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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360911

RESUMO

Pyrimethamine (Pyri) is being used in combination with other medications to treat serious parasitic infections of the body, brain, or eye and to also reduce toxoplasmosis infection in the patients with HIV infection. Additionally, Pyri can display significant anti-cancer potential in different tumor models, but the possible mode of its actions remains unclear. Hence, in this study, the possible anti-tumoral impact of Pyri on human chronic myeloid leukemia (CML) was deciphered. Pyri inhibited cell growth in various types of tumor cells and exhibited a marked inhibitory action on CML cells. In addition to apoptosis, Pyri also triggered sustained autophagy. Targeted inhibition of autophagy sensitized the tumor cells to Pyri-induced apoptotic cell death. Moreover, the activation of signal transducer and activator of transcription 5 (STAT5) and its downstream target gene Bcl-2 was attenuated by Pyri. Accordingly, small interfering RNA (siRNA)-mediated STAT5 knockdown augmented Pyri-induced autophagy and apoptosis and promoted the suppressive action of Pyri on cell viability. Moreover, ectopic overexpression of Bcl-2 protected the cells from Pyri-mediated autophagy and apoptosis. Overall, the data indicated that the attenuation of STAT5-Bcl-2 cascade by Pyri can regulate its growth inhibitory properties by simultaneously targeting both apoptosis and autophagy cell death mechanism(s).


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Pirimetamina/farmacologia , Apoptose/genética , Autofagia/genética , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição STAT5/deficiência , Fator de Transcrição STAT5/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células THP-1 , Transfecção , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
2.
FEBS J ; 288(10): 3164-3185, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33830641

RESUMO

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.


Assuntos
Apresentação de Antígeno/genética , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H1N1/genética , Macroautofagia/genética , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Brefeldina A/farmacologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Feminino , Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade Classe II/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Macroautofagia/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Transfecção
3.
J Mol Cell Cardiol ; 148: 78-88, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32891637

RESUMO

Second-hand smoking evokes inflammation and cardiovascular diseases. Recent evidence has revealed a pivotal role for deranged autophagy in smoke exposure-induced cardiac anomalies. This study evaluated the impact of haploinsufficiency of the mTOR-independent autophagy protein Beclin1 on side-stream smoke exposure-induced cardiac anomalies and mechanism(s) involved. Adult WT and Beclin1 haploinsufficiency (Becn+/-) mice were exposed to cigarette smoke for 1 h daily for 90 days. Echocardiographic, cardiomyocyte function, intracellular Ca2+, autophagy, mitophagy, apoptosis and inflammation were examined. DHE staining was employed to evaluate O2- level. Our data revealed that Beclin1 deficiency exacerbated smoke exposure-induced myocardial anomalies in geometry, fractional shortening, cardiomyocyte function, intracellular Ca2+ handling, TEM ultrastructure, and inflammation along with pronounced apoptosis and O2- production. Side-stream smoke provoked excessive autophagy/mitophagy, mtDNA release, and activation of innate immune response signals cyclic GMP-AMP synthase (cGAS) and its effector - stimulator of interferon genes (STING), the effect was abolished or unaffected by Becn haploinsufficiency. STING phosphorylation was overtly promoted by smoke exposure in Becn+/- mice. Smoke exposure also suppressed phosphorylation of mTOR although it facilitated that of ULK1 in both groups. In vitro data revealed that inhibition of cGAS or STING failed to affect smoke extract-induced mitophagy although they abrogated smoke extract-induced cardiomyocyte dysfunction except cGAS inhibition in Becn+/- mice. These data suggest that Beclin1 is integral in the maintenance of cardiac homeostasis under side-stream smoke exposure via a STING-mediated mechanism.


Assuntos
Proteína Beclina-1/genética , Haploinsuficiência/genética , Proteínas de Membrana/metabolismo , Contração Miocárdica , Miocárdio/patologia , Poluição por Fumaça de Tabaco , Remodelação Ventricular , Animais , Animais Recém-Nascidos , Apoptose , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/deficiência , Biomarcadores/metabolismo , Fenômenos Biomecânicos , DNA Mitocondrial/metabolismo , Eletrocardiografia , Inflamação/patologia , Camundongos , Mitofagia , Miocárdio/ultraestrutura , Miócitos Cardíacos/metabolismo , Nucleotidiltransferases/metabolismo , Biogênese de Organelas , Fosforilação , Transdução de Sinais , Superóxidos/metabolismo , Serina-Treonina Quinases TOR/metabolismo
4.
Am J Respir Cell Mol Biol ; 63(3): 338-348, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32374619

