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1.
Nat Commun ; 12(1): 1955, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782410

RESUMO

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.


Assuntos
Autofagia/genética , Proteínas de Ciclo Celular/genética , Chaperonas Moleculares/genética , Agregados Proteicos , Proteína Sequestossoma-1/genética , Superóxido Dismutase-1/genética , Animais , Autofagia/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Transgenes , Xenopus laevis
2.
Nat Commun ; 12(1): 16, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397898

RESUMO

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.


Assuntos
Autofagossomos/metabolismo , Estresse Oxidativo , Proteína Sequestossoma-1/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/ultraestrutura , Autofagia , Linhagem Celular , Géis , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fígado/lesões , Fígado/patologia , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica , Lipossomas Unilamelares
3.
Hum Genet ; 140(4): 649-666, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33389129

RESUMO

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.


Assuntos
Catarata/genética , Mutação de Sentido Incorreto , Sinais de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Peroxissomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico Ativo , Catarata/congênito , Catarata/metabolismo , Cromossomos Humanos Par 12 , Consanguinidade , Feminino , Ligação Genética , Humanos , Cristalino/metabolismo , Masculino , Camundongos , Camundongos Knockout , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Proteína Sequestossoma-1/metabolismo , Sequenciamento Completo do Exoma
4.
Cell Prolif ; 54(2): e12975, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33372336

RESUMO

OBJECTIVES: Diabetes is a risk factor for intervertebral disc degeneration (IVDD). Studies have demonstrated that diabetes may affect IVDD through transcriptional regulation; however, whether post-transcriptional regulation is involved in diabetic IVDD (DB-IVDD) is still unknown. This study was performed to illustrate the role of HuR, an RNA-binding protein, in DB-IVDD development and its mechanism. MATERIALS AND METHODS: The expression of HuR was evaluated in nucleus pulposus (NP) tissues from diabetic IVDD patients and in high glucose-treated NP cells. Senescence and autophagy were assessed in HuR over-expressing and downregulation NP cells. The mRNAs that were regulated by HuR were screened, and immunoprecipitation was applied to confirm the regulation of HuR on targeted mRNAs. RESULTS: The results showed that the expression of HuR was decreased in diabetic NP tissues and high glucose-treated NP cells. Downregulation of HuR may lead to increased senescence in high glucose-treated NP cells, while autophagy activation attenuates senescence in HuR deficient NP cells. Mechanistic study showed that HuR prompted Atg7 mRNA stability via binding to the AU-rich elements. Furthermore, overexpression of Atg7, but not HuR, may ameliorate DB-IVDD in rats in vivo. CONCLUSIONS: In conclusion, HuR may suppress senescence through autophagy activation via stabilizing Atg7 in diabetic NP cells; while Atg7, but not HuR, may serve as a potential therapeutic target for DB-IVDD.


Assuntos
Proteína 7 Relacionada à Autofagia/metabolismo , Autofagia , Senescência Celular , Proteína Semelhante a ELAV 1/metabolismo , Degeneração do Disco Intervertebral/patologia , Regiões 3' não Traduzidas , Animais , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/genética , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Glucose/farmacologia , Humanos , Degeneração do Disco Intervertebral/etiologia , Degeneração do Disco Intervertebral/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Sequestossoma-1/metabolismo
5.
Life Sci ; 266: 118884, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310038

RESUMO

AIMS: Growing evidence indicates insufficient autophagy is crucial to airway remodeling in asthma. However, it is uncertain whether p62, an autophagy major regulator, mediates the airway remodeling process. This study aimed to evaluate the role and underlying mechanism of p62 in airway remodeling in asthma. MATERIALS AND METHODS: Airway remodeling was confirmed via histopathology. Western blotting and RT-PCR were used to detect the expression of autophagic and glycolytic proteins, as well as glycolytic genes. Glycolysis was measured by glucose consumption and lactate production. Cell proliferation was analyzed by CCK8 assays while and the scratch test and transwell method were used for cell migration. KEY FINDINGS: We found that insufficient autophagic flux and increased p62 expression existed in chronic asthma mice. Additionally, knockdown of p62 inhibited asthmatic human bronchial smooth muscle cells (BSMCs) proliferation and migration in vitro. To elucidate the underlying mechanism of p62-mediated autophagy flux in directing BSMCs function, we demonstrated that knockdown of p62 decreased the glucose consumption and lactate production in BSMCs, whereas p62 overexpression had the opposite effect. Furthermore, we showed that p62 regulated glycolysis in BSMCs by the mTOR/c-Myc/hexokinase 2 (HK2) pathway. SIGNIFICANCE: Our findings suggest that p62 is involved in BSMCs proliferation and migration via the mTOR/c-Myc/HK2-mediated glycolysis, thereby providing a new target for airway remodeling treatment.


