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1.
Cells ; 11(12)2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35740989

RESUMO

Alzheimer's disease (AD) is the most prevalent age-associated neurodegenerative disease. A decrease in autophagy during aging contributes to brain disorders by accumulating potentially toxic substrates in neurons. Rubicon is a well-established inhibitor of autophagy in all cells. However, Rubicon participates in different pathways depending on cell type, and little information is currently available on neuronal Rubicon's role in the AD context. Here, we investigated the cell-specific expression of Rubicon in postmortem brain samples from AD patients and 5xFAD mice and its impact on amyloid ß burden in vivo and neuroblastoma cells. Further, we assessed Rubicon levels in human-induced pluripotent stem cells (hiPSCs), derived from early-to-moderate AD and in postmortem samples from severe AD patients. We found increased Rubicon levels in AD-hiPSCs and postmortem samples and a notable Rubicon localization in neurons. In AD transgenic mice lacking Rubicon, we observed intensified amyloid ß burden in the hippocampus and decreased Pacer and p62 levels. In APP-expressing neuroblastoma cells, increased APP/amyloid ß secretion in the medium was found when Rubicon was absent, which was not observed in cells depleted of Atg5, essential for autophagy, or Rab27a, required for exosome secretion. Our results propose an uncharacterized role of Rubicon on APP/amyloid ß homeostasis, in which neuronal Rubicon is a repressor of APP/amyloid ß secretion, defining a new way to target AD and other similar diseases therapeutically.


Assuntos
Doença de Alzheimer , Proteínas Relacionadas à Autofagia , Neuroblastoma , Doenças Neurodegenerativas , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Neuroblastoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo
2.
Mol Psychiatry ; 27(9): 3708-3718, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35705634

RESUMO

Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. Long-standing clinical observations and molecular data have pointed to a possible vascular deficiency that could be acting synergistically with neuronal dysfunction in SZ. As SZ is a neurodevelopmental disease, the use of human-induced pluripotent stem cells (hiPSC) allows disease biology modeling while retaining the patient's unique genetic signature. Previously, we reported a VEGFA signaling impairment in SZ-hiPSC-derived neural lineages leading to decreased angiogenesis. Here, we present a functional characterization of SZ-derived brain microvascular endothelial-like cells (BEC), the counterpart of the neurovascular crosstalk, revealing an intrinsically defective blood-brain barrier (BBB) phenotype. Transcriptomic assessment of genes related to endothelial function among three control (Ctrl BEC) and five schizophrenia patients derived BEC (SZP BEC), revealed that SZP BEC have a distinctive expression pattern of angiogenic and BBB-associated genes. Functionally, SZP BEC showed a decreased angiogenic response in vitro and higher transpermeability than Ctrl BEC. Immunofluorescence staining revealed less expression and altered distribution of tight junction proteins in SZP BEC. Moreover, SZP BEC's conditioned media reduced barrier capacities in the brain microvascular endothelial cell line HCMEC/D3 and in an in vivo permeability assay in mice. Overall, our results describe an intrinsic failure of SZP BEC for proper barrier function. These findings are consistent with the hypothesis tracing schizophrenia origins to brain development and BBB dysfunction.


Assuntos
Células-Tronco Pluripotentes Induzidas , Esquizofrenia , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Barreira Hematoencefálica/metabolismo , Esquizofrenia/metabolismo , Encéfalo , Linhagem Celular
3.
Cells ; 11(5)2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35269421

RESUMO

Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4-7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2-3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.


Assuntos
Células-Tronco Pluripotentes Induzidas , Técnicas de Cultura de Células , Criopreservação , Células Alimentadoras , Congelamento
4.
Neurobiol Aging ; 64: 123-138, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29458840

RESUMO

The occurrence of mutations of TDP-43, FUS, and C9ORF72 in amyotrophic lateral sclerosis (ALS) suggests pathogenic alterations to RNA metabolism and specifically to microRNA (miRNA) biology. Moreover, several ALS-related proteins impact stress granule dynamics affecting miRNA biogenesis and cellular miRNA levels. miRNAs are present in different biological fluids and have been proposed as potential biomarkers. Here we used next-generation sequencing to perform a comparative analysis of the expression profile of circulating miRNAs in the serum of 2 mutant superoxide dismutase 1 transgenic mice. Top hit candidates were then validated using quantitative real-time polymerase chain reaction, confirming significant changes for 6 miRNAs. In addition, one of these miRNAs was also altered in mutant TDP-43 mice. Then, we tested this set of miRNAs in the serum from sporadic ALS patients, observing a significant deregulation of hsa-miR-142-3p and hsa-miR-1249-3p. A negative correlation between the revised ALS functional rating scale and hsa-miR-142-3p levels was found. Bioinformatics analysis of the regulatory network governed by hsa-miR-142-3p identified TDP-43 and C9orf72 as possible targets, suggesting a connection with ALS pathogenesis. This study identifies miRNAs that are altered in ALS that may serve as potentials biomarkers.


