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1.
Nat Commun ; 12(1): 476, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473107

RESUMO

Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.


Assuntos
Autofagia/fisiologia , Dano ao DNA/fisiologia , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Apoptose , Caenorhabditis elegans , Linhagem Celular , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Drosophila , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fosforilação , Transcriptoma , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
2.
Nat Commun ; 12(1): 339, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436626

RESUMO

Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/química , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/química , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Biocatálise , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteína 1 do Complexo Esclerose Tuberosa/ultraestrutura , Proteína 2 do Complexo Esclerose Tuberosa/ultraestrutura
3.
J Ethnopharmacol ; 266: 113404, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32976970

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Danqi Pill, composed of the root of Salvia miltiorrhiza Bunge and the root of Panax notoginseng, is effective in the clinical treatment of myocardial ischemia in coronary heart diseases. A number of studies have shown that autophagy plays an essential role in cardiac function and energy metabolism, and disordered autophagy is associated with the progression of heart failure. However, the effect and mechanism of Danqi pill on autophagy have not been reported yet. AIM OF THE STUDY: This study aims to elucidate whether Danqi pill restores autophagy to protect against HF and its potential mechanism. MATERIALS AND METHODS: Left anterior descending ligation was established to induce an HF rat model, H2O2-stimulated H9C2 cells model was conducted to clarify the effects and potential mechanism of Danqi pill. In vivo, Danqi pill (1.5 g/kg) were orally administered for four weeks and Fenofibrate (10 mg/kg) was selected as a positive group. In vitro, Danqi pill (10-200 µg/mL) was pre-cultured for 24 h and co-cultured with H2O2 stimulation for 4 h. Importantly, transmission electron microscopy and fluorescence GPF-mRFP-LC3 reporter system were combined to monitor autophagy flux. Furtherly, we utilized Compound C, a specific AMPK inhibitor, to validate whether the autophagy was mediated by AMPK-TSC2-mTOR pathway. RESULTS: Danqi pill significantly improved cardiac function and myocardial injury in HF rats. Intriguingly, Danqi pill potently regulated autophagy mainly by promoting the formation of autophagosomes in vivo. Further results demonstrated that expressions of p-AMPK (P < 0.001) and p-TSC2 (P < 0.001) in cardiac tissue were upregulated by Danqi pill, accompanied with downregulation of p-mTOR (P < 0.01) and p-ULK1(P < 0.01). In parallel with the vivo experiment, in vitro study indicated that Danqi pill dramatically restored autophagy flux and regulated expressions of critical autophagy-related molecules. Finally, utilization of Compound C abrogated the effects of Danqi pill on autophagy flux and the expressions of p-TSC2 (P < 0.05), p-mTOR (P < 0.01) and p-ULK1 (P < 0.05). CONCLUSION: Danqi pill could improve cardiac function and protect against cardiomyocytes injury by restoring autophagy via regulating the AMPK-TSC2-mTOR signaling pathway.


Assuntos
Autofagia/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Insuficiência Cardíaca/prevenção & controle , Infarto do Miocárdio/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Insuficiência Cardíaca/etiologia , Masculino , Infarto do Miocárdio/complicações , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
4.
Gene ; 757: 144943, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32652105

RESUMO

The growth of animal skeletal muscle is mainly determined by the synthesis processes of total proteins in skeletal muscle cells, which has a significant impact on the postnatal growth of young animals. An increasing number of studies are focusing on the functions of Tuberous sclerosis complex 2 (TSC2) during the process of cell protein synthesis and growth. However, it is still unclear the effect of whether and how TSC2 on goat myoblasts proliferation and differentiation. Here, we found that TSC2 gene has opposite expression patterns in proliferation and differentiation of myoblasts. An expression vector containing goat TSC2 cDNA sequences linked with pcDNA3.1 plasmid was constructed. Myoblasts proliferation activity was significantly inhibited and cell cycle transition slowed down after the transfection of pcDNA3.1-TSC2 plasmid into goat primary myoblasts by EdU staining, CCK-8 and flow cytometry. Mechanically, we further confirmed that the overexpression TSC2 was able to down-regulate the mRNA and protein expression of mechanistic target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 (p70S6K) and some cell cycle related genes. In addition, the expression of myogenic genes and myotube formation were attenuated. Collectively, all our results of the experiment demonstrate that TSC2 could regulate myoblasts cells proliferation and differentiation via the activation of the mTOR/p70S6K signaling pathway.


