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1.
Nat Metab ; 3(3): 410-427, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33758420

RESUMO

TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca2+-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB-GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB-GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Resistência à Insulina , Lisossomos/metabolismo , Obesidade/prevenção & controle , Estresse Fisiológico , Tecido Adiposo/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Obesidade/metabolismo
2.
Sci Total Environ ; 776: 145950, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33647641

RESUMO

Environmental BaP (benzo(a)pyrene) and its metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) inhibit proliferation of human villous trophoblast cells, which might further induce recurrent miscarriage (RM). However, the underlying mechanisms remain largely unknown. In this work, we identified a novel lncRNA HZ01 (lnc-HZ01) that is up-regulated in both RM tissues and BPDE-exposed trophoblast cells. Lnc-HZ01 inhibits trophoblast cell proliferation and induces miscarriage. Mechanistically, lnc-HZ01 promotes MXD1 mRNA transcription by up-regulating its transcription factor c-JUN and also enhances MXD1 protein stability by up-regulating its deubiquitin enzyme USP36. Reversely, MXD1 up-regulates lnc-HZ01 level by enhancing its RNA stability due to the increased level of m6A RNA methylation on lnc-HZ01, the first example that m6A modification regulates trophoblast cell functions. Thus, lnc-HZ01 and MXD1 comprise a positive self-feedback loop, which is up-regulated in both RM tissues and BPDE-exposed trophoblast cells. Once this loop is activated by BaP or BPDE exposure, both pathways in this loop would be up-regulated, promote EIF4E transcription, inhibit trophoblast cell proliferation, and further induce miscarriage. This work provides new clinical and scientific understanding in unexplained miscarriage.


Assuntos
Aborto Espontâneo , RNA Longo não Codificante , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proliferação de Células , Retroalimentação , Feminino , Humanos , Metilação , Gravidez , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Trofoblastos/metabolismo
3.
PLoS One ; 16(1): e0244794, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33439865

RESUMO

Understanding how fate specification of distinct cell-types from multipotent progenitors occurs is a fundamental question in embryology. Neural crest stem cells (NCSCs) generate extraordinarily diverse derivatives, including multiple neural, skeletogenic and pigment cell fates. Key transcription factors and extracellular signals specifying NCSC lineages remain to be identified, and we have only a little idea of how and when they function together to control fate. Zebrafish have three neural crest-derived pigment cell types, black melanocytes, light-reflecting iridophores and yellow xanthophores, which offer a powerful model for studying the molecular and cellular mechanisms of fate segregation. Mitfa has been identified as the master regulator of melanocyte fate. Here, we show that an Mitf-related transcription factor, Tfec, functions as master regulator of the iridophore fate. Surprisingly, our phenotypic analysis of tfec mutants demonstrates that Tfec also functions in the initial specification of all three pigment cell-types, although the melanocyte and xanthophore lineages recover later. We show that Mitfa represses tfec expression, revealing a likely mechanism contributing to the decision between melanocyte and iridophore fate. Our data are consistent with the long-standing proposal of a tripotent progenitor restricted to pigment cell fates. Moreover, we investigate activation, maintenance and function of tfec in multipotent NCSCs, demonstrating for the first time its role in the gene regulatory network forming and maintaining early neural crest cells. In summary, we build on our previous work to characterise the gene regulatory network governing iridophore development, establishing Tfec as the master regulator driving iridophore specification from multipotent progenitors, while shedding light on possible cellular mechanisms of progressive fate restriction.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Crista Neural/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diferenciação Celular , Linhagem da Célula , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Mutagênese , Crista Neural/citologia , Pigmentação/genética , RNA Guia/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Nucleic Acids Res ; 49(4): 1886-1899, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33476385

RESUMO

Methylglyoxal (MG) is a byproduct of glycolysis that functions in diverse mammalian developmental processes and diseases and in plant responses to various stresses, including salt stress. However, it is unknown whether MG-regulated gene expression is associated with an epigenetic modification. Here we report that MG methylglyoxalates H3 including H3K4 and increases chromatin accessibility, consistent with the result that H3 methylglyoxalation positively correlates with gene expression. Salt stress also increases H3 methylglyoxalation at salt stress responsive genes correlated to their higher expression. Following exposure to salt stress, salt stress responsive genes were expressed at higher levels in the Arabidopsis glyI2 mutant than in wild-type plants, but at lower levels in 35S::GLYI2 35S::GLYII4 plants, consistent with the higher and lower MG accumulation and H3 methylglyoxalation of target genes in glyI2 and 35S::GLYI2 35S::GLYII4, respectively. Further, ABI3 and MYC2, regulators of salt stress responsive genes, affect the distribution of H3 methylglyoxalation at salt stress responsive genes. Thus, MG functions as a histone-modifying group associated with gene expression that links glucose metabolism and epigenetic regulation.