Assuntos
Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/genética , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Estudo de Associação Genômica Ampla , Transcriptoma/genética , Adulto , Idoso , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Transgênicos
6.
Biochem J ; 473(11): 1563-78, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27029347

RESUMO

Amyloid beta (Aß) peptides, in particular Aß42 and Aß40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target for treatments of Alzheimer's disease (AD). We describe the engineering of cognate Anticalins as a novel type of neutralizing protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 (Lcn2) with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulted in three different Anticalins. Biochemical characterization of the purified proteins produced by periplasmic secretion in Escherichia coli revealed high folding stability in a monomeric state, with Tm values ranging from 53.4°C to 74.5°C, as well as high affinities for Aß40, between 95 pM and 563 pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Aß sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability-with varying extent-to inhibit Aß aggregation in vitro according to the thioflavin-T fluorescence assay and, furthermore, they abolished Aß42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of AD but they also show potential as alternative drug candidates compared with antibodies.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Lipocalinas/química , Engenharia de Proteínas/métodos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Lipocalinas/farmacologia , Lipocalinas/uso terapêutico , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica
7.
Neurobiol Aging ; 33(4): 831.e21-31, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21920632

RESUMO

Inflammation is believed to be integral to the pathogenesis of Alzheimer's disease (AD). Arachidonic acid (AA) is the most important omega-6 fatty acid and a mediator of inflammatory pathways. High-sensitivity enzyme linked immunosorbent assay shows that AA and its various metabolites; prostaglandins, thromboxanes, and leukotriene B4 resulted in significantly higher secretion of both Abeta40 and 42 peptides. A combination of identical number of alternate cis and trans double bonds either at positions Δ5 or 7Z,13 or 15E (such as PGE(2), PGF(2α), THXB2 and PGF(2α)EA) or at positions Δ6Z,8E,10E,14Z (such as LB4) built in the 3-dimensional structure of 20-carbon fatty acyl chains believed to be responsible for their detrimental action. CP 24,879 and sesamin, 2 inhibitors of the AA pathway suppressed the production of amyloid-beta (Aß) peptides. Immunoblotting experiments and use of SP-C99 transfected COS-7 cells suggested that AA and its metabolites-driven altered production of Aß is mediated through gamma-secretase cleavage of amyloid precursor protein (APP). An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings by showing higher levels of Abeta and amyloid plaques in the brains, when they were fed chow supplemented with 2% AA. Our work not only supports that AA and its metabolites are involved in the production of Aß and in the pathogenesis of AD but also contributes to clarify aspects of structure-activity relationship helpful for future nonsteroidal anti-inflammatory drugs (NSAIDs) research.


Assuntos
Doença de Alzheimer/dietoterapia , Ácido Araquidônico/administração & dosagem , Ácido Araquidônico/metabolismo , Suplementos Nutricionais , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Araquidônico/química , Vias Biossintéticas/efeitos dos fármacos , Biotinilação , Células COS/efeitos dos fármacos , Células COS/metabolismo , Moduladores de Receptores de Canabinoides/farmacologia , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia , Prostaglandinas/farmacologia , Tromboxanos/farmacologia , Transfecção
8.
J Neurosci Res ; 89(11): 1795-801, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21748777

RESUMO

Several studies have shown the protective and/or deleterious effects of dietary enrichment of single fatty acids (FAs) in several animal and cell-culture models of Alzheimer's disease (AD). However, potential interactions among dietary fatty acids are traditionally ignored. None of these studies has examined and compared the differential effects of FAs in combination, as well as alone, for their effects on amyloid ß production or AD. Here we investigated the effects of omega-9 (oleic acid) and omega-6 (linoleic and arachidonic acids) fatty acids, either alone or combined, on Aß production by APP-695 and SP-C99 transfected COS-7 cells. Overall, our results are the first to demonstrate that mixtures of FAs alter the production of Aß40 and Aß42 peptides and consequently the Aß40:42 ratio differently from individual FAs. Here we show that the effects of a single lipid on Aß production are not attributed to that single FA alone. Rather, the overall lipid composition influences the specificity and level of the regulated intramembranous proteolysis of APP by the γ-secretase complex. Our results reinforce the importance of studying composite lipids/nutrients rather than single lipids or nutrients.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Ácidos Graxos/farmacologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Animais , Ácido Araquidônico/farmacologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Ácido Linoleico/farmacologia , Ácido Oleico/farmacologia
9.
J Biol Chem ; 286(8): 6100-7, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-20971855

