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1.
Nat Rev Cancer ; 24(3): 165, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37968378
2.
Int J Mol Sci ; 24(21)2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37958846

RESUMO

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with few effective treatment strategies. The research on the development of new treatments is often constrained by the limitations of preclinical models, which fail to accurately replicate the disease's essential characteristics. Herein, we describe the obtention, molecular, and functional characterization of the GBM33 cell line. This cell line belongs to the GBM class according to the World Health Organization 2021 Classification of Central Nervous System Tumors, identified by methylation profiling. GBM33 expresses the astrocytic marker GFAP, as well as markers of neuronal origin commonly expressed in GBM cells, such as ßIII-tubulin and neurofilament. Functional assays demonstrated an increased growth rate when compared to the U87 commercial cell line and a similar sensitivity to temozolamide. GBM33 cells retained response to serum starvation, with reduced growth and diminished activation of the Akt signaling pathway. Unlike LN-18 and LN-229 commercial cell lines, GBM33 is able to produce primary cilia upon serum starvation. In summary, the successful establishment and comprehensive characterization of this GBM cell line provide researchers with invaluable tools for studying GBM biology, identifying novel therapeutic targets, and evaluating the efficacy of potential treatments.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Adulto , Humanos , Glioblastoma/metabolismo , Brasil , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Tubulina (Proteína)/metabolismo
3.
J Bras Pneumol ; 47(6): e20210129, 2021.
Artigo em Inglês, Português | MEDLINE | ID: mdl-34909922

RESUMO

Malignant mesotheliomas are rare types of cancers that affect the mesothelial surfaces, usually the pleura and peritoneum. They are associated with asbestos exposure, but due to a latency period of more than 30 years and difficult diagnosis, most cases are not detected until they reach advanced stages. Treatment options for this tumor type are very limited and survival ranges from 12 to 36 months. This review discusses the molecular physiopathology, current diagnosis, and latest therapeutic options for this disease.


Assuntos
Amianto , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurais , Amianto/toxicidade , Humanos , Mesotelioma/terapia , Pleura , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/terapia
4.
J Mol Diagn ; 22(7): 957-966, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32380172

RESUMO

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is currently classified in four main molecular subgroups with different clinical outcomes: sonic hedgehog, wingless, group 3, and group 4 (MBSHH, MBWNT, MBGRP3, or MBGRP4). Presently, a 22-gene expression panel has been efficiently applied for molecular subgrouping using nCounter technology. In this study, formalin-fixed, paraffin-embedded samples from 164 Brazilian medulloblastomas were evaluated, applying the 22-gene panel, and subclassified into the low and high expression of nine key medulloblastoma-related genes. In addition, TP53 mutation status was assessed using TruSight Tumor 15 Panel, and its correlation with expression and prognostic impact was evaluated. Samples from 149 of 164 patients (90%) were classified into MBSHH (47.7%), MBWNT (16.1%), MBGRP3 (15.4%), and MBGRP4 (20.8%). GNAS presented the highest expression levels, with higher expression in MBSHH. TP53, MYCN, SOX2, and MET were also up-regulated in MBSHH, whereas PTEN was up-regulated in MBGRP4. GNAS, TP53, and PTEN low expression was associated with the unfavorable patient outcome only for MBSHH (P = 0.04, P = 0.01, and P = 0.02, respectively). TP53 mutations were detected in 28.57% of MBSHH cases and exhibited association with lower expression and worse clinical outcome, although not statistically significant. The 22-gene panel for molecular classification of medulloblastoma associated with the expression of GNAS, TP53, and PTEN improves the patient prognostication in MBSHH subgroup and can be easily incorporated in the 22-gene panel without any additional costs.


