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1.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209301

RESUMO

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Hepatócitos/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transporte Biológico Ativo , Membrana Celular/genética , Células HEK293 , Células HeLa , Humanos , Fosfatidilcolinas/genética , Proteínas rab de Ligação ao GTP/genética
2.
Immunol Lett ; 228: 129-134, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33096140

RESUMO

Abnormal activation of the transcriptional factor STAT3 (signal transducer and activator of transcription 3) was recently associated with Alzheimer Disease (AD). STAT3 phosphorylation is critical for cytokine secretion linked to neuroinflammation. Moreover, STAT3 may act as a transcriptional regulator of BACE1 (ß-APP cleaving enzyme-1), the key enzyme in amyloid ß (Aß) production. We have previously shown that neuroinflammation and increased brain BACE1 levels triggered by LPS-induced systemic inflammation in wild-type mice are associated with an enhanced STAT3 activation. Using this LPS model, the goal of this study was to investigate if a STAT3 inhibitor administration could be protective against neuroinflammation and abnormal BACE1 regulation. Our results show that intraperitoneal injection of Stattic, a molecule that selectively inhibits the activation of STAT3, decreases LPS-induced microglial activation in the hippocampus. In addition, STAT3 inhibition reduced brain levels of cytokines IL-6, IL-1ß and TNF-α triggered by LPS systemic administration. A significant reduction of BACE1 levels was observed in the hippocampus of mice treated with LPS and Stattic compared to those exposed to LPS alone. Taking together, our results show that Stattic can protect hippocampus against two pathological hallmarks of AD, and pave the way for further explorations of the therapeutic potential of STAT3 inhibition in AD.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Anti-Inflamatórios/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Óxidos S-Cíclicos/farmacologia , Hipocampo/efeitos dos fármacos , Inflamação/tratamento farmacológico , Microglia/efeitos dos fármacos , Neuroimunomodulação/efeitos dos fármacos , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Hipocampo/enzimologia , Hipocampo/imunologia , Inflamação/induzido quimicamente , Inflamação/enzimologia , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Microglia/enzimologia , Microglia/imunologia , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
3.
Cell Death Dis ; 10(3): 190, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30804327

RESUMO

Fas-associated death domain (FADD) is a key adaptor molecule involved in numerous physiological processes including cell death, proliferation, innate immunity and inflammation. Therefore, changes in FADD expression have dramatic cellular consequences. In mice and humans, FADD regulation can occur through protein secretion. However, the molecular mechanisms accounting for human FADD secretion were still unknown. Here we report that canonical, non-canonical, but not alternative, NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion. NLRP3 inflammasome activation by the bacterial toxin nigericin led to the proinflammatory interleukin-1ß (IL-1ß) release and to the induction of cell death by pyroptosis. However, we showed that FADD secretion could occur in absence of increased IL-1ß release and pyroptosis and, reciprocally, that IL-1ß release and pyroptosis could occur in absence of FADD secretion. Especially, FADD, but not IL-1ß, secretion following NLRP3 inflammasome activation required extracellular glucose. Thus, FADD secretion was an active process distinct from unspecific release of proteins during pyroptosis. This FADD secretion process required K+ efflux, NLRP3 sensor, ASC adaptor and CASPASE-1 molecule. Moreover, we identified FADD as a leaderless protein unconventionally secreted through microvesicle shedding, but not exosome release. Finally, we established human soluble FADD as a new marker of joint inflammation in gout and rheumatoid arthritis, two rheumatic diseases involving the NLRP3 inflammasome. Whether soluble FADD could be an actor in these diseases remains to be determined. Nevertheless, our results advance our understanding of the mechanisms contributing to the regulation of the FADD protein expression in human cells.