Assuntos
Regulação da Expressão Gênica de Plantas , Código das Histonas , Aldeído Pirúvico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Epigênese Genética , Estresse Salino/genética , Fatores de Transcrição/metabolismo
5.
J Agric Food Chem ; 69(2): 686-697, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33369397

RESUMO

Targeting autophagy and lysosome may serve as a promising strategy for cancer therapy. Tea polysaccharide (TP) has shown promising antitumor effects. However, its mechanism remains elusive. Here, TP was found to have a significant inhibitory effect on the proliferation of colon cancer line HCT116 cells. RNA-seq analysis showed that TP upregulated autophagy and lysosome signal pathways, which was further confirmed through experiments. Immunofluorescence experiments indicated that TP activated transcription factor EB (TFEB), a key nuclear transcription factor modulating autophagy and lysosome biogenesis. In addition, TP inhibited the activity of mTOR, while it increased the expression of Lamp1. Furthermore, TP ameliorated the lysosomal damage and autophagy flux barrier caused by Baf A1 (lysosome inhibitor). Hence, our data suggested that TP repressed the proliferation of HCT116 cells by targeting lysosome to induce cytotoxic autophagy, which might be achieved through mTOR-TFEB signaling. In summary, TP may be used as a potential drug to overcome colon cancer.


Assuntos
Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Camellia sinensis/química , Neoplasias do Colo/fisiopatologia , Lisossomos/metabolismo , Extratos Vegetais/farmacologia , Polissacarídeos/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Morte Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Células HCT116 , Humanos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
6.
Int J Mol Sci ; 21(24)2020 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-33322239

RESUMO

MYC, a well-studied proto-oncogene that is overexpressed in >20% of tumors across all cancers, is classically known as "undruggable" due to its crucial roles in cell processes and its lack of a drug binding pocket. Four decades of research and creativity led to the discovery of a myriad of indirect (and now some direct!) therapeutic strategies targeting Myc. This review explores the various mechanisms in which Myc promotes cancer and highlights five key therapeutic approaches to disrupt Myc, including transcription, Myc-Max dimerization, protein stability, cell cycle regulation, and metabolism, in order to develop more specific Myc-directed therapies.


Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Dimerização , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Mutações Sintéticas Letais/genética , Regulação para Cima
7.
Elife ; 92020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33317695

RESUMO

Vaccines are powerful tools to develop immune memory to infectious diseases and prevent excess mortality. In older adults, however vaccines are generally less efficacious and the molecular mechanisms that underpin this remain largely unknown. Autophagy, a process known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show that autophagy is specifically induced in vaccine-induced antigen-specific CD8+ T cells in healthy human volunteers. In addition, reduced IFNγ secretion by RSV-induced T cells in older vaccinees correlates with low autophagy levels. We demonstrate that levels of the endogenous autophagy-inducing metabolite spermidine fall in human T cells with age. Spermidine supplementation in T cells from old donors recovers their autophagy level and function, similar to young donors' cells, in which spermidine biosynthesis has been inhibited. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. In summary, we have provided evidence for the importance of autophagy in vaccine immunogenicity in older humans and uncovered two novel drug targets that may increase vaccination efficiency in the aging context.


Assuntos
Envelhecimento/imunologia , Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Espermidina/farmacologia , Adjuvantes Imunológicos/farmacologia , Adulto , Idoso , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Humanos , Memória Imunológica/imunologia , Interferon gama/sangue , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Vírus Sinciciais Respiratórios/imunologia , Espermidina/sangue , Vacinação , Adulto Jovem
8.
Cell Death Dis ; 11(12): 1067, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318473

