo , Placa Amiloide/metabolismo , RNA não Traduzido/metabolismo , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Amiloidogênicas/metabolismo , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Placa Amiloide/patologia , Proteínas tau/metabolismo
3.
Mol Ther ; 23(7): 1234-1247, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25903473

RESUMO

Using in silico analysis of The Cancer Genome Atlas (TCGA), we identified microRNAs associated with glioblastoma (GBM) survival, and predicted their functions in glioma growth and progression. Inhibition of two "risky" miRNAs, miR-148a and miR-31, in orthotopic xenograft GBM mouse models suppressed tumor growth and thereby prolonged animal survival. Intracranial tumors treated with uncomplexed miR-148a and miR-31 antagomirs exhibited reduced proliferation, stem cell depletion, and normalized tumor vasculature. Growth-promoting functions of these two miRNAs were, in part, mediated by the common target, the factor inhibiting hypoxia-inducible factor 1 (FIH1), and the downstream pathways involving hypoxia-inducible factor HIF1α and Notch signaling. Therefore, miR-31 and miR-148a regulate glioma growth by maintaining tumor stem cells and their niche, and providing the tumor a way to activate angiogenesis even in a normoxic environment. This is the first study that demonstrates intratumoral uptake and growth-inhibiting effects of uncomplexed antagomirs in orthotopic glioma.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , MicroRNAs/biossíntese , Oligonucleotídeos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Camundongos , MicroRNAs/antagonistas & inibidores , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
PLoS One ; 9(4): e93891, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24705917

RESUMO

In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.


Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/genética , Agregação Patológica de Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição/genética , Animais , Ensaios de Triagem em Larga Escala , Proteína Huntingtina , Camundongos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Patológica de Proteínas/metabolismo , Fatores de Transcrição/metabolismo
5.
Angiogenesis ; 17(2): 419-27, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24201897

RESUMO

Radiation therapy after lymph node dissection increases the risk of developing painful and incurable lymphedema in breast cancer patients. Lymphedema occurs when lymphatic vessels become unable to maintain proper fluid balance. The sensitivity of lymphatic endothelial cells (LECs) to ionizing radiation has not been reported to date. Here, the radiosensitivity of LECs in vitro has been determined using clonogenic survival assays. The ability of various growth factors to alter LEC radiosensitivity was also examined. Vascular endothelial growth factor (VEGF)-C enhanced radiosensitivity when LECs were treated prior to radiation. VEGF-C-treated LECs exhibited higher levels of entry into the cell cycle at the time of radiation, with a greater number of cells in the S and G2/M phases. These LECs showed higher levels of γH2A.X-an indicator of DNA damage-after radiation. VEGF-C did not increase cell death as a result of radiation. Instead, it increased the relative number of quiescent LECs. These data suggest that abundant VEGF-C or lymphangiogenesis may predispose patients to radiation-induced lymphedema by impairing lymphatic vessel repair through induction of LEC quiescence.


Assuntos
Células Endoteliais/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/farmacologia , Adulto , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Células Endoteliais/efeitos dos fármacos , Humanos , Linfangiogênese/efeitos dos fármacos , Substâncias Protetoras/farmacologia
6.
Hum Mol Genet ; 22(15): 3077-92, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23585551

RESUMO

Alzheimer's disease (AD) is a multifactorial and fatal neurodegenerative disorder for which the mechanisms leading to profound neuronal loss are incompletely recognized. MicroRNAs (miRNAs) are recently discovered small regulatory RNA molecules that repress gene expression and are increasingly acknowledged as prime regulators involved in human brain pathologies. Here we identified two homologous miRNAs, miR-132 and miR-212, downregulated in temporal cortical areas and CA1 hippocampal neurons of human AD brains. Sequence-specific inhibition of miR-132 and miR-212 induces apoptosis in cultured primary neurons, whereas their overexpression is neuroprotective against oxidative stress. Using primary neurons and PC12 cells, we demonstrate that miR-132/212 controls cell survival by direct regulation of PTEN, FOXO3a and P300, which are all key elements of AKT signaling pathway. Silencing of these three target genes by RNAi abrogates apoptosis caused by the miR-132/212 inhibition. We further demonstrate that mRNA and protein levels of PTEN, FOXO3a, P300 and most of the direct pro-apoptotic transcriptional targets of FOXO3a are significantly elevated in human AD brains. These results indicate that the miR-132/miR-212/PTEN/FOXO3a signaling pathway contributes to AD neurodegeneration.


