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1.
Nat Chem Biol ; 13(12): 1239-1244, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28991239

RESUMO

Tim17 and Tim23 are the main subunits of the TIM23 complex, one of the two major essential mitochondrial inner-membrane protein translocon machineries (TIMs). No chemical probes that specifically inhibit TIM23-dependent protein import were known to exist. Here we show that the natural product stendomycin, produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of the TIM23 complex in yeast and mammalian cells. Furthermore, stendomycin-mediated blockage of the TIM23 complex does not alter normal processing of the major regulatory mitophagy kinase PINK1, but TIM23 is required to stabilize PINK1 on the outside of mitochondria to initiate mitophagy upon membrane depolarization.


Assuntos
Proteínas Mitocondriais/metabolismo , Peptídeos/farmacologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Peptídeos Catiônicos Antimicrobianos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Estrutura Molecular , Peptídeos/química , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
2.
Mol Aspects Med ; 82: 101018, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34489092

RESUMO

Autophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity.


Assuntos
Lisossomos , Doenças Neurodegenerativas , Autofagia/genética , Humanos , Doenças Neurodegenerativas/genética , Neurônios
3.
Sci Rep ; 11(1): 19396, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588551

RESUMO

Fibrosis is characterized by the excessive production of collagen and other extracellular matrix (ECM) components and represents a leading cause of morbidity and mortality worldwide. Previous studies of nonalcoholic steatohepatitis (NASH) with fibrosis were largely restricted to bulk transcriptome profiles. Thus, our understanding of this disease is limited by an incomplete characterization of liver cell types in general and hepatic stellate cells (HSCs) in particular, given that activated HSCs are the major hepatic fibrogenic cell population. To help fill this gap, we profiled 17,810 non-parenchymal cells derived from six healthy human livers. In conjunction with public single-cell data of fibrotic/cirrhotic human livers, these profiles enable the identification of potential intercellular signaling axes (e.g., ITGAV-LAMC1, TNFRSF11B-VWF and NOTCH2-DLL4) and master regulators (e.g., RUNX1 and CREB3L1) responsible for the activation of HSCs during fibrogenesis. Bulk RNA-seq data of NASH patient livers and rodent models for liver fibrosis of diverse etiologies allowed us to evaluate the translatability of candidate therapeutic targets for NASH-related fibrosis. We identified 61 liver fibrosis-associated genes (e.g., AEBP1, PRRX1 and LARP6) that may serve as a repertoire of translatable drug target candidates. Consistent with the above regulon results, gene regulatory network analysis allowed the identification of CREB3L1 as a master regulator of many of the 61 genes. Together, this study highlights potential cell-cell interactions and master regulators that underlie HSC activation and reveals genes that may represent prospective hallmark signatures for liver fibrosis.


Assuntos
Células Estreladas do Fígado , Hepatopatia Gordurosa não Alcoólica , Transcriptoma , Animais , Voluntários Saudáveis , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Análise de Célula Única
4.
Sci Rep ; 8(1): 1799, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29379065

RESUMO

We generated induced pluripotent stem cells (iPSCs) from patient fibroblasts to yield cell lines containing varying degrees of heteroplasmy for a m.13514 A > G mtDNA point mutation (2 lines) and for a ~6 kb single, large scale mtDNA deletion (3 lines). Long term culture of the iPSCs containing a single, large-scale mtDNA deletion showed consistent increase in mtDNA deletion levels with time. Higher levels of mtDNA heteroplasmy correlated with increased respiratory deficiency. To determine what changes occurred in deletion level during differentiation, teratomas comprising all three embryonic germ layers were generated from low (20%) and intermediate heteroplasmy (55%) mtDNA deletion clones. Regardless of whether iPSCs harbouring low or intermediate mtDNA heteroplasmy were used, the final levels of heteroplasmy in all teratoma germ layers increased to a similar high level (>60%). Thus, during human stem cell division, cells not only tolerate high mtDNA deletion loads but seem to preferentially replicate deleted mtDNA genomes. This has implications for the involvement of mtDNA deletions in both disease and ageing.


Assuntos
DNA Mitocondrial/genética , Deleção de Sequência/genética , Diferenciação Celular/genética , Linhagem Celular , Células Clonais/metabolismo , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/genética , Mutação Puntual/genética
5.
Oncogene ; 24(14): 2277-88, 2005 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-15688020

RESUMO

Arsenite trioxide (As2O3) induces apoptosis in several cell lines by disturbing key signal transduction pathways through its oxidative properties. Here, we report that As2O3 also induces the phosphorylation of the retinoid receptor RXRalpha, subsequent to oxidative damages and the activation of the stress-activated protein kinases cascade (JNKs). We also report that RA amplifies both As2O3-induced phosphorylation of RXRalpha and apoptosis. Taking advantage of 'rescue' F9 cell lines expressing RXRalpha mutated at its phosphorylation sites, in an RXRalpha null background, we provide evidence that RXRalpha is a key element involved in that potentiating effect. Finally, we demonstrate that As2O3 also abrogates the transactivation of RA-target genes.


