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1.
PLoS One ; 15(3): e0229796, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134970

RESUMO

Chaperones and autophagy are components of the protein quality control system that contribute to the management of proteins that are misfolded and aggregated. Here, we use yeast prions, which are self-perpetuating aggregating proteins, as a means to understand how these protein quality control systems influence aggregate loss. Chaperones, such as Hsp104, fragment prion aggregates to generate more prion seeds for propagation. While much is known about the role of chaperones, little is known about how other quality control systems contribute to prion propagation. We show that the aprotic solvent dimethyl sulfoxide (DMSO) cures a range of [PSI+] prion variants, which are related to several misfolded aggregated conformations of the Sup35 protein. Our studies show that DMSO-mediated curing is quicker and more efficient than guanidine hydrochloride, a prion curing agent that inactivates the Hsp104 chaperone. Instead, DMSO appears to induce Hsp104 expression. Using the yTRAP system, a recently developed transcriptional reporting system for tracking protein solubility, we found that DMSO also rapidly induces the accumulation of soluble Sup35 protein, suggesting a potential link between Hsp104 expression and disassembly of Sup35 from the prion aggregate. However, DMSO-mediated curing appears to also be associated with other quality control systems. While the induction of autophagy alone does not lead to curing, we found that DMSO-mediated curing is dramatically impaired in autophagy related (atg) gene mutants, suggesting that other factors influence this DMSO mechanism of curing. Our data suggest that DMSO-mediated curing is not simply dependent upon Hsp104 overexpression alone, but may further depend upon other aspects of proteostasis.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Dimetil Sulfóxido/farmacologia , Proteínas de Choque Térmico/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Mutação , Fatores de Terminação de Peptídeos/antagonistas & inibidores , Príons/antagonistas & inibidores , Agregados Proteicos/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
2.
Nature ; 578(7794): 301-305, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025038

RESUMO

Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.


Assuntos
Autofagossomos/química , Autofagossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Mutação Puntual , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo
3.
Toxicol Lett ; 322: 77-86, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31931077

RESUMO

Failure of all-trans-retinal (atRAL) clearance contributes to retina degeneration. However, whether autophagy can be activated by excess atRAL accumulation in retinal pigment epithelial (RPE) cells is not known. This study showed that atRAL provoked mitochondria-associated reactive oxygen species (ROS) production, activated the nuclear factor (erythroid-derived 2)-like 2 and apoptosis in a human RPE cell line, ARPE-19 cells. Moreover, we found that autophagic flux was functionally activated after atRAL treatment. The antioxidant N-acetylcysteine attenuated the expression of autophagy markers, suggesting that ROS triggered atRAL-activated autophagy. In addition, autophagic cell death was observed in atRAL-treated RPE cells, while inhibition of autophagy with 3-methyladenine or LC3, Beclin1, p62 silencing ameliorated atRAL-induced cytotoxicity. Suppression of autophagy quenched mitochondrial ROS and inhibited HO-1 and γ-GCSh expression, indicating that atRAL-activated autophagy enhances intracellular oxidative stress, thereby promoting RPE cell apoptosis. Furthermore, we found that inhibiting endoplasmic reticulum (ER) stress suppressed atRAL-induced mitochondrial ROS generation, subsequently attenuated autophagy and apoptosis in RPE cells. Taken together, these results suggest that atRAL-induced oxidative stress and ER stress modulate autophagy, which may contribute to RPE degeneration. There may be positive feedback regulatory mechanisms between atRAL-induced oxidative stress and autophagy or ER stress.