Assuntos
Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/genética , Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Proteínas Hedgehog/genética , Meduloblastoma/classificação , Meduloblastoma/genética , PTEN Fosfo-Hidrolase/genética , Transcriptoma , Proteína Supressora de Tumor p53/genética , Adolescente , Brasil/epidemiologia , Neoplasias Cerebelares/epidemiologia , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA/métodos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Masculino , Meduloblastoma/epidemiologia , Mutação , Prognóstico , Adulto Jovem
5.
Mol Oncol ; 14(1): 159-179, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31701625

RESUMO

The p90 ribosomal S6 kinase (RSK) family, a downstream target of Ras/extracellular signal-regulated kinase signaling, can mediate cross-talk with the mammalian target of rapamycin complex 1 pathway. As RSK connects two oncogenic pathways in gliomas, we investigated the protein levels of the RSK isoforms RSK1-4 in nontumoral brain (NB) and grade I-IV gliomas. When compared to NB or low-grade gliomas (LGG), a group of glioblastomas (GBMs) that excluded long-survivor cases expressed higher levels of RSK1 (RSK1hi ). No difference was observed in RSK2 median-expression levels among NB and gliomas; however, high levels of RSK2 in GBM (RSK2hi ) were associated with worse survival. RSK4 expression was not detected in any brain tissues, whereas RSK3 expression was very low, with GBM demonstrating the lowest RSK3 protein levels. RSK1hi and, to a lesser extent, RSK2hi GBMs showed higher levels of phosphorylated RSK, which reveals RSK activation. Transcriptome analysis indicated that most RSK1hi GBMs belonged to the mesenchymal subtype, and RSK1 expression strongly correlated with gene expression signature of immune infiltrates, in particular of activated natural killer cells and M2 macrophages. In an independent cohort, we confirmed that RSK1hi GBMs exclude long survivors, and RSK1 expression was associated with high protein levels of the mesenchymal subtype marker lysosomal protein transmembrane 5, as well as with high expression of CD68, which indicated the presence of infiltrating immune cells. An RSK1 signature was obtained based on differentially expressed mRNAs and validated in public glioma datasets. Enrichment of RSK1 signature followed glioma progression, recapitulating RSK1 protein expression, and was associated with worse survival not only in GBM but also in LGG. In conclusion, both RSK1 and RSK2 associate with glioma malignity, but displaying isoform-specific peculiarities. The progression-dependent expression and association with immune infiltration suggest RSK1 as a potential progression marker and therapeutic target for gliomas.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Membrana/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transcriptoma/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/imunologia , Glioma/secundário , Humanos , Imuno-Histoquímica , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/genética , Gradação de Tumores , Fosforilação , Isoformas de Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/genética
6.
J Neuropathol Exp Neurol ; 78(9): 788-790, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31403685

RESUMO

Medulloblastoma is the most frequent malignant brain tumor in children, representing 20% of all childhood brain tumors. Currently, medulloblastomas are molecularly classified in 4 subgroups that are associated with distinctive clinicopathological features. KBTBD4 mutations were recently described in a subset of MBGRP3 and MBGRP4 medulloblastomas subgroups. However, no other studies reported KBTBD4 mutations in medulloblastomas. Thus, our aim was to investigate KBTBD4 mutations in a Brazilian series of medulloblastoma. We evaluated 128 medulloblastoma patients molecularly classified from 4 Brazilian reference centers. DNA from formalin-fixed, paraffin-embedded samples was screened for KBTBD4 hotspot mutations by Sanger sequencing. Most of the patients were male, average age was 16.5 years old and average overall survival was 55.9 months. The predominant histological subtype was the classic subtype, followed by nodular/desmoplastic, and the predominant medulloblastoma molecular subtype was the MBSHH subgroup (46%), followed by MBGRP3 and MBGRP4 (19%/each), and MBWNT (16%). Among the 128 samples, 111 were successfully sequenced. No KBTBD4 mutations were identified in 111 samples. Our findings suggest that KBTBD4 mutations are uncommon in Brazilian MBGRP3 and MBGRP4 medulloblastomas subgroups. Further studies in a larger series of MBGRP3 and MBGRP4 medulloblastomas are warranted to better assess role of KBTBD4 mutations.


