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1.
Nat Commun ; 13(1): 3426, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35701499

RESUMO

Regulation of endosomal Toll-like receptor (TLR) responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases, with CXCL4 proposed to potentiate TLR responses by binding to nucleic acid TLR ligands and facilitating their endosomal delivery. Here we report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively amplifying inflammatory gene transcription and interleukin (IL)-1ß production, while partially attenuating the interferon response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activates TBK1 and IKKε, repurposes these kinases towards an inflammatory response via coupling with IRF5, and activates the NLRP3 inflammasome. CXCL4 signaling, in a cooperative and synergistic manner with TLR8, induces chromatin remodeling and activates de novo enhancers associated with inflammatory genes. Our findings thus identify new regulatory mechanisms of TLR responses relevant for cytokine storm, and suggest targeting the TBK1-IKKε-IRF5 axis may be beneficial in inflammatory diseases.


Assuntos
Quinase I-kappa B , Fatores Reguladores de Interferon , Monócitos , Fator Plaquetário 4 , Proteínas Serina-Treonina Quinases , Receptor 8 Toll-Like , Epigênese Genética , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fator Plaquetário 4/imunologia , Fator Plaquetário 4/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 8 Toll-Like/genética , Receptor 8 Toll-Like/imunologia , Receptor 8 Toll-Like/metabolismo
2.
Metallomics ; 8(9): 920-930, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27714068

RESUMO

Copper (Cu) is an important trace element required for the activity of essential enzymes. However, excess Cu compromises the redox balance in cells and tissues causing serious toxicity. The process of disposal of excess Cu from organisms relies on the activity of Cu-transporting ATPase ATP7B. ATP7B is mainly expressed in liver hepatocytes where it sequesters the potentially toxic metal and mediates its excretion into the bile. Mutations in the ATP7B gene cause Wilson disease (WD), which is characterized by the accumulation of toxic Cu in the liver due to the scarce expression of ATP7B as well as the failure of ATP7B mutants to pump Cu and/or traffic to the Cu-excretion sites. The most frequent ATP7B mutant, H1069Q, still presents a significant Cu-transporting activity, but undergoes retention within the endoplasmic reticulum (ER) where the mutant is rapidly degraded. Expression of this ATP7B mutant has been recently reported to activate the p38 and JNK stress kinase pathways, which, in turn, trigger quality control mechanisms leading to the arrest of ATP7B-H1069Q in the ER and to the acceleration of its degradation. However, the main molecular players operating in these p38/JNK-dependent ER quality control pathways remain to be discovered. By using a combination of RNAseq, bioinformatics and RNAi approaches, we found a cluster of ER quality control genes whose expression is controlled by p38 and JNK and is required for the efficient retention of the ATP7B-H1069Q mutant in the ER. Silencing these genes reduced the accumulation of the ATP7B mutant in the ER and facilitated the mutant sorting and export to the Golgi and post-Golgi copper excretion sites. In sum, our findings reveal the ER-associated genes that could be utilized for the correction of ATP7B mutants and, hence, for the normalization of Cu homeostasis in Wilson disease.


Assuntos
Biomarcadores/análise , ATPases Transportadoras de Cobre/genética , Cobre/efeitos adversos , Retículo Endoplasmático/patologia , Degeneração Hepatolenticular/etiologia , Mutação , Biologia de Sistemas , Transporte Biológico , Cobre/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Células HeLa , Células Hep G2 , Degeneração Hepatolenticular/metabolismo , Degeneração Hepatolenticular/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
3.
Dis Model Mech ; 9(1): 25-38, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26747866

RESUMO

The deleterious effects of a disrupted copper metabolism are illustrated by hereditary diseases caused by mutations in the genes coding for the copper transporters ATP7A and ATP7B. Menkes disease, involving ATP7A, is a fatal neurodegenerative disorder of copper deficiency. Mutations in ATP7B lead to Wilson disease, which is characterized by a predominantly hepatic copper accumulation. The low incidence and the phenotypic variability of human copper toxicosis hamper identification of causal genes or modifier genes involved in the disease pathogenesis. The Labrador retriever was recently characterized as a new canine model for copper toxicosis. Purebred dogs have reduced genetic variability, which facilitates identification of genes involved in complex heritable traits that might influence phenotype in both humans and dogs. We performed a genome-wide association study in 235 Labrador retrievers and identified two chromosome regions containing ATP7A and ATP7B that were associated with variation in hepatic copper levels. DNA sequence analysis identified missense mutations in each gene. The amino acid substitution ATP7B:p.Arg1453Gln was associated with copper accumulation, whereas the amino acid substitution ATP7A:p.Thr327Ile partly protected against copper accumulation. Confocal microscopy indicated that aberrant copper metabolism upon expression of the ATP7B variant occurred because of mis-localization of the protein in the endoplasmic reticulum. Dermal fibroblasts derived from ATP7A:p.Thr327Ile dogs showed copper accumulation and delayed excretion. We identified the Labrador retriever as the first natural, non-rodent model for ATP7B-associated copper toxicosis. Attenuation of copper accumulation by the ATP7A mutation sheds an interesting light on the interplay of copper transporters in body copper homeostasis and warrants a thorough investigation of ATP7A as a modifier gene in copper-metabolism disorders. The identification of two new functional variants in ATP7A and ATP7B contributes to the biological understanding of protein function, with relevance for future development of therapy.


Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Cobre/toxicidade , Modelos Animais de Doenças , Degeneração Hepatolenticular/genética , Síndrome dos Cabelos Torcidos/genética , Sequência de Aminoácidos , Animais , ATPases Transportadoras de Cobre , Cães , Retículo Endoplasmático/metabolismo , Feminino , Variação Genética , Estudo de Associação Genômica Ampla , Genótipo , Células Hep G2 , Humanos , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fenótipo , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
4.
Hepatology ; 63(6): 1842-59, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26660341

RESUMO

UNLABELLED: Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans-Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum-retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c-Jun N-terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7B(H1069Q) (as well as that of several other WD-causing mutants) from the endoplasmic reticulum to the trans-Golgi network compartment, in recovery of its Cu-dependent trafficking, and in reduction of intracellular Cu levels. CONCLUSION: Our findings indicate p38 and c-Jun N-terminal kinase as intriguing targets for correction of WD-causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842-1859).


Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Degeneração Hepatolenticular/genética , Sistema de Sinalização das MAP Quinases , Cobre/metabolismo , ATPases Transportadoras de Cobre , Células HeLa , Células Hep G2 , Degeneração Hepatolenticular/metabolismo , Humanos , Fígado/metabolismo , Mutação , Via Secretória
5.
Dev Cell ; 29(6): 686-700, 2014 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-24909901

RESUMO

Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Exocitose/fisiologia , Complexo de Golgi/metabolismo , Homeostase/fisiologia , Lisossomos/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Bile/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Células Cultivadas , ATPases Transportadoras de Cobre , Complexo Dinactina , Imunofluorescência , Células HeLa , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Transporte Proteico , RNA Interferente Pequeno/genética
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