Assuntos
Remodelação das Vias Aéreas , Asma/patologia , Autofagia , Reprogramação Celular , Modelos Animais de Doenças , Miócitos de Músculo Liso/patologia , Proteína Sequestossoma-1/metabolismo , Animais , Asma/induzido quimicamente , Asma/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Glicólise , Hexoquinase/genética , Hexoquinase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/metabolismo , Ovalbumina/toxicidade , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Sequestossoma-1/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
6.
Int J Oral Sci ; 12(1): 34, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318468

RESUMO

Epithelial-mesenchymal transition (EMT) is involved in both physiological and pathological processes. EMT plays an essential role in the invasion, migration and metastasis of tumours. Autophagy has been shown to regulate EMT in a variety of cancers but not in head and neck squamous cell carcinoma (HNSCC). Herein, we investigated whether autophagy also regulates EMT in HNSCC. Analyses of clinical data from three public databases revealed that higher expression of fibronectin-1 (FN1) correlated with poorer prognosis and higher tumour pathological grade in HNSCC. Data from SCC-25 cells demonstrated that rapamycin and Earle's balanced salt solution (EBSS) promoted autophagy, leading to increased FN1 degradation, while 3-methyladenine (3-MA), bafilomycin A1 (Baf A1) and chloroquine (CQ) inhibited autophagy, leading to decreased FN1 degradation. On the other hand, autophagic flux was blocked in BECN1 mutant HNSCC Cal-27 cells, and rapamycin did not promote autophagy in Cal-27 cells; also in addition, FN1 degradation was inhibited. Further, we identified FN1 degradation through the lysosome-dependent degradation pathway using the proteasome inhibitor MG132. Data from immunoprecipitation assays also showed that p62/SQSTM1 participated as an autophagy adapter in the autophagy-lysosome pathway of FN1 degradation. Finally, data from immunoprecipitation assays demonstrated that the interaction between p62 and FN1 was abolished in p62 mutant MCF-7 and A2780 cell lines. These results indicate that autophagy significantly promotes the degradation of FN1. Collectively, our findings clearly suggest that FN1, as a marker of EMT, has adverse effects on HNSCC and elucidate the autophagy-lysosome degradation mechanism of FN1.


Assuntos
Fibronectinas , Neoplasias Ovarianas , Autofagia , Linhagem Celular Tumoral , Feminino , Humanos , Lisossomos/metabolismo , Proteína Sequestossoma-1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço
7.
Autophagy ; 16(11): 2114-2116, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32960686

RESUMO

Proteasome inhibition (PSMI) is known to activate macroautophagy (autophagy hereafter), but the underlying mechanisms remain to be fully delineated. Here we discuss our recent work identifying an important PPP3/calcineurin-TFEB-SQSTM1/p62 pathway in mediating activation of autophagy by PSMI, a compensatory process for the heart with proteasome malfunction. Through increasing PPP3/calcineurin activity and inhibiting MTOR signaling, PSMI promotes the dephosphorylation and thereby nuclear translocation of TFEB, resulting in transactivation of genes in the autophagic-lysosomal pathway (ALP) such as Mcoln1 and Sqstm1. We have discovered that SQSTM1 is required for not only induction of autophagy but also cardiac activation of TFEB by PSMI, unveiling a novel feedforward role for SQSTM1 in TFEB activation.