RESUMO

MircoRNA-21 (miR-21) was found to be highly expressed in various solid tumors, and its oncogenic properties have been extensively studied in recent years. However, the reason why miR-21 is highly expressed in various tumors remains elusive. Here, we found that the expression of miR-21 was negatively correlated with the expression of vacuole membrane protein-1 (VMP1) in colorectal cancer. Transcription of VMP1 activated either by small activating RNA (saRNA) or transcriptional activator GLI3 inhibited miR-21 expression through reducing its transcripts of VMP1-miR-21 and pri-miR-21, while no significant change in miR-21 expression after exogenous overexpression VMP1 in colorectal cancer cell HCT116. Considering the overlapping location of VMP1 and miR-21 gene in genome, the result suggested that the transcription of miR-21 was inhibited by the endogenous transcriptional activation of VMP1. Furthermore, we identified that miR-21 inhibited the activation and nuclear translocation of transcription factor EB (TFEB) through reducing the inhibitory of PTEN on AKT phosphorylation, which can directly activate the transcription of VMP1. Loss of miR-21 significantly increased VMP1 expression, which could be blocked by PTEN inhibitor (VO-Ohpic) or TFEB siRNA. These results showed that miR-21 negatively regulated VMP1 transcription through the PTEN/AKT/TFEB pathway, and TFEB-induced transcriptional activation of VMP1 could inhibit miR-21 expression, thus forming a feedback regulatory loop of miR-21/VMP1. We further found that disrupting the miR-21/VMP1 feedback loop will decrease the expression of miR-21, reduce the malignancy, and increase their sensitivity to 5-fluorouracil in colorectal cancer cells. Taken together, our results revealed a novel regulatory mechanism of miR-21 expression, and targeting the miR-21/VMP1 feedback loop may provide a new approach to inhibit miR-21 expression in colorectal cancer cells.


Assuntos
Neoplasias Colorretais/genética , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Autofagia/genética , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/ultraestrutura , Humanos , Proteínas de Membrana/genética , MicroRNAs/genética , Modelos Biológicos , PTEN Fosfo-Hidrolase/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transcrição Genética , Ativação Transcricional/genética
9.
Sci Rep ; 10(1): 22157, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33335227

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Although many drugs have entered clinical trials, few have shown effectiveness in the treatment of ALS. Other studies have shown that the stimulation of α7 nicotinic acetylcholine receptor (nAChR) can have neuroprotective effects in some models of neurodegenerative disease, as well as prevent glutamate-induced motor neuronal death. However, the effect of α7 nAChR agonists on ALS-associated mutant copper-zinc superoxide dismutase 1 (SOD1) aggregates in motor neurons remains unclear. In the present study, we examined whether α7 nAChR activation had a neuroprotective effect against SOD1G85R-induced toxicity in a cellular model for ALS. We found that α7 nAChR activation by PNU282987, a selective agonist of α7 nAChR, exhibited significant neuroprotective effects against SOD1G85R-induced toxicity via the reduction of intracellular protein aggregates. This reduction also correlated with the activation of autophagy through the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, the activation of α7 nAChRs was found to increase the biogenesis of lysosomes by inducing translocation of the transcription factor EB (TFEB) into the nucleus. These results support the therapeutic potential of α7 nAChR activation in diseases that are characterized by SOD1G85R aggregates, such as ALS.


Assuntos
Neurônios/metabolismo , Superóxido Dismutase-1/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cálcio/metabolismo , Humanos , Espaço Intracelular , Lisossomos/metabolismo , Mutação , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Fármacos Neuroprotetores , Agregados Proteicos , Ligação Proteica , Transporte Proteico , Transdução de Sinais , Superóxido Dismutase-1/genética , Serina-Treonina Quinases TOR/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/agonistas
10.
Postepy Biochem ; 66(1): 30-37, 2020 03 31.
Artigo em Polonês | MEDLINE | ID: mdl-33320482

RESUMO

Transcription factor ChREBP, in complex with MLX, binds to carbohydrate-response element (ChoRE) located in the promoters of genes related to glycolysis, gluconeogenesis, pentosephosphate pathway and lipogenesis, activating their transcription following stimulation with glucose, insulin-independently. In this article the mechanisms of ChREBP regulation and ChREBP functions under both physiological and pathophysiological conditions are described in detail. The possible use of ChREBP activity modulation as a therapeutic tool, e.g. in case of nonalcoholic fatty liver disease, diabetes type 2 and cancers, is also discussed.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Metabolismo dos Carboidratos , Metabolismo dos Lipídeos , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Fígado/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo
11.
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
12.
PLoS Pathog ; 16(8): e1008801, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866183