RESUMO

Several studies have shown the protective effects of dietary enrichment of various lipids in several late-onset animal models of Alzheimer Disease (AD); however, none of the studies has determined which structure within a lipid determines its detrimental or beneficial effects on AD. High-sensitivity enzyme-linked immunosorbent assay (ELISA) shows that saturated fatty acids (SFAs), upstream omega-3 FAs, and arachidonic acid (AA) resulted in significantly higher secretion of both Aß 40 and 42 peptides compared with long chain downstream omega-3 and monounsaturated FAs (MUFA). Their distinct detrimental action is believed to be due to a structural template found in their fatty acyl chains that lack SFAs, upstream omega-3 FAs, and AA. Immunoblotting experiments and use of APP-C99-transfected COS-7 cells suggest that FA-driven altered production of Aß is mediated through γ-secretase cleavage of APP. An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein (APP); Swedish (K670N/M671L) and Indiana (V717F), corroborated in vitro findings by showing lower levels of Aß and amyloid plaques in the brain, when they were fed a low fat diet enriched in DHA. Our work contributes to the clarification of aspects of structure-activity relationships.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/biossíntese , Amiloide/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Peptídeos/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloide/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Células COS , Chlorocebus aethiops , Modelos Animais de Doenças , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Peptídeos/genética
10.
Neurosci Lett ; 481(2): 73-7, 2010 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-20600609

RESUMO

Amyloid-beta peptides (Abeta) generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases play an important role in the pathogenesis of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of Abeta peptides into senile plaques, one of the hallmarks of AD. With the aim to clarify the molecular basis of the interaction between Abeta and cellular membranes, we investigated the effects of various phospholipids (PLs) and a PL-rich diet on Abeta production. Here we show that modulation of Abeta production and Abeta42:40 ratio is not limited to individual fatty acids, rather it is the composition of the PLs of the membrane bilayer, that influences the specificity and level of the regulated intramembranous proteolysis of APP by the gamma-secretase complex. We show that Abeta levels in the conditioned media, in response to some of the PL supplements, is increased in the center and decreased on either side of a graph that resembles bell-shaped distribution. This means that the PLs have less of a tendency to produce unusually extreme effects on Abeta production in SP-C99 transfected Cos-7 cultured cells. We proposed a mechanism-based hypothesis to rationalize PLs' effects on Abeta production.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/farmacologia , Esfingomielinas/farmacologia , Animais , Células COS , Cardiolipinas/farmacologia , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Fosfatidilinositóis/farmacologia
11.
PLoS One ; 4(8): e6779, 2009 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-19707560

RESUMO

Alzheimer's disease (AD) is characterized by neuronal degeneration and cell loss. Abeta(42), in contrast to Abeta(40), is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Abeta(40) and Abeta(42) levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids) in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Abeta(40) and Abeta(42) levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2) and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Abeta(42)/Abeta(40) ratio. Importantly however, an increased Abeta(42)/Abeta(40) ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Abeta(42)/Abeta(40) ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Abeta(42)/Abeta(40) ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes.


Assuntos
Peptídeos beta-Amiloides/genética , Neuroblastoma/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas do Tecido Nervoso/genética , Neuroblastoma/genética , Reação em Cadeia da Polimerase
12.
BMC Med ; 6: 38, 2008 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-19087254