Assuntos
Diferenciação Celular , Proliferação de Células , Desenvolvimento Muscular , Mioblastos/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cabras , Mioblastos/citologia , Mioblastos/fisiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Regulação para Cima
5.
Nature ; 585(7826): 597-602, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32612235

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.


Assuntos
Síndrome de Birt-Hogg-Dubé/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/deficiência , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patologia , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Células HeLa , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Especificidade por Substrato , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
6.
Life Sci ; 256: 117914, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512010

RESUMO

Hyperglycemia and oxidative stress are the primary stressors that elicit mitochondria specific cell stress in diabetes. Here we hypothesized that elevated level of ROS in high glucose (HG) environment, trigger mitochondrial stress by damaging mitochondrial DNA (mtDNA), altering inflammatory mediators, and neurodegenerative markers via stress signalling pathway in retinal ganglion cells (RGC-5). Mechanistically, our findings illustrated that the HG environment increases the ROS production in retinal cells leading to the disruption of antioxidant defence mechanism, and altering mitochondrial machinery such as an increase in loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, and increase in mtDNA fragmentation. Furthermore, fragmented mtDNA escape from mitochondria into the cytosol, where it engaged with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN gene (STING) phosphorylation and activate interferon regulatory factor 3 (IRF3) via ERK1/2-Akt-tuberin-mTOR dependent pathways. Our results further indicate that siRNA-mediated gene silencing of tuberin suppresses the strong downregulation of tuberin-mTOR-IRF3 activation. HG environment resulted in activation of IRF3, coinciding with the increased expression of inflammatory mediators and neurodegenerative markers. Pre-treatment of N-acetyl-l-cysteine (NAC) or ERK1/2 or phosphoinositide3-kinase (PI3-K)/Akt inhibitors in RGC-5 cells significantly reduced the HG-induced IRF3 expression and declined the expression of neurodegenerative markers. Collectively, our results demonstrates that HG-induced over production of ROS, disrupts the antioxidant defence mechanism and mitochondrial dysfunction, leading to alterations of inflammatory mediators and neurodegenerative markers through the ERK1/2-Akt-tuberin-mTOR dependent signalling pathway in RGC-5 cells.


Assuntos
Inibidores Enzimáticos/metabolismo , Glucose/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Ganglionares da Retina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Acetilcisteína/metabolismo , Animais , DNA Mitocondrial , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperglicemia/metabolismo , Inflamação/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
7.
PLoS One ; 15(2): e0228894, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32078667

RESUMO

Lymphangioleiomyomatosis (LAM) is a devastating lung disease caused by inactivating gene mutations in either TSC1 or TSC2 that result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). As LAM occurs predominantly in women during their reproductive age and is exacerbated by pregnancy, the female hormonal environment, and in particular estrogen, is implicated in LAM pathogenesis and progression. However, detailed underlying molecular mechanisms are not well understood. In this study, utilizing human pulmonary LAM specimens and cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we tested the hypothesis that estrogen promotes the growth of mTORC1-hyperactive cells through pyruvate kinase M2 (PKM2). Estrogen increased the phosphorylation of PKM2 at Ser37 and induced the nuclear translocation of phospho-PKM2. The estrogen receptor antagonist Faslodex reversed these effects. Restoration of TSC2 inhibited the phosphorylation of PKM2 in an mTORC1 inhibitor-insensitive manner. Finally, accumulation of phosphorylated PKM2 was evident in pulmonary nodule from LAM patients. Together, our data suggest that female predominance of LAM might be at least in part attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. Targeting metabolic regulators of PKM2 might have therapeutic benefits for women with LAM and other female-specific neoplasms.