Assuntos
Proteínas de Transporte/genética , Neoplasias Cerebelares/genética , Meduloblastoma/genética , Adolescente , Adulto , Brasil , Neoplasias Cerebelares/mortalidade , Neoplasias Cerebelares/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Meduloblastoma/mortalidade , Meduloblastoma/patologia , Pessoa de Meia-Idade , Mutação , Taxa de Sobrevida , Adulto Jovem
7.
Appl. cancer res ; 39: 1-5, 2019. ilus, tab
Artigo em Inglês | LILACS, Inca | ID: biblio-1015230

RESUMO

Background: Human biological material has become an important resource for biomedical research. Tumor Biobanks are facilities that collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. mRNA-translation has been demonstrated to modulate protein levels and is considered a fundamental post-transcriptional mechanism of gene expression regulation. Thus, determining translation efficiencies of individual mRNAs in human tumors may add another layer of information that contributes to the understanding of tumorigenic pathways. To analyze the RNAs actively engaged in translation, RNAs associated with ribosomes (polysomes) are isolated, identified and compared to total RNA. However, the application of this technique in human tumors depends on the stability of the polysomal structure under Biobank storage conditions that usually consists of ultra-low temperature. Since the effect of freezing on the stability of the polysomal structure in stored tumor samples is not known, it is essential to evaluate this factor in the frozen samples, validating the use of biobank samples in studies of translational efficiency. Methods: Xenograft tumors were divided in two parts, half was subject to immediate processing, and half was frozen for posterior analysis. Both parts were subject to polysomal separation, RNA extraction and identification through RNAseq. Results: It was possible to successfully extract and identify total and polysomal RNA from both fresh and frozen tumoral tissue. The quantification of the polysome profile indicated no difference in the translational efficiency estimated in fresh versus frozen tissue. Gene expression data from the fresh versus frozen tissues were compared and the correlation between the polysome associated fresh x frozen (R = 0,89) and total fresh x frozen (0,90) mRNAs was calculated. No difference was identified between the two conditions. Conclusions: We demonstrated that tissue freezing does not affect the polysomal structure, consequently validating the viability of the use of biobank stored tissue for polysome associated RNA analysis (AU)


Assuntos
Humanos , Polirribossomos , RNA , Expressão Gênica , Regulação da Expressão Gênica , Neoplasias
8.
Biochem J ; 474(17): 2981-2991, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28739602

RESUMO

Prion protein (PrPC) was initially described due to its involvement in transmissible spongiform encephalopathies. It was subsequently demonstrated to be a cell surface molecule involved in many physiological processes, such as vesicle trafficking. Here, we investigated the roles of PrPC in the response to insulin and obesity development. Two independent PrPC knockout (KO) and one PrPC overexpressing (TG20) mouse models were fed high-fat diets, and the development of insulin resistance and obesity was monitored. PrPC KO mice fed high-fat diets presented all of the symptoms associated with the development of insulin resistance: hyperglycemia, hyperinsulinemia, and obesity. Conversely, TG20 animals fed high-fat diets showed reduced weight and insulin resistance. Accordingly, the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was reduced in PrPC KO mice and increased in TG20 animals. PrPC KO cells also presented reduced glucose uptake upon insulin stimulation, due to reduced translocation of the glucose transporter Glut4. Thus, our results suggest that PrPC reflects susceptibility to the development of insulin resistance and metabolic syndrome.


Assuntos
Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Obesidade/metabolismo , PPAR gama/metabolismo , Proteínas PrPC/metabolismo , Proteínas Priônicas/metabolismo , Células 3T3-L1 , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Cultivadas , Cruzamentos Genéticos , Dieta Hiperlipídica/efeitos adversos , Embrião de Mamíferos/patologia , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Obesidade/etiologia , Obesidade/patologia , PPAR gama/genética , Proteínas PrPC/genética , Proteínas Priônicas/genética , Transporte Proteico , Aumento de Peso
9.
Cell Mol Life Sci ; 70(17): 3211-27, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23543276