RESUMO

BACKGROUND: Alzheimer's disease (AD) is characterized by neurodegeneration and changes in cellular processes, including neurogenesis. Proteolytic processing of the amyloid precursor protein (APP) plays a central role in AD. Owing to varying APP processing, several beta-amyloid peptides (Abeta) are generated. In contrast to the form with 40 amino acids (Abeta40), the variant with 42 amino acids (Abeta42) is thought to be the pathogenic form triggering the pathological cascade in AD. While total-Abeta effects have been studied extensively, little is known about specific genome-wide effects triggered by Abeta42 or Abeta40 derived from their direct precursor C99. METHODS: A combined transcriptomics/proteomics analysis was performed to measure the effects of intracellularly generated Abeta peptides in human neuroblastoma cells. Data was validated by real-time polymerase chain reaction (real-time PCR) and a functional validation was carried out using RNA interference. RESULTS: Here we studied the transcriptomic and proteomic responses to increased or decreased Abeta42 and Abeta40 levels generated in human neuroblastoma cells. Genome-wide expression profiles (Affymetrix) and proteomic approaches were combined to analyze the cellular response to the changed Abeta42- and Abeta40-levels. The cells responded to this challenge with significant changes in their expression pattern. We identified several dysregulated genes and proteins, but only the cellular retinoic acid binding protein 1 (CRABP1) was up-regulated exclusively in cells expressing an increased Abeta42/Abeta40 ratio. This consequently reduced all-trans retinoic acid (RA)-induced differentiation, validated by CRABP1 knock down, which led to recovery of the cellular response to RA treatment and cellular sprouting under physiological RA concentrations. Importantly, this effect was specific to the AD typical increase in the Abeta42/Abeta40 ratio, whereas a decreased ratio did not result in up-regulation of CRABP1. CONCLUSION: We conclude that increasing the Abeta42/Abeta40 ratio up-regulates CRABP1, which in turn reduces the differentiation potential of the human neuroblastoma cell line SH-SY5Y, but increases cell proliferation. This work might contribute to the better understanding of AD neurogenesis, currently a controversial topic.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores do Ácido Retinoico/biossíntese , Regulação para Cima , Diferenciação Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neurônios/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/análise , Receptores do Ácido Retinoico/genética
13.
Exp Hematol ; 35(12): 1858-71, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17697743

RESUMO

OBJECTIVE: Studies in animal models have indicated that hematopoietic progenitor cells (HPC) migrate and home to the central nervous system and might acquire neural features under specific circumstances. The interaction between HPC and the neural environment and the functional effect on hematopoiesis have not yet been defined. METHODS: CD34(+)133(+) cells from mobilized peripheral blood were cocultured with primary murine neurons or astrocytes. Chemotaxis and adhesive interactions were studied by applying beta(1)- and beta(2)-integrin function-blocking anibodies. The impact of neural feeder layers on integrin expression of HPC and the presence of appropriate adhesion ligands on neural cells were determined by immunostaining and flow cytometry. The hematopoietic long-term fate was monitored by time-lapse microscopy of individual cell-division history followed by long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) assays. Neural differentiation was assessed by immunostaining against specific neuronal and glial antigens. RESULTS: The 23.0% +/- 4.9% of HPC showed stromal cell-derived factor-1-induced migration toward neural cells, and 20.2% +/- 1.6% displayed firm beta(1)-integrin-mediated adhesion to astrocytes. The latter expressed appropriate adhesion ligands, stabilized beta(1)-integrin expression, and increased beta(2)-integrin expression of HPC. Neural differentiation of HPC could not be identified but astrocytes were able to induce limited self-renewing cell divisions of HPC and thus maintain 25.8% +/- 3.4% of the initial LTC-IC and 80.7% +/- 1.9% of the initial CFC. CONCLUSION: Human HPC are able to interact with neural cells and interaction maintains, albeit to a limited extent, the self-renewal capability of HPC.


Assuntos
Divisão Celular , Células-Tronco Hematopoéticas/citologia , Animais , Astrócitos/citologia , Linhagem da Célula , Movimento Celular , Humanos , Camundongos
14.
Mol Cell Biol ; 23(6): 1994-2008, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12612073

RESUMO

In breast cancer cells, estrogens activate the Src/Erk pathway through an interaction of the estrogen receptor alpha (ERalpha) with the SH2 domain of c-Src. Progestins have been reported to activate also this pathway either via an interaction of the progesterone receptor isoform B (PRB) with ERalpha, which itself activates c-Src, or by direct interaction of PRB with the SH3 domain of c-Src. Here we identify two domains of PRB, ERID-I and -II, mediating a direct interaction with the ligand-binding domain of ERalpha. ERID-I and ERID-II flank a proline cluster responsible for binding of PRB to c-Src. In mammalian cells, the interaction of PRB with ERalpha and the progestin activation of the Src/Erk cascade are abolished by deletion of either ERID-I or ERID-II. These regions are not required for transactivation of a progesterone-responsive reporter gene. Mutations in the proline cluster of PRB that prevent a direct interaction with c-Src do not affect the strong activation of c-Src by progestins in the presence of ERalpha. Thus, in cells with ERalpha, ERID-I and ERID-II are necessary and sufficient for progestin activation of the endogenous Src/Erk pathway.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Progesterona/farmacologia , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/química , Animais , Neoplasias da Mama/patologia , Células COS , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Receptor alfa de Estrogênio , Genes Reporter , Humanos , Proteína Quinase 3 Ativada por Mitógeno , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/metabolismo , Relação Estrutura-Atividade , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de src
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