Assuntos
Autofagia , Complexo de Endopeptidases do Proteassoma , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Calcineurina/metabolismo , Macroautofagia , Proteína Sequestossoma-1/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(32): 19190-19200, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723828

RESUMO

The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.


Assuntos
Macroautofagia , Organelas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Humanos , Organelas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
9.
Autophagy ; 16(10): 1915-1917, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32686580

RESUMO

Hepatocellular carcinoma (HCC) is the consequence of chronic liver damage caused by the excessive generation of reactive oxygen species (ROS). To mitigate the deleterious effects of ROS, cells activate the transcription factor NFE2L2/NRF2, which is constitutively degraded through its partner KEAP1. The inactivation of KEAP1 by ROS results in the upregulation of NFE2L2, which leads to the upregulation of critical detoxifying molecules that serve to keep ROS at tolerable levels in order to maintain cell viability. It is thought that this mechanism allows cells to accumulate mutations, which together with the additional pro-tumorigenic and pro-survival effects of NFE2L2 activation, promote cancer initiation and progression. Germane to this phenomenon is macroautophagy/autophagy, which under homeostatic conditions has also been proposed to serve as a detoxifying mechanism by clearing up toxic aggregates and damaged organelles. Our recent data establish a new paradigm for the role that autophagy plays in HCC development.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Autofagia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo , Proteína Sequestossoma-1/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32513711

RESUMO

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
11.
Nat Commun ; 11(1): 2306, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385399

RESUMO

During ß-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62Δ69-251 mice, global p62-/- and Ucp1-Cre p62flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Proteína Sequestossoma-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adipogenia/fisiologia , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/diagnóstico por imagem , Tecido Adiposo Branco/metabolismo , Animais , Núcleo Celular/metabolismo , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Desacopladora 1/metabolismo
12.
Mol Carcinog ; 59(8): 967-979, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32424979

RESUMO

Drug resistance is the leading cause for rapid progression and relapse in small-cell lung cancer (SCLC) patients. Thus overcoming drug resistance still remains to be urgently resolved during SCLC treatment. Here, we found p62/SQSTM1 was enriched in SCLC spheroids, a subpopulation possessing cancer stem-like properties, which is responsible for cancer relapse and metastasis. Subsequent functional assays in vitro showed that short hairpin RNA (shRNA)-mediated p62 knockdown increased sensitivity of SCLC cell lines to cisplatin (DDP), whereas lentivirus-mediated p62 ectopic overexpression diminished DDP-induced cytotoxicity in both NCI-H446 and NCI-H1688 cell lines. Moreover, ectopic p62 overexpression promoted DDP resistance of NCI-H446 cells-derived tumor xenografts in immunodeficient mice in vivo, as indicated by accelerated tumor growth rate and reduced fluorescent activity of cleaved caspase-3. Gene expression profiling analysis revealed that p62 was positively correlated with neuronal precursor cell-expressed, developmentally downregulated gene 9 (NEDD9) expression level. Consistently, NEDD9 messenger RNA (mRNA) level was decreased upon p62 suppression by small interfering RNA (siRNA) and increased with p62 transient overexpression in SCLC cell lines, suggesting that p62 positively regulated NEDD9 mRNA. Depletion of NEDD9 by siRNA, to a large extent, reversed p62-overexpressed SCLC cells to DDP-induced cytotoxicity, implying NEDD9 might act as a downstream target which was in charge of p62-mediated DDP resistance. Taken together, our findings uncovered a previously unknown role of p62 in the regulation of SCLC drug resistance, assigning p62 as an attractive target for SCLC treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Sequestossoma-1/antagonistas & inibidores , Carcinoma de Pequenas Células do Pulmão/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Interferente Pequeno/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Chem Biol Interact ; 329: 109094, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32278738