Assuntos
Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Vitamina A/toxicidade , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais
4.
Nat Cell Biol ; 22(2): 187-199, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932738

RESUMO

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Autofagossomos/química , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Biotinilação , Vesículas Extracelulares/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteômica/métodos , Células RAW 264.7 , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo
5.
Endocrinology ; 161(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31875883

RESUMO

Uterine receptivity is critical for establishing and maintaining pregnancy. For the endometrium to become receptive, stromal cells must differentiate into decidual cells capable of secreting factors necessary for embryo survival and placental development. Although there are multiple reports of autophagy induction correlated with endometrial stromal cell (ESC) decidualization, the role of autophagy in decidualization has remained elusive. To determine the role of autophagy in decidualization, we utilized 2 genetic models carrying mutations to the autophagy gene Atg16L1. Although the hypomorphic Atg16L1 mouse was fertile and displayed proper decidualization, conditional knockout in the reproductive tract of female mice reduced fertility by decreasing the implantation rate. In the absence of Atg16L1, ESCs failed to properly decidualize and fewer blastocysts were able to implant. Additionally, small interfering RNA knock down of Atg16L1 was detrimental to the decidualization response of human ESCs. We conclude that Atg16L1 is necessary for decidualization, implantation, and overall fertility in mice. Furthermore, considering its requirement for human endometrial decidualization, these data suggest Atg16L1 may be a potential mediator of implantation success in women.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Decídua/metabolismo , Endométrio/metabolismo , Mutação , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Decídua/citologia , Implantação do Embrião/genética , Endométrio/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Interferência de RNA , Células Estromais/citologia , Células Estromais/metabolismo
6.
Biomed Pharmacother ; 121: 109580, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704614

RESUMO

Colorectal cancer (CRC) is a malignant tumor with a high incidence and death rate in the world. Molecular interactions inside cells or tissues during tumor occurrence, development, and drug resistance are important for disease prevention and treatment. The long non-coding RNA SNHG14 has been proven to exert its oncogenic function in multiple cancers. However, there is no study regarding the role of SNHG14 in CRC research. In the present study, we applied RT-qPCR and western blot to determine the gene expression levels. MTT and TUNEL assays were used to detect cell proliferation and apoptosis rate. Cell migration and invasion abilities were determined by wound healing and transwell assays, respectively. StarBase was used to predict the potential binding sites and luciferase reporter assay was applied to confirm the direct interactions. Besides, we conducted a xenograft experiment to detect tumor growth rate in vivo. Our results showed that SNHG14 and ATG14 were both significantly higher in CRC tumor tissues than the normal ones, while miR-186 was decreased. The similar results were also observed in CRC cell lines. We confirmed that SNHG14 could directly interact with miR-186 and inhibited its expression. Meanwhile, miR-186 could directly bind ATG14 to inhibit its expression level. In vitro experiments showed that higher expression of SNHG14 led to higher cell proliferation, migration and invasion, while miR-186 significantly inhibited these tumor phenotypes. Furthermore, overexpression of ATG14 could strongly recover the CRC phenotypes attenuated by shSNHG14 or miR-186 mimics. Interestingly, we constructed cisplatin-resistant CRC cells and found that overexpression of ATG14 significantly enhanced the cell proliferation rate and inhibited cell apoptosis. Our research indicated that the novel axis of SNHG14/miR-186/ATG14 could play a vital role in regulating CRC cell progression. Moreover, this axis showed its clinical potential in regulating cisplatin resistance during CRC treatment.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Relacionadas à Autofagia/genética , Cisplatino/farmacologia , Neoplasias Colorretais/tratamento farmacológico , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Autofagia/genética , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Immunol Res ; 2019: 5071687, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815154