RESUMO

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Vesículas Secretórias/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Hipocampo/citologia , Immunoblotting , Camundongos , Proteínas PrPC/metabolismo , Vesículas Secretórias/ultraestrutura
10.
J Biol Chem ; 288(15): 10860-9, 2013 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-23447528

RESUMO

The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos/metabolismo , Neurogênese/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Fator 4 Ativador da Transcrição/biossíntese , Fator 4 Ativador da Transcrição/genética , Animais , Comportamento Animal/fisiologia , Células Cultivadas , Regulação para Baixo/fisiologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Comportamento Alimentar/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Memória/fisiologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Ribossomos/genética , Ribossomos/metabolismo , Sinapses/genética , Sinapses/metabolismo
11.
J Histochem Cytochem ; 61(4): 272-82, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23340270

RESUMO

Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT.


Assuntos
Automação , Proteínas Proto-Oncogênicas c-akt/análise , Serina-Treonina Quinases TOR/análise , Análise Serial de Tecidos , Humanos , Imuno-Histoquímica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/biossíntese , Serina-Treonina Quinases TOR/metabolismo
12.
J Neurochem ; 124(2): 210-23, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23145988

RESUMO

Prion protein (PrP(C)) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP(C) interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP(C) co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross-talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP(C)-mediated axonogenesis in peripheral neurons in response to STI1 and laminin-γ1 chain-derived peptide (Ln-γ1). STI1 and Ln-γ1 promoted robust axonogenesis in wild-type neurons, whereas no effect was observed in neurons from PrP(C) -null mice. PrP(C) binding to Ln-γ1 or STI1 led to an increase in intracellular Ca(2+) levels via distinct mechanisms: STI1 promoted extracellular Ca(2+) influx, and Ln-γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln-γ1, but depends on, C-type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP(C)-mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP(C). These results suggest a role for PrP(C) as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.


Assuntos
Sinalização do Cálcio/fisiologia , Gânglios Espinais/fisiologia , Proteínas de Choque Térmico/fisiologia , Laminina/fisiologia , Proteínas PrPC/fisiologia , Receptor Cross-Talk/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Axônios/química , Axônios/fisiologia , Sobrevivência Celular/fisiologia , Líquido Extracelular/química , Líquido Extracelular/fisiologia , Gânglios Espinais/química , Proteínas de Choque Térmico/química , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Laminina/metabolismo , Camundongos , Camundongos Knockout , Cultura Primária de Células , Ligação Proteica/fisiologia , Células Receptoras Sensoriais/química , Regulação para Cima/fisiologia
13.
FASEB J ; 25(1): 265-79, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20876210

RESUMO

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln γ1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln γ1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln γ1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.


Assuntos
Laminina/metabolismo , Neuritos/fisiologia , Proteínas PrPC/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Animais , Benzoatos/farmacologia , Cálcio/metabolismo , Células Cultivadas , Feminino , Glicina/análogos & derivados , Glicina/farmacologia , Células HEK293 , Humanos , Immunoblotting , Laminina/genética , Laminina/farmacologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuritos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas PrPC/genética , Ligação Proteica , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/genética , Fosfolipases Tipo C/metabolismo
14.
Curr Issues Mol Biol ; 12(2): 63-86, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19767651

RESUMO

PrP(C) is highly expressed in both the central and peripheral nervous systems from early stages of development and in adulthood. Its major conformational change and conversion into an abnormal form (PrP(Sc)) has been associated with the generation of prions, the infectious agent of transmissible spongiform encephalopathies (TSEs). The massive neurodegeneration presented by individuals suffering from these diseases has been associated with the gain of neurotoxic activity of PrP(Sc). On the other hand, major neurodegeneration is also observed in transgenic mice expressing PrP(C) molecules deleted of specific domains, which points to important functional domains within this molecule, and supports the hypothesis that loss-of PrP(C) function may contribute to the pathogenesis of TSEs. Furthermore, a large body of data demonstrates direct or indirect interaction of PrP(C) with extracellular matrix proteins, soluble factors, transmembrane proteins, G-protein coupled receptors and ions channels. The ability of PrP(C) to drive the assembly of multi-component complexes at the cell surface is likely the basis for its neurotrophic functions. These properties indicate that PrP(C) may be relevant for not only the spongiform encephalopathies, but also as an ancillary component of the pathogenesis of other neurodegenerative diseases, and therefore amenable to therapeutic targeting.