Assuntos
Doença de Alzheimer/genética , Apoptose/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Regulação para Baixo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Interferência de RNA , Ratos , Transdução de Sinais , Fatores de Transcrição de p300-CBP/metabolismo
7.
Neoplasia ; 14(2): 84-94, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22431917

RESUMO

Neurofibromatosis type 2 (NF2) is an autosomal-dominant multiple neoplasia syndrome that results from mutations in the NF2 tumor suppressor gene. Patients with NF2 develop hallmark schwannomas that require surgery or radiation, both of which have significant adverse effects. Recent studies have indicated that the tumor microenvironment-in particular, tumor blood vessels-of schwannomas may be an important therapeutic target. Furthermore, although much has been done to understand how merlin, the NF2 gene product, functions as a tumor suppressor gene in schwannoma cells, the functional role of merlin in the tumor microenvironment and the mechanism(s) by which merlin regulates angiogenesis to support schwannoma growth is largely unexplored. Here we report that the expression of semaphorin 3F (SEMA3F) was specifically downregulated in schwannoma cells lacking merlin/NF2. When we reintroduced SEMA3F in schwannoma cells, we observed normalized tumor blood vessels, reduced tumor burden, and extended survival in nude mice bearing merlin-deficient brain tumors. Next, using chemical inhibitors and gene knockdown with RNA interference, we found that merlin regulated expression of SEMA3F through Rho GTPase family member Rac1. This study shows that, in addition to the tumor-suppressing activity of merlin, it also functions to maintain physiological angiogenesis in the nervous system by regulating antiangiogenic factors such as SEMA3F. Restoring the relative balance of proangiogenic and antiangiogenic factors, such as increases in SEMA3F, in schwannoma microenvironment may represent a novel strategy to alleviate the clinical symptoms of NF2-related schwannomas.


Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Proteínas de Membrana/metabolismo , Neovascularização Patológica , Proteínas do Tecido Nervoso/metabolismo , Neurilemoma/irrigação sanguínea , Neurofibromina 2/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Estimativa de Kaplan-Meier , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas do Tecido Nervoso/genética , Neurilemoma/metabolismo , Neurilemoma/patologia , Neurofibromatose 2/metabolismo , Neurofibromatose 2/patologia , Neurofibromina 2/genética , Permeabilidade , Transdução de Sinais , Trombospondinas/genética , Trombospondinas/metabolismo
8.
Proc Natl Acad Sci U S A ; 108(28): 11596-601, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21709229

RESUMO

Antivascular agents have become a standard of treatment for many malignancies. However, most of them target the VEGF pathway and lead to refractoriness. To improve the diversity of options for antivascular therapy, we applied a high-throughput screen for small molecules targeting cell adhesion. We then assayed the resulting antiadhesion hits in a transgenic zebrafish line with endothelial expression of EGFP (Tg(fli1:EGFP)(y1)) to identify nontoxic molecules with antivascular activity selective to neovasculature. This screen identified dehydro-α-lapachone (DAL), a natural plant product. We found that DAL inhibits vessel regeneration, interferes with vessel anastomosis, and limits plexus formation in zebrafish. Furthermore, DAL induces vascular pruning and growth delay in orthotopic mammary tumors in mice. We show that DAL targets cell adhesion by promoting ubiquitination of the Rho-GTPase Rac1, which is frequently up-regulated in many different cancers.


