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1.
Biochem Cell Biol ; 97(1): 68-72, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29879361

RESUMO

Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the regulation and attenuation of cell signaling. Efficient sorting of endocytosed receptors is performed by the ESCRT machinery, which targets receptors for degradation by the sequential establishment of protein complexes. These events are tightly regulated and malfunction of ESCRT components can lead to abnormal trafficking and sustained signaling and promote tumor formation or progression. In this review, we analyze the modular domain organization of the alternative ESCRT protein HD-PTP and its role in receptor trafficking and tumorigenesis.


Assuntos
Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Neoplasias/fisiopatologia , Proteínas Tirosina Fosfatases não Receptoras/química , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Humanos , Transporte Proteico , Relação Estrutura-Atividade
2.
Clin Immunol ; 181: 9-15, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28536054

RESUMO

We aimed to investigate the immunologic effects of vitamin D replacement in RRMS patients. In a controlled single center study, patients deficient in 25-hydroxyvitamin D (serum level<25ng/ml) received 10,000IU/week cholecalciferol for 3months. Sufficient vitamin D patients (serum level>35ng/ml) were followed for the same period. Assessments were performed at baseline and at 3months. 25-hydroxyvitamin D levels increased significantly from baseline to month-3 in the deficient group after treatment and remained stable in the sufficient group. We observed a decreased interferon-γ (IFNγ) secretion by CD4+ T cells in vitamin D deficient group but not in the sufficient group, and a negative correlation between baseline serum vitamin D and IFNγ production. There was no change in the frequency of T helper or regulatory T cell subsets in either group. Increasing serum levels of 25-hydroxyvitamin D are associated with decreased production of IFNγ by CD4+ T cells.


Assuntos
Colecalciferol/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/imunologia , Deficiência de Vitamina D/tratamento farmacológico , Vitaminas/uso terapêutico , Adulto , Linfócitos T CD4-Positivos/imunologia , Estudos Controlados Antes e Depois , Citocinas/imunologia , Feminino , Humanos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/complicações , Resultado do Tratamento , Vitamina D/análogos & derivados , Vitamina D/sangue , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/imunologia , Adulto Jovem
3.
Biochem Soc Trans ; 45(3): 845-854, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28620046

RESUMO

Sustained cellular signalling originated from the receptors located at the plasma membrane is widely associated with cancer susceptibility. Endosomal sorting and degradation of the cell surface receptors is therefore crucial to preventing chronic downstream signalling and tumorigenesis. Since the Endosomal Sorting Complexes Required for Transport (ESCRT) controls these processes, ESCRT components were proposed to act as tumour suppressor genes. However, the bona fide role of ESCRT components in tumorigenesis has not been clearly demonstrated. The ESCRT member HD-PTP/PTPN23 was recently identified as a novel haplo-insufficient tumour suppressor in vitro and in vivo, in mice and humans. In this mini-review, we outline the role of the ESCRT components in cancer and summarize the functions of HD-PTP/PTPN23 in tumorigenesis.


Assuntos
Carcinogênese/metabolismo , Neoplasias/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Animais , Genes Supressores de Tumor , Humanos , Proteínas Tirosina Fosfatases não Receptoras/metabolismo
4.
Clin Immunol ; 166-167: 59-71, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27041081

RESUMO

Vitamin D is a secosteroid hormone that plays an important regulatory role in calcium homeostasis and bone metabolism. Immune cells can both produce and respond to 1,25(OH)2D3. CD4+ T cells from vitamin D receptor (VDR) KO mice produce higher levels of IFN-γ and IL-17 than their wild type counterparts, and play a crucial role in the pathogenesis of autoimmune diseases (AID). We are particularly interested in studying the effect of vitamin D on pathogenic Th17 cells in humans. We investigated the in vitro effect of 1,25(OH)2D3 and 25(OH)D3 on the differentiation and cytokine production of primary CD4+ T cells from normal donors, and cultured in Th17 polarizing conditions. Both forms of vitamin D reduced the expression of pathogenic Th17 markers and their secretion of pro-inflammatory cytokines (IL-17A, IFN-γ). Furthermore, both vitamin D forms induced an expansion of CD25hi cells and upregulated their expression of CTLA-4 and Foxp3 regulatory markers.