Assuntos
Príons/metabolismo , Animais , Humanos , Camundongos , Modelos Genéticos , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Príons/genética
15.
J Comp Neurol ; 517(3): 371-84, 2009 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-19760599

RESUMO

Prion protein (PrP(C)) is the normal isoform of PrP(Sc), a protein involved in neurodegenerative disorders. PrP(C) participates in neuritogenesis, neuroprotection, and memory consolidation through its interaction with the secreted protein stress-inducible protein 1 (STI1) and the extracellular matrix protein vitronectin (Vn). Although PrP(C) mRNA expression has been documented during embryogenesis, its protein expression patterns have not been evaluated. Furthermore, little is known about either Vn or STI protein expression. In this study, PrP(C), STI1, and Vn protein expression was explored throughout mouse embryonic life. We found that the distributions of the three proteins were spatiotemporally related. STI1 and Vn expression became evident at E8, earlier than PrP(C), in the nervous system and heart. At E10, we observed, in the spinal cord, a gradient of expression of the three proteins, more abundant in the notochord and floor plate, suggesting that they can have a role in axonal growth. As development proceeded, the three proteins were detected in other organs, suggesting that they may play a role in the development of nonneural tissues as well. Finally, although STI1 and Vn are PrP(C) ligands, their expression was not altered in PrP(C)-null mice.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas PrPC/metabolismo , Vitronectina/metabolismo , Animais , Axônios/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Gânglios Espinais/embriologia , Gânglios Espinais/metabolismo , Coração/embriologia , Rim/embriologia , Rim/metabolismo , Fígado/embriologia , Fígado/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Notocorda/embriologia , Notocorda/metabolismo , Proteínas PrPC/genética , Medula Espinal/embriologia , Medula Espinal/metabolismo , Fatores de Tempo
16.
Int J Cancer ; 125(7): 1523-31, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19444918

RESUMO

Cellular Prion Protein (PrP(C)) is a cell surface protein highly expressed in the nervous system, and to a lesser extent in other tissues. PrP(C) binds to the extracellular matrix laminin and vitronectin, to mediate cell adhesion and differentiation. Herein, we investigate how PrP(C) expression modulates the aggressiveness of transformed cells. Mesenchymal embryonic cells (MEC) from wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were immortalized and transformed by co-expression of ras and myc. These cells presented similar growth rates and tumor formation in vivo. When injected in the tail vein, Prnp(0/0)ras/myc cells exhibited increased lung colonization compared with Prnp(+/+)ras/myc cells. Additionally, Prnp(0/0)ras/myc cells form more aggregates with blood components than Prnp(+/+)ras/myc cells, facilitating the arrest of Prnp(0/0)ras/myc cells in the lung vasculature. Integrin alpha(v)beta(3) is more expressed and activated in MEC and in transformed Prnp(0/0) cells than in the respective Prnp(+/+) cells. The blocking of integrin alpha(v)beta(3) by RGD peptide reduces lung colonization in transformed Prnp(0/0) cells to similar levels of those presented by transformed Prnp(+/+) cells. Our data indicate that PrP(C) negatively modulates the expression and activation of integrin alpha(v)beta(3) resulting in a more aggressive phenotype. These results indicate that PrP(C) may have main implications in modulating metastasis formation.


Assuntos
Agregação Celular , Integrina alfaV/metabolismo , Integrina alfaVbeta3/metabolismo , Neoplasias Pulmonares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Metástase Neoplásica , Proteínas PrPC/metabolismo , Análise de Variância , Animais , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Knockout , Proteínas PrPC/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas ras/metabolismo
17.
Glia ; 57(13): 1439-49, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19243016

RESUMO

Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.