Assuntos
Inibidores da Angiogênese/farmacologia , Naftoquinonas/farmacologia , Inibidores da Angiogênese/isolamento & purificação , Animais , Animais Geneticamente Modificados , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde/genética , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos SCID , Naftoquinonas/isolamento & purificação , Plantas Medicinais/química , Tabebuia/química , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
9.
Cancer Res ; 70(9): 3483-93, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20406973

RESUMO

Patients with bilateral vestibular schwannomas associated with neurofibromatosis type 2 (NF2) experience significant morbidity such as complete hearing loss. We have recently shown that treatment with bevacizumab provided tumor stabilization and hearing recovery in a subset of NF2 patients with progressive disease. In the current study, we used two animal models to identify the mechanism of action of anti-vascular endothelial growth factor (VEGF) therapy in schwannomas. The human HEI193 and murine Nf2(-/-) cell lines were implanted between the pia and arachnoid meninges as well as in the sciatic nerve to mimic central and peripheral schwannomas. Mice were treated with bevacizumab (10 mg/kg/wk i.v.) or vandetanib (50 mg/kg/d orally) to block the VEGF pathway. Using intravital and confocal microscopy, together with whole-body imaging, we measured tumor growth delay, survival rate, as well as blood vessel structure and function at regular intervals. In both models, tumor vessel diameter, length/surface area density, and permeability were significantly reduced after treatment. After 2 weeks of treatment, necrosis in HEI193 tumors and apoptosis in Nf2(-/-) tumors were significantly increased, and the tumor growth rate decreased by an average of 50%. The survival of mice bearing intracranial schwannomas was extended by at least 50%. This study shows that anti-VEGF therapy normalizes the vasculature of schwannoma xenografts in nude mice and successfully controls the tumor growth, probably by reestablishing a natural balance between VEGF and semaphorin 3 signaling.


Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neurilemoma/tratamento farmacológico , Piperidinas/farmacologia , Quinazolinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Bevacizumab , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neurilemoma/irrigação sanguínea , Neurilemoma/genética , Neurofibromatose 2/tratamento farmacológico , Neurofibromatose 2/genética , Neurofibromina 2/deficiência , Neurofibromina 2/genética
10.
Nat Biotechnol ; 28(3): 256-63, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20190739

RESUMO

Huntington's Disease (HD) is a dominantly inherited pathology caused by the accumulation of mutant huntingtin protein (HTT) containing an expanded polyglutamine (polyQ) tract. As the polyglutamine binding peptide 1 (QBP1) is known to bind an expanded polyQ tract but not the polyQ motif found in normal HTT, we selectively targeted mutant HTT for degradation by expressing a fusion molecule comprising two copies of QBP1 and copies of two different heat shock cognate protein 70 (HSC70)-binding motifs in cellular and mouse models of HD. Chaperone-mediated autophagy contributed to the specific degradation of mutant HTT in cultured cells expressing the construct. Intrastriatal delivery of a virus expressing the fusion molecule ameliorated the disease phenotype in the R6/2 mouse model of HD. Similar adaptor molecules comprising HSC70-binding motifs fused to an appropriate structure-specific binding agent(s) may have therapeutic potential for treating diseases caused by misfolded proteins other than those with expanded polyQ tracts.


Assuntos
Autofagia/fisiologia , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Motivos de Aminoácidos , Animais , Dependovirus/genética , Modelos Animais de Doenças , Proteínas de Choque Térmico HSC70/genética , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Ratos , Proteínas Recombinantes de Fusão/genética
11.
J Biol Chem ; 284(19): 13153-64, 2009 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-19278999

RESUMO

Huntington disease (HD) is a fatal hereditary neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Whereas the pathological significance of the expanded polyQ has been clearly established and a tremendous effort to develop therapeutic tools for HD has been exerted, there is yet no effective cure. Whereas many molecules able to reduce the polyQ accumulation and aggregation have been identified, including several Rho kinase (ROCK) inhibitors, it remains very important to determine the mechanism of action of the potential drugs. ROCK inhibitors, including Y-27632 were reported to decrease aggregation of htt and androgen receptor (AR) through ROCK1 and protein kinase C-related protein kinase-2 (PRK-2). A downstream effector of ROCK1, actin-binding factor profilin, was shown to inhibit the mutant htt aggregation but not AR by direct interaction. We found that the anti-aggregation effect of ROCK inhibitors was not limited to the mutant htt and AR and that Y-27632 was also able to reduce the aggregation of ataxin-3 and atrophin-1 with expanded polyQ. These results suggested that in addition to the mechanism reported for htt and AR, there might also be other common mediators involved in the reduced aggregation of different polyQ proteins. In this study, we show that Y-27632 not only reduced the mutant htt aggregation by enhancing its degradation, but surprisingly was able to activate the main cellular degradation pathways, proteasome, and macroautophagy. We also show that this unique effect was mediated by ROCK1 and ROCK2.