RESUMO

Bronchopulmonary dysplasia (BPD) is a common and serious complication associated with preterm birth. The pathogenesis of BPD is incompletely understood, and there is an unmet clinical need for effective treatments. The role of autophagy as a potential cytoprotective mechanism in BPD remains to be fully elucidated. In the present study, we investigated the role and regulation of autophagy in experimental models of BPD. Regulation and cellular distribution of autophagic activity during postnatal lung development and in neonatal hyperoxia-induced lung injury (nHILI) were assessed in the autophagy reporter transgenic GFP-LC3 (GFP-microtubule-associated protein 1A/1B-light chain 3) mouse model. Autophagic activity and its regulation were also examined in a baboon model of BPD. The role of autophagy in nHILI was determined by assessing lung morphometry, injury, and inflammation in autophagy-deficient Beclin 1 heterozygous knockout mice (Becn1+/-). Autophagic activity was induced during alveolarization in control murine lungs and localized primarily to alveolar type II cells and macrophages. Hyperoxia exposure of neonatal murine lungs and BPD in baboon lungs resulted in impaired autophagic activity in association with insufficient AMPK (5'-AMP-activated protein kinase) and increased mTORC1 (mTOR complex 1) activation. Becn1+/- lungs displayed impaired alveolarization, increased alveolar septal thickness, greater neutrophil accumulation, and increased IL-1ß concentrations when exposed to nHILI. Becn1+/- alveolar macrophages isolated from nHILI-exposed mice displayed increased expression of proinflammatory genes. In conclusion, basal autophagy is induced during alveolarization and disrupted during progression of nHILI in mice and BPD in baboons. Becn1+/- mice are more susceptible to nHILI, suggesting that preservation of autophagic activity may be an effective protective strategy in BPD.


Assuntos
Autofagia/genética , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patologia , Hiperóxia/patologia , Células Epiteliais Alveolares/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína Beclina-1/deficiência , Displasia Broncopulmonar/metabolismo , Modelos Animais de Doenças , Humanos , Hiperóxia/genética , Hiperóxia/metabolismo , Pulmão/patologia , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Macrófagos Alveolares/metabolismo , Camundongos Knockout , Pneumonia/patologia
5.
Nat Commun ; 11(1): 260, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937766

RESUMO

Transmission from an infected mosquito to a host is an essential process in the life cycle of mosquito-borne flaviviruses. Numerous studies have demonstrated that mosquito saliva facilitates viral transmission. Here we find that a saliva-specific protein, named Aedes aegypti venom allergen-1 (AaVA-1), promotes dengue and Zika virus transmission by activating autophagy in host immune cells of the monocyte lineage. The AG6 mice (ifnar1-/-ifngr1-/-) bitten by the virus-infected AaVA-1-deficient mosquitoes present a lower viremia and prolonged survival. AaVA-1 intracellularly interacts with a dominant negative binder of Beclin-1, known as leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), and releases Beclin-1 from LRPPRC-mediated sequestration, thereby enabling the initialization of downstream autophagic signaling. A deficiency in Beclin-1 reduces viral infection in mice and abolishes AaVA-1-mediated enhancement of ZIKV transmission by mosquitoes. Our study provides a mechanistic insight into saliva-aided viral transmission and could offer a potential prophylactic target for reducing flavivirus transmission.


Assuntos
Aedes/metabolismo , Autofagia , Infecções por Flavivirus/transmissão , Flavivirus/fisiologia , Proteínas de Insetos/metabolismo , Mosquitos Vetores/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Aedes/virologia , Animais , Proteína Beclina-1/deficiência , Proteína Beclina-1/metabolismo , Vírus da Dengue/fisiologia , Infecções por Flavivirus/virologia , Humanos , Proteínas de Insetos/deficiência , Proteínas de Insetos/genética , Camundongos , Mosquitos Vetores/virologia , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteínas e Peptídeos Salivares/deficiência , Proteínas e Peptídeos Salivares/genética , Células THP-1 , Replicação Viral , Zika virus/fisiologia
6.
FASEB J ; 34(2): 3129-3150, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908069

RESUMO

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.