Assuntos
Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Proteínas de Choque Térmico/metabolismo , Proteínas PrPC/metabolismo , Animais , Astrócitos/citologia , Encéfalo/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Nestina , Proteínas PrPC/genética , Proteína Quinase C/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Vimentina/metabolismo
18.
J Neurosci ; 28(26): 6691-702, 2008 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-18579743

RESUMO

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrP(C)) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrP(C) trafficking and tested whether this process controls PrP(C)-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrP(C), induced PrP(C) endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrP(C); however, heterologous expression of PrP(C) reconstituted both PKA and ERK1/2 activation. In contrast, a PrP(C) mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrP(C) endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.


Assuntos
Encéfalo/metabolismo , Endocitose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinaminas/metabolismo , Ativação Enzimática/fisiologia , Proteínas de Choque Térmico/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Técnicas de Cultura de Órgãos , Proteínas PrPC/genética , Transporte Proteico/fisiologia
19.
J Cell Sci ; 120(Pt 11): 1915-26, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17504807

RESUMO

The physiological functions of the cellular prion protein, PrP(C), as a cell surface pleiotropic receptor are under debate. We report that PrP(C) interacts with vitronectin but not with fibronectin or collagen. The binding sites mediating this PrP(C)-vitronectin interaction were mapped to residues 105-119 of PrP(C) and the residues 307-320 of vitronectin. The two proteins were co-localized in embryonic dorsal root ganglia from wild-type mice. Vitronectin addition to cultured dorsal root ganglia induced axonal growth, which could be mimicked by vitronectin peptide 307-320 and abrogated by anti-PrP(C) antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrP(C)-null mice. Functional assays demonstrated that relative to wild-type cells, PrP(C)-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater alphavbeta3 activity. Our findings indicate that PrP(C) plays an important role in axonal growth, and this function may be rescued in PrP(C)-knockout animals by integrin compensatory mechanisms.


Assuntos
Axônios/metabolismo , Integrinas/metabolismo , Proteínas PrPC/metabolismo , Vitronectina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Humanos , Camundongos , Proteínas PrPC/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Vitronectina/química
20.
Neurobiol Dis ; 26(1): 282-90, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17329112

RESUMO

Cellular prion protein (PrP(C)) is a cell surface glycoprotein that interacts with several ligands such as laminin, NCAM (Neural-Cell Adhesion Molecule) and the stress-inducible protein 1 (STI1). PrP(C) association with these proteins in neurons mediates adhesion, differentiation and protection against programmed cell death. Herein, we used an aversively motivated learning paradigm in rats to investigate whether STI1 interaction with PrP(C) affects short-term memory (STM) formation and long-term memory (LTM) consolidation. Blockage of PrP(C)-STI1 interaction with intra-hippocampal infusion of antibodies against PrP(C) or STI1 immediately after training impaired both STM and LTM. Furthermore, infusion of PrP(C) peptide 106-126, which competes for PrP(C)-STI1 interaction, also inhibited both forms of memory. Remarkably, STI1 peptide 230-245, which includes the PrP(C) binding site, had a potent enhancing effect on memory performance, which could be blocked by co-treatment with the competitive PrP(C) peptide 106-126. Taken together, these results demonstrate that PrP(C)-STI1 interaction modulates both STM and LTM and suggests a potential use of ST11 peptide 230-245 as a pharmacological agent.


Assuntos
Proteínas de Choque Térmico/fisiologia , Memória de Curto Prazo/fisiologia , Memória/fisiologia , Proteínas PrPC/fisiologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/fisiologia , Western Blotting , Proteínas de Choque Térmico/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Indicadores e Reagentes , Masculino , Plasticidade Neuronal/fisiologia , Proteínas PrPC/antagonistas & inibidores , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Ratos , Ratos Wistar , Sinapses/fisiologia
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