Assuntos
Estrogênios/metabolismo , Piruvato Quinase/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Animais , Linhagem Celular Tumoral , Estrogênios/fisiologia , Feminino , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/fisiopatologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosforilação , Piruvato Quinase/fisiologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/genética
8.
Genet Test Mol Biomarkers ; 24(1): 1-5, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31855466

RESUMO

Objective: Tuberous sclerosis complex (TSC) is a multisystem disease. Variants in the TSC1 and TSC2 genes have been reported to be associated with TSC and are considered pathogenic. The purpose of this study was to determine the genetic mutations and expression patterns of TSC1 and TSC2 in 21 Chinese patients suffering from TSC who were clinically characterized by epilepsy. Methods: Peripheral blood samples were taken from 21 patients, their parents, and other family members. Their TSC1 and TSC2 genes were sequenced through next-generation sequencing to identify all variants. Results: We identified variants in 17/21 patients in either their TSC1 or TSC2 genes: 6 patients had TSC1 mutations and 11 had TSC2 mutations. There were 13 spontaneous mutations, and 3 that had been inherited from a parent. The mutations were classified by types: there were three missense mutations, five frameshift mutations, two splice site mutations, four nonsense mutations, two single codon deletions resulting the loss of an amino acid, and one large fragment deletion. Six of the mutations have not been previously reported. Conclusion: The genotypic analysis of Chinese TSC patients who are clinically characterized by epilepsy can potentially be useful for genetic counseling and prenatal diagnoses for patients and their families.


Assuntos
Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Esclerose Tuberosa/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , China/epidemiologia , Códon sem Sentido , Análise Mutacional de DNA/métodos , Epilepsia/genética , Éxons , Família , Feminino , Mutação da Fase de Leitura , Humanos , Masculino , Mutação , Mutação de Sentido Incorreto , Linhagem , Transcriptoma/genética , Esclerose Tuberosa/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/genética
9.
Dig Dis Sci ; 65(6): 1689-1699, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31863340

RESUMO

BACKGROUND: Gastrointestinal motility disorder is an important pathological basis for functional dyspepsia (FD). Epigastric ache and discomfort are the main symptoms of FD, and ghrelin deficiency is closely related to the occurrence and development of FD. While electroacupuncture (EA) alleviated the symptoms of FD patients and improved their quality of life, there is a lack of sufficient mechanistic evidence to support these beneficial effects. METHODS: An in vivo FD model was established in wild-type and mammalian target of rapamycin (mTOR) knockout (-/-) rats. FD rats were subjected to EA with or without mTOR agonists or inhibitors. Gastric emptying and intestinal propulsion were assessed, and pathological changes in the hypothalamus, gastric antrum, and small intestine were examined histologically. In addition, ghrelin expression and AMPK/TSC2/Rheb/mTOR activation were detected by quantitative reverse transcription polymerase chain reaction and western blot. RESULTS: EA alone or in combination with mTOR inhibitors improved gastrointestinal function in FD rats by increasing the rates of intestinal propulsion and gastric emptying, and pathological changes in the hypothalamus, gastric antrum, and small intestine were alleviated. This may be related to the significant upregulation of ghrelin expression and the effective activation of the AMPK/TSC2/Rheb/mTOR signaling pathway. Interestingly, EA also improved gastrointestinal function and ghrelin expression in mTOR (-/-) KO FD rats. CONCLUSION: Altering the level of ghrelin by regulating AMPK/TSC2/Rheb-mediated mTOR inhibition is an important way through which EA treats FD. The complex EA-mediated regulatory mechanisms of the brain-gut axis still require further exploration.


Assuntos
Adenilato Quinase/metabolismo , Dispepsia/terapia , Eletroacupuntura , Grelina/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Adenilato Quinase/genética , Animais , Dispepsia/metabolismo , Esvaziamento Gástrico , Deleção de Genes , Regulação da Expressão Gênica , Grelina/genética , Humanos , Hipotálamo , Intestino Delgado/patologia , Leucina/farmacologia , Masculino , Distribuição Aleatória , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Ratos , Ratos Sprague-Dawley , Estômago/patologia , Estresse Psicológico , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Regulação para Cima
10.
J Biol Chem ; 295(1): 263-274, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31767684

RESUMO

Mammalian target of rapamycin complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of tuberous sclerosis complex subunit 2 (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.