Assuntos
Inibidores Enzimáticos/farmacologia , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Amidas/farmacologia , Animais , Ataxina-3 , Autofagia , Western Blotting , Morte Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína Huntingtina , Camundongos , Proteínas do Tecido Nervoso/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Nucleares/genética , Profilinas/genética , Profilinas/metabolismo , Ligação Proteica , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Hum Mol Genet ; 17(20): 3223-35, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18658163

RESUMO

Huntington's disease (HD) is a fatal neurodegenerative disorder. Despite a tremendous effort to develop therapeutic tools in several HD models, there is no effective cure at present. Acidosis has been observed previously in cellular and in in vivo models as well as in the brains of HD patients. Here we challenged HD models with amiloride (Ami) derivative benzamil (Ben), a chemical agent used to rescue acid-sensing ion channel (ASIC)-dependent acidotoxicity, to examine whether chronic acidosis is an important part of the HD pathomechanism and whether these drugs could be used as novel therapeutic agents. Ben markedly reduced the huntingtin-polyglutamine (htt-polyQ) aggregation in an inducible cellular system, and the therapeutic value of Ben was successfully recapitulated in the R6/2 animal model of HD. To reveal the mechanism of action, Ben was found to be able to alleviate the inhibition of the ubiquitin-proteasome system (UPS) activity, resulting in enhanced degradation of soluble htt-polyQ specifically in its pathological range. More importantly, we were able to demonstrate that blocking the expression of a specific isoform of ASIC (asic1a), one of the many molecular targets of Ben, led to an enhancement of UPS activity and this blockade also decreased htt-polyQ aggregation in the striatum of R6/2 mice. In conclusion, we believe that chemical compounds that target ASIC1a or pharmacological alleviation of UPS inhibition would be an effective and promising approach to combat HD and other polyQ-related disorders.


Assuntos
Doença de Huntington/tratamento farmacológico , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais Iônicos Sensíveis a Ácido , Adulto , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Proteínas do Tecido Nervoso/genética , Peptídeos/química , Peptídeos/genética , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/genética , Solubilidade
13.
Biochem Biophys Res Commun ; 361(1): 43-8, 2007 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-17644063

RESUMO

Processing of APP by BACE1 plays a crucial role in the pathogenesis of Alzheimer disease (AD). Recently, the voltage-gated sodium channel (Na(v)) beta4 subunit (beta4), an auxiliary subunit of Na(v) that is supposed to serve as a cell adhesion molecule, has been identified as a substrate for BACE1. However, the biological consequence of BACE1 processing of beta4 remains illusive. Here, we report the biological effects of beta4 processing by BACE1. Overexpression of beta4 in Neuro2a cells promoted neurite extension and increased the number of F-actin rich filopodia-like protrusions. While coexpression of BACE1 together with beta4 further accelerated neurite extension, the number of filopodia-like protrusions was reduced. Overexpression of C-terminal fragment of beta4 that was generated by BACE1 (beta4-CTF) partially recapitulated the results obtained with BACE1 overexpression. These results suggest that the processing of beta4 by BACE1 regulates neurite length and filopodia-like protrusion density in neurons.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Neuritos/ultraestrutura , Pseudópodes/ultraestrutura , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Camundongos , Dados de Sequência Molecular , Canais de Sódio/química , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem
14.
Mol Cell Biol ; 25(19): 8732-47, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16166651