Assuntos
Envelhecimento/metabolismo , Autofagia , Glucuronidase/metabolismo , Fosfatos/metabolismo , Animais , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Feminino , Glucuronidase/genética , Células HEK293 , Humanos , Rim/metabolismo , Proteínas Klotho , Masculino , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
7.
Nat Microbiol ; 5(2): 272-281, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959973

RESUMO

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens1,2. However, if left uncontrolled, these responses may lead to detrimental inflammation3. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism4. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation5-12. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200-but not of other essential autophagy components ATG5, ATG16L1 or ATG7-in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection.


Assuntos
Autofagia/genética , Listeria monocytogenes/patogenicidade , Macrófagos Peritoneais/imunologia , Animais , Autofagia/imunologia , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proliferação de Células , Suscetibilidade a Doenças/imunologia , Feminino , Predisposição Genética para Doença , Interferon gama/imunologia , Listeria monocytogenes/imunologia , Listeriose/etiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
FEBS Lett ; 594(7): 1155-1165, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31823348

RESUMO

Beclin 1/Atg6 is an essential autophagy gene, and deficiency of this gene in organisms leads to impaired autophagic flux, usually with cell apoptosis; however, the causative mechanism of cell apoptosis is not clear. Here, we knocked out the beclin 1 gene in zebrafish and found that autophagic flux is disrupted in mutants. Beclin 1-deficient zebrafish live through embryogenesis but die at larval stage. We found accumulated protein aggregates and vigorous apoptosis in mutant larvae, predominantly in the liver. The hepatic cell apoptosis in mutants results from an endoplasmic reticulum (ER) stress response; however, it is not the leading cause of mutant larval lethality. Our work proposes that ER stress induces cell apoptosis in Beclin 1-deficient organisms.


Assuntos
Apoptose , Proteína Beclina-1/deficiência , Estresse do Retículo Endoplasmático , Hepatócitos/citologia , Larva/citologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Autofagia , Proteína Beclina-1/genética , Deleção de Genes , Genes Essenciais , Humanos , Larva/genética , Fígado/citologia , Fígado/metabolismo , Agregados Proteicos , Peixe-Zebra/genética
9.
Eur J Pharmacol ; 858: 172338, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31029709

RESUMO

Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) has been validated as a potent protective agent of Atherosclerosis (AS). Here, we explored the molecular mechanism of MALAT1 exerting protective function in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical vein endothelial cells (HUVECs). qRT-PCR assay was used to assess the expression of MALAT1 and miR-216a-5p. Western blot analysis was performed to detect LC3-I, LC3-II, Beclin-1 and p62 levels. The autophagosome formation was analyzed by immunofluorescence analysis. Cell apoptosis was measured by flow cytemotry and caspase 3 activity. Dual-luciferase reporter assay or RNA immunorecipitation assay was performed to verify the targeted interrelation between MALAT1 and miR-216a-5p, or miR-216a-5p and Beclin-1. Our data revealed that MALAT1 was upregulated and miR-216a-5p was downregulated in serum of AS patients and ox-LDL-treated HUVECs. ox-LDL treatment induced HUNECs autophagy. Moreover, MALAT1 enhanced autophagy and survival in ox-LDL-treated HUVECs. MALAT1 directly binded to miR-216a-5p and MALAT1 enhanced Beclin-1 expression by sponging miR-216a-5p. Also, miR-216-5p-mediated repressive effect on autophagy and survival was abrogated by MALAT1. Additionally, Beclin-1 knockdown inhibited autophagy and survival in ox-LDL-treated HUVECs. In all, our data suggested that MALAT1 might play a protective role at least partly by sponging miR-216a-5p and regulating Beclin-1, highlighting that MALAT1 might be a potential therapeutic target of AS.


Assuntos
Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Aterosclerose/genética , Aterosclerose/patologia , Autofagia/genética , Proteína Beclina-1/deficiência , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade
10.
Nature ; 567(7747): 262-266, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842662

RESUMO

Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects infections or tissue damage by binding to microbial or self DNA in the cytoplasm1. Upon binding DNA, cGAS produces cGAMP that binds to and activates the adaptor protein STING, which then activates the kinases IKK and TBK1 to induce interferons and other cytokines2-6. Here we report that STING also activates autophagy through a mechanism that is independent of TBK1 activation and interferon induction. Upon binding cGAMP, STING translocates to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and the Golgi in a process that is dependent on the COP-II complex and ARF GTPases. STING-containing ERGIC serves as a membrane source for LC3 lipidation, which is a key step in autophagosome biogenesis. cGAMP induced LC3 lipidation through a pathway that is dependent on WIPI2 and ATG5 but independent of the ULK and VPS34-beclin kinase complexes. Furthermore, we show that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Interestingly, STING from the sea anemone Nematostella vectensis induces autophagy but not interferons in response to stimulation by cGAMP, which suggests that induction of autophagy is a primordial function of the cGAS-STING pathway.