Assuntos
Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Óxidos/farmacologia , Receptor X Retinoide alfa/metabolismo , Tretinoína/farmacologia , Animais , Trióxido de Arsênio , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Primers do DNA , Dimerização , Camundongos , Fosforilação , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Cell Signal ; 17(10): 1229-39, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16038797

RESUMO

The retinoid response is mediated by two classes of nuclear receptors, the retinoic acid receptors (RARalpha, beta, and gamma) and the retinoid X receptors (RXRalpha, beta, and gamma) which act as ligand-dependent heterodimeric RAR/RXR transcription activators. Like most transcription factors, RARs and RXRs are regulated by phosphorylation processes. Here, we report that stress agents induce RXRalpha phosphorylation, subsequently to the activation of the stress-activated protein kinases cascade (JNKs). This phosphorylation process concerns three residues located in the N-terminal AF-1 domain of RXRalpha and one located in the omega loop of the Ligand Binding Domain. To decipher how stress-induced RXRalpha phosphorylation influences the transcription of RA-target genes, we used a ribotoxic stress agent, anisomycin, which activates signaling kinases without promoting DNA or protein damages, at subinhibitory concentrations. Taking advantage of vectors expressing recombinant RXRalpha mutated at its phosphorylation sites and of F9 cell lines re-expressing the same RXRalpha mutants in an RXRalpha null background, we provide evidence that stress signaling modulates RAR/RXRalpha-mediated transcription, through the phosphorylation of RXRalpha at the residue located in the Omega loop, in a promoter context-dependent manner.


Assuntos
Regiões Promotoras Genéticas/genética , Receptor X Retinoide alfa/fisiologia , Tretinoína/farmacologia , Animais , Anisomicina/farmacologia , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Sistema Enzimático do Citocromo P-450/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Mutação , Fosforilação/efeitos dos fármacos , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/genética , Ácido Retinoico 4 Hidroxilase , Receptor alfa de Ácido Retinoico , Receptor X Retinoide alfa/agonistas , Receptor X Retinoide alfa/metabolismo , Serina/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptor gama de Ácido Retinoico
7.
Gene ; 328: 1-16, 2004 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-15019979

RESUMO

The pleiotropic effects of retinoids are mediated by nuclear retinoid receptors (RARs and RXRs) which are ligand-activated transcription factors. In response to retinoid binding, RAR/RXR heterodimers undergo major conformational changes and orchestrate the transcription of specific gene networks, through binding to specific DNA response elements and recruiting cofactor complexes that act to modify local chromatin structure and/or engage the basal transcription machinery. Then the degradation of RARs and RXRs by the ubiquitin-proteasome controls the magnitude and the duration of the retinoid response. RARs and RXRs also integrate a variety of signaling pathways through phosphorylation events which cooperate with the ligand for the control of retinoid-target genes transcription. These different modes of regulation reveal unexpected levels of complexity in the dynamics of retinoid-dependent transcription.


Assuntos
Receptores do Ácido Retinoico/metabolismo , Retinoides/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação/genética , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica , Humanos , Modelos Genéticos , Complexos Multienzimáticos/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma , Transcrição Gênica/genética , Ubiquitina/metabolismo
8.
Nat Cell Biol ; 16(11): 1069-79, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25327288

RESUMO

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.


Assuntos
Autofagia/fisiologia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Ferritinas/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Humanos , Lisossomos/metabolismo , Camundongos , Fagossomos/metabolismo , Ligação Proteica
9.
Mol Cell Biol ; 30(2): 481-95, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19917724

RESUMO

The target of rapamycin (TOR) complex 1 (TORC1) signaling pathway is a critical regulator of translation and cell growth. To identify novel components of this pathway, we performed a kinome-wide RNA interference (RNAi) screen in Drosophila melanogaster S2 cells. RNAi targeting components of the p38 stress-activated kinase cascade prevented the cell size increase elicited by depletion of the TOR negative regulator TSC2. In mammalian and Drosophila tissue culture, as well as in Drosophila ovaries ex vivo, p38-activating stresses, such as H(2)O(2) and anisomycin, were able to activate TORC1. This stress-induced TORC1 activation could be blocked by RNAi against mitogen-activated protein kinase kinase 3 and 6 (MKK3/6) or by the overexpression of dominant negative Rags. Interestingly, p38 was also required for the activation of TORC1 in response to amino acids and growth factors. Genetic ablation either of p38b or licorne, its upstream kinase, resulted in small flies consisting of small cells. Mutants with mutations in licorne or p38b are nutrition sensitive; low-nutrient food accentuates the small-organism phenotypes, as well as the partial lethality of the p38b null allele. These data suggest that p38 is an important positive regulator of TORC1 in both mammalian and Drosophila systems in response to certain stresses and growth factors.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Alelos , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Peróxido de Hidrogênio/farmacologia , Larva/genética , Larva/metabolismo , Masculino , Mutação , Ovário/citologia , Ovário/enzimologia , Oxidantes/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Interferência de RNA/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sirolimo/farmacologia , Tamoxifeno/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genética
10.
Environ Sci Technol ; 43(23): 8908-15, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19943665