RESUMO

The c-Jun N-terminal protein kinase (JNK)/c-Jun and p53 pathways form distinct death-signaling modules in neurons that culminate in Bax-dependent apoptosis. To investigate whether this signaling autonomy is due to recruitment of particular BH3-only proteins, we searched for a toxic signal that would activate both pathways in the same set of neurons. We show that arsenite activates both the JNK/c-Jun and p53 pathways in cortical neurons, which together account for >95% of apoptosis, as determined by using the mixed-lineage kinase (JNK/c-Jun) pathway inhibitor CEP11004 and p53-null mice. Despite the coexistence of both pathways in at least 30% of the population, Bim mRNA and protein expression was increased only by the JNK/c-Jun signaling pathway, whereas Noxa and Puma mRNA and Puma protein expression was entirely JNK/c-Jun independent. About 50% of Puma/Noxa expression was p53 dependent, with the remaining signal being independent of both pathways and possibly facilitated by arsenite-induced reduction in P-Akt. However, functionally, Puma was predominant in mediating Bax-dependent apoptosis, as evidenced by the fact that more than 90% of apoptosis was prevented in Puma-null neurons, although Bim was still upregulated, while Bim- and Noxa-null neurons died similarly to wild-type neurons. Thus, the p53 and JNK/c-Jun pathways can activate mutually exclusive subclasses of BH3-only proteins in the same set of neurons. However, other factors besides expression may determine which BH3-only proteins mediate apoptosis.


Assuntos
Arsenitos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Morte Celular , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Frações Subcelulares , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
15.
J Biol Chem ; 280(24): 23009-17, 2005 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-15824102

RESUMO

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.


Assuntos
Endopeptidases/química , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/química , Animais , Ácido Aspártico Endopeptidases , Sítios de Ligação , Western Blotting , Encéfalo/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Detergentes/farmacologia , Endopeptidases/metabolismo , Fibroblastos/metabolismo , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Dados de Sequência Molecular , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção
16.
J Neurochem ; 93(3): 641-53, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15836623

RESUMO

Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the down-regulation of specific genes in this region of the brain.


Assuntos
Regulação para Baixo/genética , Proteínas de Fluorescência Verde/genética , Doença de Huntington/metabolismo , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/antagonistas & inibidores , Proteínas Nucleares/genética , Peptídeos/metabolismo , Animais , Química Encefálica/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Hipotálamo/química , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/metabolismo , Ocitocina/antagonistas & inibidores , Ocitocina/biossíntese , Ocitocina/genética , Peptídeos/genética , Regiões Promotoras Genéticas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Vasopressinas/antagonistas & inibidores , Vasopressinas/biossíntese , Vasopressinas/genética
17.
Plant Physiol ; 134(1): 332-8, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14671018

RESUMO

In Arabidopsis, asparagine (Asn) synthetase is encoded by a small gene family (ASN1, ASN2, and ASN3). It has been shown that ASN1 and ASN2 exhibit reciprocal gene expression patterns toward light and metabolites. Moreover, changes in total free Asn levels parallel the expression of ASN1, but not ASN2. In this study, we show that ASN2 expression correlates with ammonium metabolism. We demonstrate that the light induction of ASN2 is ammonium dependent. The addition and removal of ammonium exerted fast and reciprocal effects on the levels of ASN2 mRNA, specifically under light-grown conditions. NaCl and cold stress increased cellular free ammonium and ASN2 mRNA levels in a coordinated manner, suggesting that the effects of stress on ASN2 expression may be mediated via accumulation of ammonium. The correlation between ASN2 and cellular ammonium metabolism was further demonstrated by analysis of ASN2 transgenic plants. When plants were grown on Murashige and Skoog medium containing 50 mm ammonium, ASN2 overexpressors accumulated less endogenous ammonium compared with the wild-type Colombia-0 and ASN2 underexpressors. When plants were subjected to high-light irradiance, ammonium levels built up. Under such conditions, ASN2 underexpressors accumulated more endogenous ammonium than the wild-type Colombia-0 and ASN2 overexpressors. These results support the notion that ASN2 is closely correlated to ammonium metabolism in higher plants.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Genes de Plantas , Compostos de Amônio Quaternário/metabolismo , Arabidopsis/efeitos da radiação , Expressão Gênica/efeitos da radiação , Luz , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo
18.
Invest Ophthalmol Vis Sci ; 44(10): 4451-6, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14507892