Assuntos
Autofagia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Animais , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Citosol/virologia , Vírus de DNA/genética , Vírus de DNA/metabolismo , DNA Viral/metabolismo , Retículo Endoplasmático/metabolismo , Evolução Molecular , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Interferons/biossíntese , Interferons/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Proteínas de Ligação a Fosfato , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Anêmonas-do-Mar , Proteínas de Transporte Vesicular/metabolismo
11.
Virulence ; 9(1): 1176-1194, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30067475

RESUMO

Peste des petits ruminants (PPR) is an acute and highly contagious disease in small ruminants that causes significant economic losses in developing countries. An increasing number of studies have demonstrated that both autophagy and apoptosis are important cellular mechanisms for maintaining homeostasis, and they participate in the host response to pathogens. However, the crosstalk between apoptosis and autophagy in host cells during PPRV infection has not been clarified. In this study, autophagy was induced upon virus infection in caprine endometrial epithelial cells (EECs), as determined by the appearance of double- and single-membrane autophagy-like vesicles, LC3-I/LC3-II conversion, and p62 degradation. We also found that PPRV infection triggered a complete autophagic response, most likely mediated by the non-structural protein C and nucleoprotein N. Moreover, our results suggest that autophagy not only promotes the replication of PPRV in EECs but also provides a potential mechanism for inhibiting PPRV-induced apoptosis. Inhibiting autophagosome formation by wortmannin and knocking down the essential autophagic proteins Beclin-1 and ATG7 induces caspase-dependent apoptosis in EECs in PPRV infection. However, inhibiting autophagosome and lysosome fusion by NH4Cl and chloroquine did not increase the number of apoptotic cells. Collectively, these data are the first to indicate that PPRV-induced autophagy inhibits caspase-dependent apoptosis and thus contributes to the enhancement of viral replication and maturity in host cells.


Assuntos
Apoptose , Autofagia , Caspases/metabolismo , Células Epiteliais/virologia , Vírus da Peste dos Pequenos Ruminantes/patogenicidade , Replicação Viral , Cloreto de Amônio/farmacologia , Androstadienos/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Cloroquina/farmacologia , Endométrio/citologia , Feminino , Cabras , Interações Hospedeiro-Patógeno , Humanos , Técnicas In Vitro , Lisossomos/efeitos dos fármacos , Lisossomos/fisiologia , Nucleoproteínas/metabolismo , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/fisiologia , Proteína C/metabolismo , Wortmanina
12.
Proc Natl Acad Sci U S A ; 115(16): 4176-4181, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29610308

RESUMO

Allelic loss of the autophagy gene, beclin 1/BECN1, increases the risk of patients developing aggressive, including human epidermal growth factor receptor 2 (HER2)-positive, breast cancers; however, it is not known whether autophagy induction may be beneficial in preventing HER2-positive breast tumor growth. We explored the regulation of autophagy in breast cancer cells by HER2 in vitro and the effects of genetic and pharmacological strategies to increase autophagy on HER2-driven breast cancer growth in vivo. Our findings demonstrate that HER2 interacts with Beclin 1 in breast cancer cells and inhibits autophagy. Mice with increased basal autophagy due to a genetically engineered mutation in Becn1 are protected from HER2-driven mammary tumorigenesis, and HER2 fails to inhibit autophagy in primary cells derived from these mice. Moreover, treatment of mice with HER2-positive human breast cancer xenografts with the Tat-Beclin 1 autophagy-inducing peptide inhibits tumor growth as effectively as a clinically used HER2 tyrosine kinase inhibitor (TKI). This inhibition of tumor growth is associated with a robust induction of autophagy, a disruption of HER2/Beclin 1 binding, and a transcriptional signature in the tumors distinct from that observed with HER2 TKI treatment. Taken together, these findings indicate that the HER2-mediated inhibition of Beclin 1 and autophagy likely contributes to HER2-mediated tumorigenesis and that strategies to block HER2/Beclin 1 binding and/or increase autophagy may represent a new therapeutic approach for HER2-positive breast cancers.