Assuntos
Calcifediol/farmacologia , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Antígeno CTLA-4/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Vitaminas/farmacologia
5.
Expert Rev Proteomics ; 12(6): 637-50, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26479122

RESUMO

Multiple sclerosis (MS) is a complex disease characterized by extensive phenotypic variability. Biomarkers to capture the different aspects of MS heterogeneity, and to help make a diagnosis and monitor disease progression, while providing insights into etiopathogenesis and response to treatment, are urgently needed. Omics technologies and research efforts with microRNAs have provide unparalleled opportunities for exploring altered protein profiles associated with molecular mechanisms of disease, substantially expanding the list of candidate biomarkers for MS. This review presents evidence from proteomic studies that have focused on identification of biomarkers released in biofluids as a result of the different pathophysiological processes of MS. Also discussed is the emerging role of miRNAs as complementary biomarkers related to cellular processes occurring in MS patients. Also provided is an overview of candidate biomarkers that have been proposed for elucidating pathophysiological processes and disease activity and for guiding clinical diagnosis and/or therapeutic interventions in MS.


Assuntos
MicroRNAs/metabolismo , Esclerose Múltipla/diagnóstico , Proteoma/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Humanos , Linfócitos/metabolismo , MicroRNAs/sangue , Esclerose Múltipla/metabolismo , Proteínas do Tecido Nervoso/metabolismo
6.
STAR Protoc ; 3(3): 101475, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-35755125

RESUMO

EGFR cell surface density, stability, internalization, and recycling can be measured by cell surface ELISA (cs-ELISA). Performing this experiment on ice impedes receptor internalization; thus the physiological cell surface receptor levels can be measured by cs-ELISA. Cell surface EGFR levels are detected by measuring Amplex Red fluorescence intensity. Although cell surface receptor levels can be measured by flow cytometry, cs-ELISA does not include cell dissociation steps that might affect cell surface receptor levels. For complete details on the use and execution of this protocol, please refer to Kazan et al. (2019).


Assuntos
Receptores ErbB , Receptores de Superfície Celular , Membrana Celular/metabolismo , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/metabolismo , Citometria de Fluxo , Receptores de Superfície Celular/metabolismo
7.
iScience ; 24(11): 103274, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34761192

RESUMO

Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery.

8.
Methods Mol Biol ; 1998: 93-103, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250296

RESUMO

The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5ß1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5ß1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Integrina alfa5beta1/metabolismo , Microscopia Intravital/métodos , Análise de Célula Única/métodos , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/métodos , Integrina alfa5beta1/química , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Imagem Molecular/métodos , Sondas Moleculares/química
9.
Data Brief ; 22: 635-638, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30671510

RESUMO

Bevacizumab or Avastin® (Av), the recombinant antibody targeting VEGF, improves progression-free but not overall survival of metastatic breast cancer patients due to development of Av resistance. We showed that Av-therapy-induced inflammatory microenvironment contributes to the refractoriness to Av treatment. Here we present data regarding the effect of Av treatment on migration of a non-invasive breast cancer cell line, MCF-7. The data presented hereis related to the research article "Bevacizumab induces inflammation in MDA-MB-231 breast cancer cell line and in a mouse model" (Hajjar et al., 2018).

10.
Cell Signal ; 53: 400-412, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30445167

RESUMO

BACKGROUND: Bevacizumab or Avastin® (Av) is an anti-vascular endothelial growth factor agent. It does not improve survival of breast cancer patients due to development of refractoriness. Av treatment was shown to increase inflammation in a diabetic mouse model, and also to induce epithelial-to-mesenchymal transition of non-transformed breast epithelia. This study aimed to understand if the Av-induced inflammatory microenvironment could be a mechanism of Av refractoriness. Expression profiles of inflammatory mediators, in vitro in MDA-MB-231 cells, in vivo in a mouse model xenografted with MDA-MB-231 cells and from archived cases of human breast carcinoma tissues were evaluated. Gap junctions are also crucial for angiogenesis and tumor cell extravasation. The effect of connexin 43 (Cx43) overexpression on the expression of inflammatory markers in MDA-MB-231 cells treated with Av was assessed. METHODS: MDA-MB-231 cells, control or overexpressing Cx43, were used in this study. Proliferation and invasion assays were performed. Quantitative PCR, ELISA and western blotting were performed to assess the regulation of inflammatory mediators and other factors upon Av treatment. Immunofluorescence was performed to document the translocation of Nuclear Factor-kappa B p65. RESULTS: Breast cancer tissues had elevated transcriptional levels of inflammatory mediators. Av treatment increased expression levels of inflammatory mediators and metastatic factors in vitro and in vivo. Interestingly, overexpressing Cx43 in MDA-MB-231 cells alleviated the inflammatory effects induced by Av treatment. CONCLUSION: This study attributes Av refractoriness to the Av therapy-induced inflammatory microenvironment.