Assuntos
Artrite Reumatoide/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Gota/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/efeitos dos fármacos , Animais , Caspase 1/metabolismo , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nigericina/farmacologia , Canais de Potássio/metabolismo
4.
Cell Cycle ; 17(15): 1901-1916, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30109813

RESUMO

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the sensor inositol-requiring enzyme 1α (IRE1), an endoribonuclease that splices the mRNA of the transcription factor XBP1 (X-box-binding protein 1). To better understand the protein network that regulates the activity of the IRE1 pathway, we systematically screened the proteins that interact with IRE1 and identified a ribonuclease inhibitor called ribonuclease/angiogenin inhibitor 1 (RNH1). RNH1 is a leucine-rich repeat domains-containing protein that binds to and inhibits ribonucleases. Immunoprecipitation experiments confirmed this interaction. Docking experiments indicated that RNH1 physically interacts with IRE1 through its cytosolic RNase domain. Upon ER stress, the interaction of RNH1 with IRE1 in the ER increased at the expense of the nuclear pool of RNH1. Inhibition of RNH1 expression using siRNA mediated RNA interference upon ER stress led to an increased splicing activity of XBP1. Modulation of IRE1 RNase activity by RNH1 was recapitulated in a cell-free system, suggesting direct regulation of IRE1 by RNH. We conclude that RNH1 attenuates the activity of IRE1 by interacting with its ribonuclease domain. These findings have implications for understanding the molecular mechanism by which IRE1 signaling is attenuated upon ER stress.


Assuntos
Proteínas de Transporte/metabolismo , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Transporte/farmacologia , Linhagem Celular Transformada , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Modelos Moleculares , Domínios Proteicos , Proteoma , Splicing de RNA , Uromodulina/metabolismo
5.
Theranostics ; 7(11): 2757-2774, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28824714

RESUMO

The growing field of cardio-oncology addresses the side effects of cancer treatment on the cardiovascular system. Here, we explored the cardiotoxicity of the antiangiogenic therapy, sunitinib, in the mouse heart from a diagnostic and therapeutic perspective. We showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis and reduced left ventricular ejection fraction as demonstrated by echocardiography. The capacity of positron emission tomography with [18F]fluorodeoxyglucose to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment. Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes. Co-administration of the endothelin receptor antagonist, macitentan, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis. These results support the endothelin system in mediating the cardiotoxic effects of sunitinib and endothelin receptor antagonism as a potential therapeutic approach to prevent cardiotoxicity. Furthermore, metabolic and functional imaging can monitor the cardiotoxic effects and the benefits of endothelin antagonism in a theranostic approach.


Assuntos
Antineoplásicos/administração & dosagem , Antagonistas dos Receptores de Endotelina/metabolismo , Indóis/administração & dosagem , Miocárdio/metabolismo , Pirróis/administração & dosagem , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Anaerobiose , Animais , Antineoplásicos/efeitos adversos , Glicólise , Indóis/efeitos adversos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Proteoma/análise , Pirróis/efeitos adversos , Sunitinibe
6.
Elife ; 62017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28287395

RESUMO

p27Kip1 (p27) is a cyclin-CDK inhibitor and negative regulator of cell proliferation. p27 also controls other cellular processes including migration and cytoplasmic p27 can act as an oncogene. Furthermore, cytoplasmic p27 promotes invasion and metastasis, in part by promoting epithelial to mesenchymal transition. Herein, we find that p27 promotes cell invasion by binding to and regulating the activity of Cortactin, a critical regulator of invadopodia formation. p27 localizes to invadopodia and limits their number and activity. p27 promotes the interaction of Cortactin with PAK1. In turn, PAK1 promotes invadopodia turnover by phosphorylating Cortactin, and expression of Cortactin mutants for PAK-targeted sites abolishes p27's effect on invadopodia dynamics. Thus, in absence of p27, cells exhibit increased invadopodia stability due to impaired PAK1-Cortactin interaction, but their invasive capacity is reduced compared to wild-type cells. Overall, we find that p27 directly promotes cell invasion by facilitating invadopodia turnover via the Rac1/PAK1/Cortactin pathway.


Assuntos
Cortactina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Podossomos/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Linhagem Celular , Movimento Celular , Humanos , Camundongos
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