RESUMO

BACKGROUND: Oxidative stress in cardiac myocytes is an important pathogenesis of cardiac lipotoxicity. Autophagy is a cellular self-digestion process that can selectively remove damaged organelles under oxidative stress, and thus presents a potential therapeutic target against cardiac lipotoxicity. Globular CTRP9 (gCTRP9) is a newly identified adiponectin paralog with established metabolic regulatory properties. The aim of this work is to investigate whether autophagy participates the protection effects of gCTRP9 in neonatal rat cardiac myocytes (NRCMs) under oxidative stress and the underlying mechanism. RESULTS: NRCMs were treated with PA of various concentrations for indicated time period. Our results showed that PA enhanced intracellular ROS accumulation, decreased mitochondrial membrane potential (Δψm) and increased activation of caspases 3. These changes suggested lipotoxicity due to excessive PA. In addition, PA was observed to impair autophagic flux in NRCMs and impaired autophagosome clearance induced by PA contributes to cardiomyocyte death. Besides, we found that gCTRP9 increased the ratio of LC3II/I and the expression of ATG5 which was vital to the formation of autophagosomes and decreased the level of P62, suggesting enhanced autophagic flux in the absence or presence of PA. The result was further confirmed by the methods of infection with LC3-mRFP-GFP lentivirus and blockage of autophagosome-lysosome fusion by BafA1. Moreover, gCTRP9 reestablished the loss of mitochondrial membrane potential, suppressed ROS generation, and reduced PA -induced myocyte death. However, the protective effect of gCTRP9 on the cardiac lipotoxicity was partly abolished by blockade of autophagy by autophagy-related 5 (ATG5) siRNA, indicating that the effect of gCTRP9 on cell survival is critically mediated through regulation of autophagy. CONCLUSION: Autophagy induction by gCTRP9 could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.


Assuntos
Adiponectina/metabolismo , Autofagia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/farmacologia , Animais , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/metabolismo
14.
Eur J Med Chem ; 193: 112231, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32193054

RESUMO

p62/SQSTM1 (hereafter as p62) is a stress-inducible cellular protein, which interacts with various signaling proteins to regulate a variety of cellular functions. Growing lines of evidence supported a critical role of p62 in tumorigenesis, and p62 may become a therapeutic target for tumor. In this review, we summarize biological functions of structural domains of p62, reported bioactive molecules targeting p62, and the relationship between p62 and tumorigenesis.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Proteína Sequestossoma-1/antagonistas & inibidores , Antineoplásicos/química , Humanos , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Sequestossoma-1/metabolismo
15.
J Stroke Cerebrovasc Dis ; 29(5): 104743, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32127256

RESUMO

BACKGROUND: Bone marrow stromal cell (BMSC) transplantation is a promising therapeutic approach for cerebral ischemia, as it elicits multiple neuroprotective effects. However, it remains unclear how BMSC transplantation modulates the ubiquitin-proteasome system (UPS) and autophagy under cerebral ischemia. METHODS: In the present study, an intermediate level of cerebral ischemia (30 minutes) was chosen to examine the effect of BMSC transplantation on the molecular switch regulating UPS and autophagy. BMSC or vehicle was stereotactically injected into the penumbra 15 minutes after sham operation or transient middle cerebral artery occlusion (tMCAO). RESULTS: Thirty minutes of tMCAO artery occlusion significantly increased TUNEL-, ubiquitin-, and p62-positive cells (which peaked at 72 hours, 2 hours, and 2 hours after reperfusion, respectively) and ratios of both BAG3/BAG1 and LC3-II/LC3-I at 24 hours after reperfusion. However, intracerebral injection of BMSCs significantly reduced infarct volume and numbers of TUNEL- and p62-positive cells, and improved BAG3/BAG1 and LC3-II/LC3-I ratios. In addition, observed increases in ubiquitin-positive cells 2 hours after reperfusion were slightly suppressed by BMSC transplantation. CONCLUSIONS: These data suggest a protective role of BMSC transplantation, which drove the molecular switch from autophagy to UPS in a murine model of ischemic stroke.