Assuntos
Inibidores da Angiogênese/farmacologia , Bevacizumab/farmacologia , Neoplasias da Mama/tratamento farmacológico , Inflamação/induzido quimicamente , Inibidores da Angiogênese/efeitos adversos , Animais , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/farmacologia , Bevacizumab/efeitos adversos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neovascularização Patológica/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos
11.
Cancers (Basel) ; 11(4)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939738

RESUMO

Connexins regulate multiple cellular functions and are considered tumor suppressors. Connexin43 (Cx43) is frequently down-regulated in breast tumors. However, Cx43 regulation during cancer onset and metastasis is complex and context-dependent. We investigated the effect of Cx43 over-expression or knock-down on the metastatic potential of MDA-MB-231 breast cancer cells in vitro and in vivo and in human breast cancer tissues. MDA-MB-231 cells over-expressing (Cx43D) or down-regulating Cx43 (shCx43) were generated and used in proliferation, migration, and invasion assays. The regulation of genes/proteins implicated in progression, invasion and metastasis was assessed in vitro and in immune-compromized mice injected with MDA-MB-231, Cx43D or shCx43 cells. Primary tumor onset/growth, metastasis and overall survival of these animals was monitored and evaluated. In addition, Cx43 expression in human breast carcinoma samples was assessed by qPCR. Cx43 over-expression increased protein levels of epithelial markers E-cadherin and zonula occludens 1 expression and resulted in the sequestration of ß-catenin at the cell membrane, while Cx43 knock-down induced protein expression of the mesenchymal marker N-cadherin and an increased invasive potential of shCx43 cells. In vivo, in mice xenografted with breast cancer cells, Cx43 over-expression decreased tumor volume, attenuated cell metastasis to lungs and liver and increased overall mice survival. Importantly, the expression of Cx43 in triple negative human breast cancer tissues is also down-regulated. Collectively, Cx43 over-expression induced an epithelial-like phenotype in MDA-MB-231 cells and suppressed tumor growth and metastasis to secondary organs in vivo. In contrast, Cx43 knock-down in MDA-MB-231 cells induced a mesenchymal phenotype with increased cell invasion leading to an enhanced metastatic phenotype. These data provide evidence for a pivotal role of Cx43 in breast cancer metastasis and support the potential targeting of connexins in breast cancer therapy.

12.
Cell Rep ; 26(13): 3613-3628.e6, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917316

RESUMO

TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK confers pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, whereas ablation of total AMPK activity abolishes this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages is observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status with innate immunity.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Imunidade Inata , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Resistência à Doença , Imunidade Inata/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL
13.
Sci Rep ; 9(1): 11945, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420572

RESUMO

The signalling output of many transmembrane receptors that mediate cell-cell communication is restricted by the endosomal sorting complex required for transport (ESCRT), but the impact of this machinery on Eph tyrosine kinase receptor function is unknown. We identified the ESCRT-associated adaptor protein HD-PTP as part of an EphB2 proximity-dependent biotin identification (BioID) interactome, and confirmed this association using co-immunoprecipitation. HD-PTP loss attenuates the ephrin-B2:EphB2 signalling-induced collapse of cultured cells and axonal growth cones, and results in aberrant guidance of chick spinal motor neuron axons in vivo. HD-PTP depletion abrogates ephrin-B2-induced EphB2 clustering, and EphB2 and Src family kinase activation. HD-PTP loss also accelerates ligand-induced EphB2 degradation, contrasting the effects of HD-PTP loss on the relay of signals from other cell surface receptors. Our results link Eph function to the ESCRT machinery and demonstrate a role for HD-PTP in the earliest steps of ephrin-B:EphB signalling, as well as in obstructing premature receptor depletion.


Assuntos
Axônios/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Efrina-B2/genética , Neurônios Motores/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Receptor EphB2/genética , Animais , Axônios/ultraestrutura , Embrião de Galinha , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/ultraestrutura , Efrina-B2/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Neurônios Motores/ultraestrutura , Cultura Primária de Células , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteólise , Receptor EphB2/metabolismo , Transdução de Sinais , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura , Quinases da Família src/genética , Quinases da Família src/metabolismo
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