Assuntos
Aminoácidos/deficiência , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ácidos Fosfatídicos/metabolismo , Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Endocitose , Humanos , Camundongos , Fosfolipase D/metabolismo , Transporte Proteico , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
11.
Autophagy ; 16(3): 435-450, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31203721

RESUMO

Particulate matter (PM) is able to induce airway epithelial injury, while the detailed mechanisms remain unclear. Here we demonstrated that PM exposure inactivated MTOR (mechanistic target of rapamycin kinase), enhanced macroautophagy/autophagy, and impaired lysosomal activity in HBE (human bronchial epithelial) cells and in mouse airway epithelium. Genetic or pharmaceutical inhibition of MTOR significantly enhanced, while inhibition of autophagy attenuated, PM-induced IL6 expression in HBE cells. Consistently, club-cell-specific deletion of Mtor aggravated, whereas loss of Atg5 in bronchial epithelium reduced, PM-induced airway inflammation. Interestingly, the augmented inflammatory responses caused by MTOR deficiency were markedly attenuated by blockage of downstream autophagy both in vitro and in vivo. Mechanistically, the dysregulation of MTOR-autophagy signaling was partially dependent on activation of upstream TSC2, and interacted with the TLR4-MYD88 to orchestrate the downstream NFKB activity and to regulate the production of inflammatory cytokines in airway epithelium. Moreover, inhibition of autophagy reduced the expression of EPS15 and the subsequent endocytosis of PM. Taken together, the present study provides a mechanistic explanation for how airway epithelium localized MTOR-autophagy axis regulates PM-induced airway injury, suggesting that activation of MTOR and/or suppression of autophagy in local airway might be effective therapeutic strategies for PM-related airway disorders.Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; ALI: air liquid interface; AP2: adaptor related protein complex 2; ATG: autophagy related; BALF: bronchoalveolar lavage fluid; COPD: chronic obstructive pulmonary disease; CXCL: C-X-C motif chemokine ligand; DOX: doxycycline; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EPS15: epidermal growth factor receptor pathway substrate 15; HBE: human bronchial epithelial; H&E: hematoxylin & eosin; IKK: IKB kinase; IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTEC: mouse tracheal epithelial cells; MTOR: mechanistic target of rapamycin kinase; MYD88: MYD88 innate immune signal transduction adaptor; NFKB: nuclear factor of kappa B; NFKBIA: NFKB inhibitor alpha; PM: particulate matter; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RELA: RELA proto-oncogene, NFKB subunit; SCGB1A1: secretoglobin family 1A member 1; siRNA: small interfering RNAs; SQSTM1: sequestosome 1; TEM: transmission electronic microscopy; TLR4: toll like receptor 4; TSC2: TSC complex subunit 2.


Assuntos
Autofagia , Células Epiteliais/patologia , Material Particulado/toxicidade , Pneumonia/induzido quimicamente , Pneumonia/patologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína 5 Relacionada à Autofagia/metabolismo , Brônquios/patologia , Linhagem Celular , Citocinas/metabolismo , Endocitose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Deleção de Genes , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
12.
Biol Res ; 52(1): 58, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783925

RESUMO

BACKGROUND: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. METHODS: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. RESULTS: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. CONCLUSION: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , Serina-Treonina Quinases TOR/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Animais , Apoptose/genética , Autofagia/genética , Western Blotting , Imunoprecipitação da Cromatina , Imunofluorescência , Camundongos , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
13.
Int J Mol Sci ; 20(22)2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703252

RESUMO

Cellular homeostasis is controlled by an evolutionary conserved cellular digestive process called autophagy. This mechanism is tightly regulated by the two sensor elements called mTORC1 and AMPK. mTORC1 is one of the master regulators of proteostasis, while AMPK maintains cellular energy homeostasis. AMPK is able to promote autophagy by phosphorylating ULK1, the key inducer of autophagosome formation, while mTORC1 downregulates the self-eating process via ULK1 under nutrient rich conditions. We claim that the feedback loops of the AMPK-mTORC1-ULK1 regulatory triangle guarantee the appropriate response mechanism when nutrient and/or energy supply changes. In our opinion, there is an essential double negative feedback loop between mTORC1 and AMPK. Namely, not only does AMPK downregulate mTORC1, but mTORC1 also inhibits AMPK and this inhibition is required to keep AMPK inactive at physiological conditions. The aim of the present study was to explore the dynamical characteristic of AMPK regulation upon various cellular stress events. We approached our scientific analysis from a systems biology perspective by incorporating both theoretical and molecular biological techniques. In this study, we confirmed that AMPK is essential to promote autophagy, but is not sufficient to maintain it. AMPK activation is followed by ULK1 induction, where protein has a key role in keeping autophagy active. ULK1-controlled autophagy is always preceded by AMPK activation. With both ULK1 depletion and mTORC1 hyper-activation (i.e., TSC1/2 downregulation), we demonstrate that a double negative feedback loop between AMPK and mTORC1 is crucial for the proper dynamic features of the control network. Our computer simulations have further proved the dynamical characteristic of AMPK-mTORC1-ULK1 controlled cellular nutrient sensing.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Morte Celular Autofágica/fisiologia , Retroalimentação Fisiológica/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Ativação Enzimática/fisiologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
14.
Toxicol Appl Pharmacol ; 384: 114772, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676321