Assuntos
Autofagia , Encéfalo/enzimologia , Infarto da Artéria Cerebral Média/cirurgia , Transplante de Células-Tronco Mesenquimais , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Encéfalo/patologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/metabolismo , Ubiquitinação
16.
Circ J ; 84(4): 616-625, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32115441

RESUMO

BACKGROUND: Ischemic postconditioning (IPostC) is an endogenous protective mechanism to reduce ischemia-reperfusion (I/R) injury. However, whether IPostC protects aged cardiomyocytes against I/R injury is not fully understood. Considering the protective function of microRNA 30a (miR-30a) against ischemia-induced injury in H9C2 cells, its role in the protective effects of IPostC on I/R injury of aged cardiomyocytes was investigated further.Methods and Results:To mimic I/R and IPostC in vitro, the aged cardiomyocyte model for hypoxia postconditioning (HPostC) treatment was established by 9 days of incubation with 8 mg/mL D-galactose and then followed by exposure to hypoxic environment. HPostC significantly alleviated hypoxia/reoxygenation (H/R) injury and reduced autophagy of aged cardiomyocytes, as evidenced by decreased LC3B-II expression and increased p62 by Western blot. Quantified by quantitative real-time polymerase chain reaction (qRT-PCR), miR-30a was increased in aged cardiomyocytes treated with HPostC compared with I/R injury group. Overexpression of miR-30a by LV3-rno-miR-30a mimic promoted cardioprotective effect of HPostC in aged cardiomyocytes by suppressing BECN1-mediated autophagy, all of which was abrogated by knockdown of miR-30a expression. Epigenetic analyses demonstrated that HPostC reduced DNA methyltransferase 3b-mediated DNA hypomethylation levels at miR-30a promoter, leading to upregulation of miR-30a. CONCLUSIONS: HPostC protected aged cardiomyocytes survival against H/R injury via DNMT3b-dependent activation of miR-30a. miR-30a could be a potential therapeutic target for ischemic myocardial infarction.


Assuntos
Autofagia , Senescência Celular , Metilação de DNA , Epigênese Genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Hipóxia Celular , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Regiões Promotoras Genéticas , Ratos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
17.
Cell Mol Life Sci ; 77(17): 3341-3350, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32193608

RESUMO

It is well-established that Lysine-specific demethylase 1 (LSD1, also known as KDM1A) roles as a lysine demethylase canonically acting on H3K4me1/2 and H3K9me1/2 for regulating gene expression. Though the discovery of non-histone substrates methylated by LSD1 has largely expanded the functions of LSD1 as a typical demethylase, recent groundbreaking studies unveiled its non-catalytic functions as a second life for this demethylase. We and others found that LSD1 is implicated in the interaction with a line of proteins to exhibit additional non-canonical functions in a demethylase-independent manner. Here, we present an integrated overview of these recent literatures charging LSD1 with unforeseen functions to re-evaluate and summarize its non-catalytic biological roles beyond the current understanding of its demethylase activity. Given LSD1 is reported to be ubiquitously overexpressed in a variety of tumors, it has been generally considered as an innovative target for cancer therapy. We anticipate that these non-canonical functions of LSD1 will arouse the consideration that extending the LSD1-based drug discovery to targeting LSD1 protein interactions non-catalytically, not only its demethylase activity, may be a novel strategy for cancer prevention.


Assuntos
Histona Desmetilases/metabolismo , Autofagia , Desmetilação , Proteína 7 com Repetições F-Box-WD/metabolismo , Histona Desmetilases/química , Histonas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitinação
18.
Cell Mol Life Sci ; 77(18): 3547-3565, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32072237

