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1.
Cell Mol Life Sci ; 76(20): 4165-4178, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31076805

RESUMO

Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination.


Assuntos
Basigina/genética , Membrana Celular/microbiologia , Interações Hospedeiro-Patógeno/genética , Listeria monocytogenes/fisiologia , Shigella flexneri/fisiologia , Células A549 , Actinas/genética , Actinas/metabolismo , Animais , Basigina/antagonistas & inibidores , Basigina/metabolismo , Células CACO-2 , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Ciclofilina A/deficiência , Ciclofilina A/genética , Endocitose , Fibroblastos/microbiologia , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/ultraestrutura , Camundongos , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Shigella flexneri/patogenicidade , Shigella flexneri/ultraestrutura , Transdução de Sinais
2.
J Infect Dis ; 219(1): 145-153, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29733369

RESUMO

Background: Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Methods: In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Results: Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Conclusions: Our results demonstrate a crucial role for CypA during Listeria infections.


Assuntos
Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/microbiologia , Ciclofilina A/metabolismo , Listeria/metabolismo , Listeriose/metabolismo , Células A549 , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actinas/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Extensões da Superfície Celular/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Listeria/patogenicidade , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Peptidilprolil Isomerase/metabolismo
3.
Anat Rec (Hoboken) ; 301(12): 2086-2094, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30312007

RESUMO

Salmonella enterica serovar Typhimurium (S. Typhimurium), enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) commandeer the actin cytoskeleton of their host cells as a crucial step in their infectious processes. These pathogens depend on the injection of their own effectors directly into target host cells in order to usurp cellular signaling pathways that lead to morphological actin rearrangements in those cells. Here we show that the PPIase Cyclophilin A (CypA) is a novel component of S. Typhimurium-induced membrane ruffles and functions to restrict bacterial invasion levels, as in cells depleted of CypA, bacterial loads increase. We also demonstrate that CypA requires the EPEC effector Tir as well as pedestal formation for its recruitment to bacterial attachment sites and that its presence at pedestals also holds during EHEC infections. Finally, we demonstrate that CypA is found at lamellipodia; actin-rich structures at the leading edge of motile cells. Our findings further establish CypA as a component of dynamic actin-rich structures formed during bacterial infections and within cells in general. Anat Rec, 301:2086-2094, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Ciclofilina A/metabolismo , Escherichia coli/metabolismo , Salmonella/metabolismo , Citoesqueleto de Actina/química , Actinas/análise , Animais , Ciclofilina A/análise , Células HeLa , Humanos , Camundongos , Potoroidae
5.
Sci Rep ; 6: 22378, 2016 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-26932182

RESUMO

CypA (Cyclophilin A) is a peptidyl-prolyl isomerase previously shown to be required for chondrogenic differentiation and endochondral ossification. However, the effects of CypA on osteoclast activity and bone maintenance are entirely unknown. Here, we show that Ppia(-/-) mice demonstrate low bone mineral density, reduced osteoblast numbers, and increased osteoclast numbers. When isolated from the calvaria, Ppia(-/-) osteoblasts demonstrate decreased osteogenic differentiation, whereas Ppia(-/-) osteoclasts derived from the long bones showed increased osteoclastic activity. Overexpression and gene silencing of CypA verified osteogenic and anti-osteoclastic effects. In osteoblasts, CypA is necessary for BMP-2 (Bone Morphogenetic Protein-2)-induced Smad phosphorylation. In osteoclasts, loss of CypA activates BtK (Bruton's tyrosine kinase) and subsequently integrates with TRAF6 (TNF receptor-associated factor 6) and/or c-fos signaling to induce NFATc1 (nuclear factors of activated T cells, cytoplasmic 1). Collectively, CypA dually exerts pro-osteogenic and anti-osteoclastic effects. Thus, modulation of CypA may be useful in future efforts targeting osteoporosis.