RESUMO

Rice stripe virus (RSV) is one of the most destructive viral diseases affecting rice production. However, so far, only one RSV resistance gene has been cloned, the molecular mechanisms underlying host-RSV interaction are still poorly understood. Here, we show that increasing levels or signaling of brassinosteroids (BR) and jasmonic acid (JA) can significantly enhance the resistance against RSV. On the contrary, plants impaired in BR or JA signaling are more susceptible to RSV. Moreover, the enhancement of RSV resistance conferred by BR is impaired in OsMYC2 (a key positive regulator of JA response) knockout plants, suggesting that BR-mediated RSV resistance requires active JA pathway. In addition, we found that RSV infection suppresses the endogenous BR levels to increase the accumulation of OsGSK2, a key negative regulator of BR signaling. OsGSK2 physically interacts with OsMYC2, resulting in the degradation of OsMYC2 by phosphorylation and reduces JA-mediated defense to facilitate virus infection. These findings not only reveal a novel molecular mechanism mediating the crosstalk between BR and JA in response to virus infection and deepen our understanding about the interaction of virus and plants, but also suggest new effective means of breeding RSV resistant crops using genetic engineering.


Assuntos
Brassinosteroides/metabolismo , Ciclopentanos/metabolismo , Oryza , Oxilipinas/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais , Tenuivirus , 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 , Oryza/genética , Oryza/metabolismo , Oryza/virologia , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/virologia , Tenuivirus/genética , Tenuivirus/metabolismo
13.
J Clin Pathol ; 73(11): 691-694, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32820043

RESUMO

Transcription factor enhancer 3 (TFE3), on the short arm of chromosome Xp11.23 and its protein, belongs to the microphthalmia transcription family (MiTF) of transcription factors. It shares close homology with another member of the family, MiTF which is involved in melanocyte development. When a cell is stressed and/or starved, TFE3 protein translocates into the nucleus. TFE3 gene fusions with multiple different partner genes occur in several tumours with resultant nuclear expression of TFE3 protein. The main tumours associated with TFE3 gene fusions are: renal cell carcinoma, alveolar soft part sarcoma, a subset of epithelioid haemangioendotheliomas (EHE), some perivascular epithelioid cell tumours and rare examples of ossifying fibromyxoid tumour and malignant chondroid syringoma. TFE3 immunohistochemistry is of use in routine diagnostic practice with the aforementioned tumours harbouring TFE3 fusions leading to nuclear staining. In addition, there are tumours lacking TFE3 fusions but also display TFE3 nuclear immunolabeling, and these include: granular cell tumour, solid pseudopapillary neoplasm of the pancreas and ovarian sclerosing stromal tumour.


Assuntos
Adenoma Pleomorfo/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Carcinoma de Células Renais/genética , Hemangioendotelioma Epitelioide/genética , Neoplasias de Células Epitelioides Perivasculares/genética , Sarcoma Alveolar de Partes Moles/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Carcinoma de Células Renais/patologia , Núcleo Celular/metabolismo , Tumores do Estroma Endometrial/patologia , Feminino , Tumor de Células Granulares/patologia , Hemangioendotelioma Epitelioide/patologia , Humanos , Imuno-Histoquímica , Melanócitos/patologia , Fusão Oncogênica , Neoplasias de Células Epitelioides Perivasculares/patologia , Transporte Proteico , Sarcoma Alveolar de Partes Moles/patologia , Estresse Fisiológico , Translocação Genética
14.
Nat Cell Biol ; 22(8): 973-985, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32753672

RESUMO

Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/fisiologia , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Calcineurina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/fisiologia , Transporte Proteico , Proteínas Qa-SNARE/metabolismo
15.
Gene ; 760: 145017, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32755655

RESUMO

Cytoplasmic vacuolization usually occurs in cells treated with different agents and substances. We found that LZ-106, an analog of enoxacin, is a potent lysosomotropic agent, contributing to the formation of cytoplasmic vacuoles in cells. Studies of LZ-106-induced vacuolization in H460 cells showed acid environment inside these vacuoles. Further study demonstrated that markers in the late endosomes and lysosomes, like LAMP1 and RAB7, on the surface of the vacuoles, implying that these vacuoles might derive from endosomes and/or lysosomes. By studying the fluorescence intensity of LZ-106, we discovered that LZ-106 tended to locate in acid organelles, and Bafilomycin A1, a V-ATPase inhibitor, was able to suppress its acid organelles localization. Also, we noticed that LZ-106 could induce lysosome stress, involving pH increment and lysosomal membrane damage. Moreover, the expression levels of some lysosome-related proteins, like LAMP1, EEA1, and Cathepsin B, were also altered upon LZ-106 treatment. At last, we confirmed LZ-106 can activate TFEB, a key regulator of lysosomes. Knockdown of TFEB could also reverse LZ-106's effect on vacuolization in H460 cells. Taken together, due to LZ-106's lysosomotropic properties, it is able to accumulate in the acid organelles and induce lysosomal dysfunction in H460 cells, leading to TFEB activation and the following cytoplasmic vacuolization.