RESUMO

Reductive stress is defined as a condition of sustained increase in cellular glutathione/glutathione disulfide and NADH/NAD+ ratios. Reductive stress is emerging as an important pathophysiological event in several diseased states, being as detrimental as is oxidative stress. Occurrence of reductive stress has been documented in several cardiomyopathies and is an important pathophysiological factor particularly in coronary artery disease and myocardial infarction. Excess activation of the transcription factor, Nrf2-the master regulator of the antioxidant response-, consequent in most cases to defective autophagy, can lead to reductive stress. In addition, hyperglycemia-induced activation of the polyol pathway can lead to increased NADH/NAD+ ratio, which might translate into increased levels of hydrogen sulfide-via enhanced activity of cystathionine ß-synthase-that would fuel reductive stress through inhibition of mitochondrial complex I. Reductive stress may be either a potential weapon against cancer priming tumor cells to apoptosis or a cancer's ally promoting tumor cell proliferation and making tumor cells resistant to reactive oxygen species-inducing drugs. In non-cancer pathological states reductive stress is definitely harmful paradoxically leading to reactive oxygen species overproduction via excess NADPH oxidase 4 activity. In face of the documented occurrence of reductive stress in several heart diseases, there is much less information about the occurrence and effects of reductive stress in skeletal muscle tissue. In the present review we describe relevant results emerged from studies of reductive stress in the heart and review skeletal muscle conditions in which reductive stress has been experimentally documented and those in which reductive stress might have an as yet unrecognized pathophysiological role. Establishing whether reductive stress has a (patho)physiological role in skeletal muscle will hopefully contribute to answer the question whether antioxidant supplementation to the general population, athletes, and a large cohort of patients (e.g. heart, sarcopenic, dystrophic, myopathic, cancer, and bronco-pulmonary patients) is harmless or detrimental.


Assuntos
Células Musculares/metabolismo , Estresse Oxidativo , Antioxidantes/farmacologia , Autofagia , Glutationa/metabolismo , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/metabolismo
19.
Toxicology ; 435: 152408, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32057834

RESUMO

PURPOSE: To investigate the effects and mechanisms of NADPH on Kainic acid (KA)-induced excitotoxicity. METHODS: KA, a non-N-methyl-d-aspartate glutamate receptor agonist, was exposed to adult SD rats via intrastriatal injection and rat primary cortical neurons to establish excitotoxic models in vivo and in vitro, respectively. To determine the effects of NADPH on KA-induced excitotoxicity, neuronal survival, neurologically behavioral score and oxidative stress were evaluated. To explore the mechanisms of neuroprotective effects of NADPH, the autophagy-lysosome pathway related proteins were detected. RESULTS: In vivo, NADPH (1 mg/kg or 2 mg/kg) diminished KA (2.5 nmol)-induced enlargement of lesion size in striatum, improved KA-induced dyskinesia and reversed KA-induced activation of glial cells. Nevertheless, the neuroprotective effect of NADPH was not significant under the condition of autophagy activation. NADPH (2 mg/kg) inhibited KA (2.5 nmol)-induced down-regulation of TP-53 induced glycolysis and apoptosis regulator (TIGAR) and p62, and up-regulation of the protein levels of LC3-II/LC3-I, Beclin-1 and Atg5. In vitro, the excitotoxic neuronal injury was induced after KA (50 µM, 100 µM or 200 µM) treatment as demonstrated by decreased cell viability. Moreover, KA (100 µM) increased the intracellular levels of calcium and reactive oxygen species (ROS) and declined the levels of the reduced form of glutathione (GSH). Pretreatment of NADPH (10 µM) effectively reversed these changes. Meanwhile NADPH (10 µM) inhibited KA (100 µM)-induced down-regulation of TIGAR and p62, and up-regulation of the ratio of LC3-II/LC3-I, Beclin-1, Atg5, active-cathepsin B and active-cathepsin D. CONCLUSIONS: Our data provide a possible mechanism that NADPH ameliorates KA-induced excitotoxicity by blocking the autophagy-lysosome pathway and up-regulating TIGAR along with its antioxidant properties.


Assuntos
Autofagia/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Caínico/toxicidade , Lisossomos/efeitos dos fármacos , NADP/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/metabolismo
20.
Open Biol ; 10(2): 190307, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32070232

RESUMO

RNA-binding proteins typically change the fate of RNA, such as stability, translation or processing. Conversely, we recently uncovered that the small non-coding vault RNA 1-1 (vtRNA1-1) directly binds to the autophagic receptor p62/SQSTM1 and changes the protein's function. We refer to this process as 'riboregulation'. Here, we discuss this newly uncovered vault RNA function against the background of three decades of vault RNA research. We highlight the vtRNA1-1-p62 interaction as an example of riboregulation of a key cellular process.


Assuntos
RNA não Traduzido/genética , Proteína Sequestossoma-1/metabolismo , Animais , Autofagia , Regulação da Expressão Gênica , Humanos , Estabilidade de RNA , RNA não Traduzido/química
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