Assuntos
Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Ciclofilina A/metabolismo , Animais , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Células RAW 264.7 , Transdução de Sinais , Microtomografia por Raio-X
6.
Mol Cell Biol ; 35(12): 2119-30, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25870110

RESUMO

Recent studies showed that cyclophilin A (CypA) promotes NF-κB/p65 nuclear translocation, resulting in enhanced NF-κB activity and altered expression of its target genes, such as the Sox9 transcriptional factor, which plays a critical role in chondrogenic differentiation and endochondral ossification. In this report, we unveil the role of CypA in signal-induced chondrogenic differentiation and endochondral ossification. Expression levels of the chondrogenic differentiation markers and transcriptional regulators Sox9 and Runx2 were all significantly lower in CypA knockdown chondrogenic cells than in wild-type cells, indicating that CypA plays a functional role in chondrogenic differentiation. In vitro differentiation studies using micromass cultures of mouse limb bud cells further supported the conclusion that CypA is needed for chondrogenic differentiation. Newborn CypA-deficient pups double stained with alcian blue and alizarin red exhibited generalized, pronounced skeletal defects, while high-resolution micro-computed tomography (microCT) analyses of the femurs and lumbar vertebrae revealed delayed or incomplete endochondral ossification. Comparative histology and immunohistochemistry (IHC) analyses further verified the effects of CypA deficiency on chondrogenic differentiation. Our results provide evidence for the important contribution of CypA as a pertinent component acting through NF-κB-Sox9 in regulation of chondrogenesis signaling. These findings are important to better understand signal-induced chondrogenesis of chondrogenic progenitors in physiological and pathophysiological contexts.


Assuntos
Condrogênese , Ciclofilina A/genética , Ciclofilina A/metabolismo , Osteogênese , Animais , Osso e Ossos/anormalidades , Osso e Ossos/metabolismo , Osso e Ossos/ultraestrutura , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Deleção de Genes , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Microtomografia por Raio-X
7.
PLoS One ; 9(8): e96211, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25119989

RESUMO

BACKGROUND: Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects. METHODOLOGY: Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes. RESULTS: GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-κB subunit p65 at the N-terminal 170-176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity. CONCLUSIONS: Our findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant activation of NF-κB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases.


Assuntos
Núcleo Celular/metabolismo , Ciclofilina A/metabolismo , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Sítios de Ligação/genética , Linhagem Celular , Cicloeximida/farmacologia , Ciclofilina A/antagonistas & inibidores , Ciclofilina A/genética , Ciclosporina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática , Fibroblastos , Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Lactonas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica , Transdução de Sinais , Compostos de Espiro/farmacologia , Transcrição Gênica , Ativação Transcricional
8.
Molecules ; 15(11): 8377-89, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21081858

RESUMO

Extract of Toona sinensis (TS) has been reported to have various effects on cultured cell lines, including anti-proliferative activity in cancer cells. We have studied the effects of TS on various human oral squamous carcinoma cell lines (HOSCC), including UM1, UM2, SCC-4, and SCC-9. These cell lines were treated with TS leaf extract and screened for viability, apoptosis, necrosis, and apoptotic gene expression. Normal human oral keratinocytes (NHOK) served as a control for cytotoxic assays. Viability of TS-treated HOSCC was reduced, whereas that of NHOK was not affected. FACScan analysis revealed that the leaf extract induced apoptosis or a combination of apoptosis and necrosis, depending on cell type. Microarray and semi-quantitative RT-PCR analysis for apoptotic-related gene expression revealed that 3,4,5-trihydroxybenzoic acid (gallic acid, one of the major bioactive compounds purified from TS extract) up-regulated pro-apoptotic genes such TNF-α, TP53BP2, and GADD45A, and down-regulated the anti-apoptotic genes Survivin and cIAP1, resulting in cell death. This study suggests that gallic acid, the major bioactive compound present, is responsible for the anti-neoplastic effect of Toona sinensis leaf extract.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ácido Gálico/uso terapêutico , Meliaceae/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma de Células Escamosas , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ácido Gálico/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Queratinócitos/efeitos dos fármacos , Neoplasias Bucais , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Extratos Vegetais/química , Fator de Necrose Tumoral alfa/metabolismo
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