Assuntos
Enoxacino/análogos & derivados , Enoxacino/farmacologia , Vacúolos/efeitos dos fármacos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/química , Macrolídeos/farmacologia
16.
Nat Commun ; 11(1): 3954, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770072

RESUMO

The systematic stabilization of protein-protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.


Assuntos
Proteínas 14-3-3/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Proteínas 14-3-3/antagonistas & inibidores , Proteínas 14-3-3/ultraestrutura , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/ultraestrutura , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
17.
Nat Commun ; 11(1): 4332, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859902

RESUMO

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.


Assuntos
Infecções por Coxsackievirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Proteoma/análise , Animais , Autofagia , 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 , Linhagem Celular , Sobrevivência Celular , Enterovirus/fisiologia , Enterovirus Humano B/fisiologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosforilação , Transdução de Sinais , Proteínas Virais/metabolismo
18.
Arterioscler Thromb Vasc Biol ; 40(9): 2054-2069, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640907

RESUMO

OBJECTIVE: Increased CTSS (cathepsin S) has been reported to play a critical role in atherosclerosis progression. Both CTSS synthesis and secretion are essential for exerting its functions. However, the underlying mechanisms contributing to CTSS synthesis and secretion in atherosclerosis remain unclear. Approach and Results: In this study, we showed that nicotine activated autophagy and upregulated CTSS expression in vascular smooth muscle cells and in atherosclerotic plaques. Western blotting and immunofluorescent staining showed that nicotine inhibited the mTORC1 (mammalian target of rapamycin complex 1) activity, promoted the nuclear translocation of TFEB (transcription factor EB), and upregulated the expression of CTSS. Chromatin immunoprecipitation-qualificative polymerase chain reaction, electrophoretic mobility shift assay, and luciferase reporter assay further demonstrated that TFEB directly bound to the CTSS promoter. mTORC1 inhibition by nicotine or rapamycin promoted lysosomal exocytosis and CTSS secretion. Live cell assays and IP-MS (immunoprecipitation-mass spectrometry) identified that the interactions involving Rab10 (Rab10, member RAS oncogene family) and mTORC1 control CTSS secretion. Nicotine promoted vascular smooth muscle cell migration by upregulating CTSS, and CTSS inhibition suppressed nicotine-induced atherosclerosis in vivo. CONCLUSIONS: We concluded that nicotine mediates CTSS synthesis and secretion through regulating the autophagy-lysosomal machinery, which offers a potential therapeutic target for atherosclerosis treatment.


Assuntos
Aterosclerose/tratamento farmacológico , Autofagia/efeitos dos fármacos , Catepsinas/biossíntese , Lisossomos/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nicotina/farmacologia , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , 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 , Catepsinas/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Exocitose , Lisossomos/enzimologia , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout para ApoE , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/ultraestrutura , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/ultraestrutura , Via Secretória , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
19.
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
20.
Nat Cell Biol ; 22(7): 815-827, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601373

RESUMO

Lysosomes serve as cellular degradation and signalling centres that coordinate metabolism in response to intracellular cues and extracellular signals. Lysosomal capacity is adapted to cellular needs by transcription factors, such as TFEB and TFE3, which activate the expression of lysosomal and autophagy genes. Nuclear translocation and activation of TFEB are induced by a variety of conditions such as starvation, lysosome stress and lysosomal storage disorders. How these various cues are integrated remains incompletely understood. Here, we describe a pathway initiated at the plasma membrane that controls lysosome biogenesis via the endocytic regulation of intracellular ion homeostasis. This pathway is based on the exo-endocytosis of NHE7, a Na+/H+ exchanger mutated in X-linked intellectual disability, and serves to control intracellular ion homeostasis and thereby Ca2+/calcineurin-mediated activation of TFEB and downstream lysosome biogenesis in response to osmotic stress to promote the turnover of toxic proteins and cell survival.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Membrana Celular/metabolismo , Endocitose , Lisossomos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/metabolismo , Clatrina/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , Trocadores de Sódio-Hidrogênio/genética
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