RESUMO

PURPOSE: Recently, a novel N-methyl-D-aspartate receptor (NMDAR) subunit, NR3A, has been discovered in the brain and shown to decrease NMDAR activity by modulating the calcium permeability of the receptor channel. The insertion of NR3A within the NMDAR complex may thus alter NMDAR properties and play a crucial role during processes of neuronal development and degeneration. The present study is the first to investigate the expression and cellular localization of NR3A on the protein level in the retina and to elucidate its putative functional roles within the retinal circuitry. METHODS: The expression of NR3A in the retina was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. Functional aspects of NR3A in the retina were addressed by measuring the NMDA-induced increase in intracellular calcium, [Ca(2+)](i), in retinal cells prepared from wild-type (NR3A(+/+)) and NR3A knockout (NR3A(+/-), and NR3A(-/-)) mice. RESULTS: NR3A protein expression was initially observed in the first postnatal week and was predominantly localized to cell bodies in the ganglion cell layer. In older animals, two bands of NR3A immunoreactivity were additionally observed in the inner plexiform layer. NMDA-evoked [Ca(2+)](i) responses were found to be significantly greater in retinal cells in NR3A(-/-) mice than in wild-type retinas. CONCLUSIONS: The data indicate that NR3A is specifically expressed in the inner retina and may modulate NMDAR-mediated calcium influx and thus [Ca(2+)](i) levels in retinal ganglion cells and amacrine cells.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Metilaspartato/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Retina/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
J Comp Neurol ; 450(4): 303-17, 2002 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-12209845

RESUMO

NR3A is a developmentally regulated N-methyl-D-aspartate receptor (NMDAR) subunit that was previously known as NMDAR-L or chi-1. Unlike other NMDAR subunits, NR3A inhibits the NMDAR-associated ion channel in a novel manner, and a role in synaptogenesis has been suggested for this subunit. Here, we report a comprehensive study to delineate the temporal and anatomic expression of NR3A protein in the mammalian brain by using a monoclonal anti-NR3A antibody. NR3A protein was found to peak at postnatal day (P) 8, and to decrease gradually from P12 to adulthood in the rat central nervous system. Moreover, NR3A protein was heavily expressed in all areas of the isocortex, portions of the amygdaloid nuclei, and selective cell layers and nuclei of the hippocampus, thalamus, hypothalamus, brainstem, and spinal cord. NR3A protein was also expressed in the cerebellar cortex, whereas only weak signal was detected in the previous in situ studies by using riboprobes. At an ultrastructural level, NR3A was associated specifically with asymmetrical synapses and localized to postsynaptic membranes. This information will facilitate future research on NMDARs by providing clues to possible inclusion of the NR3A subunit in NMDARs in many brain regions.


Assuntos
Encéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/biossíntese , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Células Cultivadas , Vértebras Cervicais/metabolismo , Humanos , Imuno-Histoquímica , Rim/metabolismo , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de N-Metil-D-Aspartato/química , Medula Espinal/citologia , Medula Espinal/metabolismo
20.
J Neurophysiol ; 87(4): 2052-63, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11929923

RESUMO

Recently, we cloned and began to characterize a new N-methyl-D-aspartate receptor (NMDAR) subunit, NR3A. Here we extend our earlier findings by showing that recombinantly expressed NR3A in COS cells is biochemically associated with both NR1 and NR2 subunits. In the oocyte or HEK 293 cell expression systems, co-injection of NR3A with NR1/NR2 subunits acts in a dominant-interfering manner, resulting in a decrease in NMDAR unitary conductance, decrease in Ca(2+) permeability, decrease in Mg(2+) sensitivity, and slight increase in mean open time compared with NR1/NR2 channels. The smaller unitary conductance channel has also been observed in primary cortical neurons cultured from wild-type rodent on postnatal day 8 (P8) and similarly found to be relatively insensitive to Mg(2+) block. Consistent with these findings, whole cell NMDA-evoked currents are larger in NR3A-deficient mice compared with wild-type mice, and this effect follows a developmental pattern similar to that of NR3A protein expression on Western blots, with peak expression at P8. Finally, a new longer splice variant of NR3A has been cloned and found to be expressed in rodent cortical neurons by single-cell RT-PCR and in situ hybridization.


Assuntos
Córtex Cerebral/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Processamento Alternativo , Animais , Células COS , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Condutividade Elétrica , Feminino , Immunoblotting , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Magnésio/farmacologia , Camundongos , Oócitos , Permeabilidade , Testes de Precipitina , Subunidades Proteicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...