RESUMO

The injury of intestinal epithelial barrier is considered as the key pathophysiological process in response to gastrointestinal infection and inflammation, and plays an important role in the initiation and development of colitis. Alpinetin has been shown to improve intestinal barrier homeostasis under colitis condition, but the mechanism is still unclear. Here, we showed that alpinetin significantly improved transepithelial electrical resistance (TEER) in TNF-α-stimulated Caco-2 cells, which was mainly mediated by inhibiting the apoptosis. Mechanistic studies demonstrated that alpinetin markedly increased the production of autophagosomes, along with obvious regulation of LC3B-II, beclin-1, p62, Atg7 and Atg5 expressions. In addition, it also markedly repressed the activation of mTORC1 signaling pathway, which was ascribed to TSC2 rather than p-AKT, p-ERK, p-AMPKα or PTEN expressions in Caco-2 and NCM460 cells. Furthermore, the enrichment of H3K9me3 at TSC2 promoter region was decreased and ubiquitin proteasome degradation of suv39h1 was increased. Additionally, alpinetin activated aryl hydrocarbon receptor (AhR) and promoted co-localization of AhR with suv39h1 in the cytoplasm. The relationship between alpinetin-regulated AhR/suv39h1/TSC2/mTORC1 signals, autophagy and apoptosis of Caco-2 and NCM460 cells was confirmed by using CH223191, siAhR, siTSC2 and chloroquine. Finally, CH223191 and leucine abolished alpinetin-mediated inhibition of intestinal epithelial cells apoptosis, improvement of intestinal epithelial barrier and amelioration of colitis.


Assuntos
Autofagia/efeitos dos fármacos , Colite Ulcerativa/tratamento farmacológico , Flavanonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Autofagia/imunologia , Células CACO-2 , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Flavanonas/uso terapêutico , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metiltransferases/metabolismo , Camundongos , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/imunologia , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
15.
Endocrinology ; 160(12): 3001-3017, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31599935

RESUMO

A growing body of evidence implies a pivotal role of sirtuin-1 (Sirt1) in chondrocyte function and homeostasis; however, its underlying mechanisms mediating chondrogenesis, which is an essential process for physiological skeletal growth, are still poorly understood. In the current study, we generated TamCartSirt1-/- [Sirt1 conditional knockout (cKO)] mice to explore the role of Sirt1 during postnatal endochondral ossification. Compared with control mice, cKO mice exhibited growth retardation associated with inhibited chondrocyte proliferation and hypertrophy, as well as activated apoptosis. These effects were regulated by hyperactivation of mammalian target of rapamycin complex 1 (mTORC1) signaling, and thereby inhibition of autophagy and induction of endoplasmic reticulum stress in growth plate chondrocytes. IP injection of the mTORC1 inhibitor rapamycin to mice with Sirt1 deletion partially neutralized such inhibitory effects of Sirt1 ablation on longitudinal bone growth, indicating the causative link between SIRT1 and mTORC1 signaling in the growth plate. Mechanistically, SIRT1 interacted with tuberous sclerosis complex 2 (TSC2), a key upstream negative regulator of mTORC1 signaling, and loss of Sirt1 inhibited TSC2 expression, resulting in hyperactivated mTORC1 signaling in chondrocytes. In conclusion, our findings suggest that loss of Sirt1 may trigger mTORC1 signaling in growth plate chondrocytes and contributes to growth retardation, thus indicating that SIRT1 is an important regulator during chondrogenesis and providing new insights into the clinical potential of SIRT1 in bone development.