Assuntos
Autofagia , Proteína Beclina-1/fisiologia , Proteínas de Neoplasias/fisiologia , Receptor ErbB-2/fisiologia , Substituição de Aminoácidos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Técnicas de Introdução de Genes , Humanos , Lapatinib , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Mutação , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/farmacologia , Distribuição Aleatória , Receptor ErbB-2/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biochem Biophys Res Commun ; 498(1): 18-24, 2018 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-29496445

RESUMO

Age-related changes of adult stem cell are crucial for tissue aging and age-related diseases. Thus, clarifying mechanisms to prevent adult stem cell aging is indispensable for healthy aging. Metformin, a drug for type 2 diabetes, has been highlighted for its anti-aging and anti-cancer effect. In Drosophila intestinal stem cell (ISC), we previously reported the inhibitory effect of metformin on age-related phenotypes of ISC. Here, we showed that knockdown of Atg6, a crucial autophagy-related factor, in ISC induces age-related phenotypes of ISC such as hyperproliferation, centrosome amplification, and DNA damage accumulation. Then, we revealed that metformin inhibits ISC aging phenotypes in Atg6-dependent manner. Taken together, our study suggests that Atg6 is required for the inhibitory effect of metformin on ISC aging, providing an intervention mechanism of metformin on adult stem cell aging.


Assuntos
Proteína Beclina-1/deficiência , Senescência Celular/efeitos dos fármacos , Proteínas de Drosophila/deficiência , Drosophila melanogaster/citologia , Intestinos/citologia , Metformina/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Proteína Beclina-1/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Paraquat/toxicidade , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos
14.
Eur J Pharmacol ; 823: 58-64, 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29408091

RESUMO

Aspirin not only reduces the incidence of hepatocellular carcinoma (HCC) but also plays a synergistic role with chemotherapy for HCC treatment. However, the underlying mechanisms remain incompletely elucidated. Given that autophagy triggers cancer cell death, the present study examined the autophagic effect of aspirin on HCC cells. Results showed that aspirin increased LC3II/LC3I ratio, decreased p62 expression, and enhanced autophagic flux (autophagosome and autolysosome puncta) in Hep3B, HepG2, or SMMC-7721 cells, reflecting the autophagy of HCC cells. The autophagic effects of aspirin depended on Beclin-1 expression. Aspirin disrupted the interaction between Bcl-2 and Beclin-1. In addition to activating the AMP-activated protein kinase, c-Jun N-terminal kinase, and Glycogen synthase kinase-3 pathways, aspirin inhibited the mammalian-target-of rapamycin-S6K1/4E-BP1 signaling. Aspirin induced autophagy of HCC cell. This study contributes to understanding the chemoprotective and inhibitory effects of aspirin on HCC development.


Assuntos
Aspirina/farmacologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Fosfoproteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo
15.
Mol Neurobiol ; 53(8): 5500-9, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26456737

RESUMO

Beclin-1 is assumed to be a critical component participating in autophagosome formation in mammals; however, the exact role of Beclin-1 in autophagy remains controversial. Here (1) we created a HT22-Beclin-1-knockdown cell line using the Q-techBECN1 technique, (2) examined the potential role of Beclin-1 in an autophagic response in hippocampal HT22 neurons challenged with rapamycin, (3) investigated the expression of several gene products involved in the autophagic pathway, and (4) checked the effects of Beclin-1 knockdown on neuronal death induced by AAS. Rapamycin induced and altered the expression of autophagy signature proteins in wild-type cultures as well as in HT22-Beclin-1-knockdown cells. However, among the examined markers, only two factors exhibited dramatic changes when comparing controls to HT22-Beclin-1-knockdown cells. The amount of LC3, an important protein for the initiation of autophagosome formation and LAMP-1, a major constituent of the lysosomal membrane, underwent a dramatic and highly significant increase in control cultures challenged with rapamycin. In contrast, rapamycin was not able to induce any significant changes in LC3 and LAMP-1 levels in HT22-Beclin-1-knockdown cells. In addition, the knockdown of Beclin-1 enhanced neuronal susceptibility to death signals induced by AAS. Our data demonstrate the essential role of Beclin-1 in the formation of autophagosomes and lysosome biogenesis and underline that deletion of this key system is deleterious for cell viability.


Assuntos
Autofagossomos/metabolismo , Proteína Beclina-1/deficiência , Hipocampo/citologia , Lisossomos/metabolismo , Neurônios/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Catepsina B/metabolismo , Contagem de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Sirolimo/farmacologia , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
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