RESUMO

Growing concern over the contribution of freshwater reservoirs to increases in atmospheric greenhouse gas (GHG) concentrations and the relevance of long-term continuous measurements has led Fisheries and Oceans Canada, in conjunction with Manitoba Hydro, to develop continuous GHG monitors. Continuous water pCO(2), pCH(4), and pO(2) measurements were gathered to estimate gas fluxes in one temperate reservoir (Riviere-des-Prairies) and two boreal reservoirs (Eastmain-1 and Robert-Bourassa) in Quebec, and in four boreal reservoirs (Grand Rapids, Jenpeg, Kettle, and McArthur Falls) in Manitoba, Canada. Mean daily CO(2) fluxes ranged between 7 and 14 mmolCO(2)*m(-2)*d(-1) in Manitoba and between 15 and 55 mmolCO(2)*m(-2)*d(-1) in Quebec. Summertime episodes of water undersaturation in CO(2) were observed at Jenpeg, Kettle, and McArthur, suggesting higher productivities of these systems compared to the other systems studied. Mean daily CH(4) fluxes ranged between 0 and 69 micromolCH(4)*m(-2)*d(-1) in Manitoba and between 9 and 48 micromolCH(4)*m(-2)*d(-1) in Quebec. Comparisons of results obtained in the Eastmain-1 area using automated monitors, floating chambers or dissolved gas analyses over multiple-station field campaigns demonstrated that a continuous GHG monitor at a single sampling station provided representative and robust results.


Assuntos
Automação/métodos , Técnicas de Química Analítica/métodos , Água Doce/química , Gases/análise , Efeito Estufa , Dióxido de Carbono/análise , Manitoba , Metano/análise , Oxigênio/análise , Quebeque , Solubilidade , Fatores de Tempo
11.
Mol Cell ; 20(5): 673-85, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16337592

RESUMO

The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kinome. Two known Ras-regulated kinases were identified, phosphoinositide 3-kinase p110alpha and ribosomal protein S6 kinase p70(S6K1), plus the MAP kinase kinase kinase kinase MINK, which had not previously been implicated in Ras signaling. MINK is activated after Ras induction via a mechanism involving reactive oxygen species and mediates stimulation of the stress-activated protein kinase p38 MAPK downstream of the Raf/ERK pathway. p38 MAPK activation is essential for Ras-induced p21(WAF1/CIP1) upregulation and cell cycle arrest. MINK is thus a distal target of Ras signaling in the induction of a growth-arrested, senescent-like phenotype that may act to oppose oncogenic transformation in HOSE cells.


Assuntos
Células Epiteliais/enzimologia , Neoplasias Ovarianas/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/farmacologia , Interferência de RNA/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
J Biol Chem ; 277(32): 28683-9, 2002 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-12032153

RESUMO

Mouse F9 embryocarcinoma cells constitute a well established cell autonomous model system for investigating retinoic acid (RA) signaling in vitro. RA induces the differentiation of F9 cells grown as monolayers into endodermal-like cells and decreases their rate of proliferation. Knock-out of the retinoic X receptor alpha (RXRalpha) gene abolishes endodermal differentiation and the induction of several endogenous RA-responsive genes. RXRalpha null cells are also drastically impaired in their antiproliferative response to RA. The role of the RXRalpha phosphorylation site located in the N-terminal A region (Ser(22)) has been investigated here by establishing cell lines re-expressing RXRalpha either wild type or mutated at the phosphorylation site (RXRalphaS22A) in a RXRalpha-null background. We show that Ser(22) is dispensable for RA-induced endodermal differentiation but is crucial for the expression of several RA-responsive genes. Ser(22) is also indispensable for the antiproliferative effect of RA and necessary for the RA-induced down-regulation of p21(CIP) and p27(KIP) CKIs proteins that are known to be involved in the control of cell cycle progression.


Assuntos
Receptores do Ácido Retinoico/química , Fatores de Transcrição/química , Tretinoína/farmacologia , Animais , Sítios de Ligação , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/metabolismo , Regulação para Baixo , Endoderma/citologia , Immunoblotting , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/química , Serina/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Tretinoína/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo
13.
J Biol Chem ; 277(28): 24859-62, 2002 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-12032135

RESUMO

Nuclear retinoic acid (RA) receptors (RARs) are phosphorylated at conserved serine residues located in their N-terminal domain. Phosphorylation of RARgamma2 at these residues is increased in response to RA subsequently to the activation of p38MAPK. We show here that this RA-induced phosphorylation of RARgamma2 resulted from the down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. By overexpressing Akt and by using agents that activated or inhibited the PI3K/Akt pathway, we also demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targeted not only the phosphorylation of RARgamma2 but also the turnover and transcriptional activity of the receptor. Altogether these data indicate that the PI3K/Akt pathway plays an important role in retinoic acid signaling.


Assuntos
Regulação para Baixo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Receptores do Ácido Retinoico/fisiologia , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Linhagem Celular , Ativação Enzimática , Hidrólise , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Transcrição Gênica/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno , Receptor gama de Ácido Retinoico
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