Assuntos
Condrócitos/fisiologia , Lâmina de Crescimento/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Osteogênese , Sirtuína 1/fisiologia , Animais , Desenvolvimento Ósseo , Condrogênese , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
16.
J Biol Chem ; 294(45): 16729-16739, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548312

RESUMO

The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser474; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model where Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.


Assuntos
Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Núcleo Celular/metabolismo , Proteína Forkhead Box O1/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Insulina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
17.
Theranostics ; 9(17): 4946-4958, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410193

RESUMO

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , MicroRNAs/genética , Paraganglioma/genética , Transcriptoma , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Metástase Neoplásica , Paraganglioma/metabolismo , Paraganglioma/patologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Células Tumorais Cultivadas
18.
Am J Physiol Renal Physiol ; 317(5): F1201-F1210, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31461347

RESUMO

Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36-37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Fibrose/patologia , Doenças Renais Policísticas/patologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linhagem da Célula , Feminino , Fibrose/genética , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Miofibroblastos/fisiologia , Doenças Renais Policísticas/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
19.
Mar Drugs ; 17(7)2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31277207

RESUMO

The purpose of the present study is to improve the endothelial progenitor cells (EPC) activation, proliferation, and angiogenesis using enzyme-aided extraction of fucoidan by amyloglucosidase (EAEF-AMG). Enzyme-aided extraction of fucoidan by AMG (EAEF-AMG) significantly increased EPC proliferation by reducing the reactive oxygen species (ROS) and decreasing apoptosis. Notably, EAEF-AMG treated EPCs repressed the colocalization of TSC2/LAMP1 and promoted perinuclear localization of mTOR/LAMP1 and mTOR/Rheb. Moreover, EAEF-AMG enhanced EPC functionalities, including tube formation, cell migration, and wound healing via regulation of AKT/Rheb signaling. Our data provided cell priming protocols to enhance therapeutic applications of EPCs using bioactive compounds for the treatment of CVD.


Assuntos
Células Progenitoras Endoteliais/efeitos dos fármacos , Glucana 1,4-alfa-Glucosidase/metabolismo , Polissacarídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Progenitoras Endoteliais/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Cicatrização/efeitos dos fármacos
20.
Zhongguo Zhong Yao Za Zhi ; 44(11): 2348-2352, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31359662

RESUMO

The aim of this paper was to investigate the effect of SIRT1/TSC_2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg_1. CD34~+CD38~- leukemia stem cells(CD34~+CD38~-LSCs) was isolated by magnetic cell sorting(MACS) and divided into two groups. The control group cells were routinely cultured, 40 µmol·L~(-1) ginsenoside Rg_1 was added to the control group for co-culture in Rg_1 group. The effect of Rg_l to induce CD34~+CD38~-LSCs senescence were evaluated by senescence-associated ß-Galactosidase(SA-ß-Gal) staining, cell cycle assay, CCK-8 and Colony-Assay. The expression of senescence associated SIRT1, TSC_2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR) and Western blot. The results showed that the CD34~+CD38~-LSCs could effectively be isolated by MACS, and the purity of CD34~+CD38~-LSCs is up to(95.86±3.04)%. Compared with the control group, the percentage of positive cells expressed SA-ß-Gal in the Rg_1 group is increased, the senescence morphological changes were observed in the CD34~+CD38~-LSCs in the Rg_1 group. The proliferation inhibition rate and the number of cells entered G_0/G_1 phase in the Rg_1 group were increased, but the colony-formed ability was decreased, Rg_1 could significantly inhibit the proliferation and self-renewal ability of CD34~+CD38~-LSCs. The expression of SIRT1 and TSC_2 mRNA and protein were down regulated in the Rg_1 group compared with the control group. Our research implied that Rg_1 may induce the senescence of CD34~+CD38~-LSCs and SIRT1/TSC_2 signal axis plays a significant role in this process.


Assuntos
Senescência Celular/efeitos dos fármacos , Ginsenosídeos/farmacologia , Leucemia Mieloide Aguda , Células-Tronco Neoplásicas/efeitos dos fármacos , Transdução de Sinais , Sirtuína 1/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Humanos , Células Tumorais Cultivadas
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