RESUMO

Autophagy is an important biology process, central to the maintenance of biology process in both physiological and pathological situations. It is regarded as a "double-edged sword"-exerting both protective and/or detrimental effects. These two-way effects are observed in immune cells as well as renal resident cells, including podocytes, mesangial cells, tubular epithelial cells, and endothelial cells of the glomerular capillaries. Mounting evidence suggests that autophagy is implicated in the pathological process of various immune-related renal diseases (IRRDs) as well as the kidney that underwent transplantation. Here, we provide an overview of the pathological role of autophagy in IRRDs, including lupus nephritis, IgA nephropathy, membrane nephropathy, ANCA-associated nephritis, and diabetic nephropathy. The understanding of the pathogenesis and regulatory mechanisms of autophagy in these renal diseases may lead to the identification of new diagnostic targets and refined therapeutic modulation.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Proteínas Relacionadas à Autofagia/imunologia , Autofagia/imunologia , Nefropatias Diabéticas/imunologia , Glomerulonefrite por IGA/imunologia , Hematúria/imunologia , Nefrite Lúpica/imunologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Anticorpos Anticitoplasma de Neutrófilos/biossíntese , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Linfócitos B/imunologia , Linfócitos B/patologia , Células Dendríticas , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Regulação da Expressão Gênica , Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/patologia , Hematúria/genética , Hematúria/patologia , Humanos , Transplante de Rim , Nefrite Lúpica/genética , Nefrite Lúpica/patologia , Macrófagos/imunologia , Macrófagos/patologia , Células Mesangiais/imunologia , Células Mesangiais/patologia , Podócitos/imunologia , Podócitos/patologia , Linfócitos T/imunologia , Linfócitos T/patologia
8.
PLoS One ; 14(11): e0223955, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31725732

RESUMO

African swine fever (ASF) is a swine disease caused by a large, structurally complex, double-stranded DNA virus, African swine fever virus (ASFV). In domestic pigs, acute infection by highly virulent ASF viruses causes hemorrhagic fever and death. Previous work has suggested that ASFV pathogenesis is primarily mediated by host cytokines produced by infected monocytes and macrophages. To better understand molecular mechanisms mediating virus pathogenesis and immune evasion, we used transcriptome analysis to identify gene expression changes after ASFV infection in ex vivo swine macrophages. Our results suggest that the cytokines of TNF family including FASLG, LTA, LTB, TNF, TNFSF4, TNFSF10, TNFSF13B and TNFSF18 are the major causative cytokine factors in ASF pathogenesis via inducing apoptosis. Other up-regulated proinflammatory cytokines (IL17F and interferons) and down-regulated anti-inflammatory cytokine (IL10) may also significantly contribute to ASF pathogenesis and cause excessive tissue inflammatory responses. The differential expression of genes also indicates that ASFV could evade both the innate and adaptive immune responses by (i) inhibiting MHC Class II antigen processing and presentation, (ii) avoiding CD8+ T effector cells and neutrophil extracellular traps via decreasing expression of neutrophil/CD8+ T effector cell-recruiting chemokines, (iii) suppressing M1 activation of macrophages, (iv) inducing immune suppressive cytokines, and (v) inhibiting the processes of macrophage autophagy and apoptosis. These results provide novel information to further investigate and better understand the mechanism of pathogenesis and immune evasion of this devastating swine disease.


Assuntos
Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/imunologia , Evasão da Resposta Imune , Macrófagos/imunologia , Macrófagos/virologia , Imunidade Adaptativa/genética , Febre Suína Africana/genética , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/imunologia , Animais , Apresentação do Antígeno/genética , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Quimiocinas/genética , Citocinas/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Evasão da Resposta Imune/genética , Imunidade Inata/genética , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Receptores de Citocinas/genética , Transdução de Sinais/genética , Sus scrofa , Suínos , Regulação para Cima
9.
Oxid Med Cell Longev ; 2019: 8426259, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31737174

RESUMO

Lack of physical exercise is considered an important risk factor for chronic diseases. On the contrary, physical exercise reduces the morbidity rates of obesity, diabetes, bone disease, and hypertension. In order to gain novel molecular and cellular clues, we analyzed the effects of physical exercise on differentiation of mesenchymal circulating progenitor cells (M-CPCs) obtained from runners. We also investigated autophagy and telomerase-related gene expression to evaluate the involvement of specific cellular functions in the differentiation process. We performed cellular and molecular analyses in M-CPCs, obtained by a depletion method, of 22 subjects before (PRE RUN) and after (POST RUN) a half marathon performance. In order to prove our findings, we performed also in vitro analyses by testing the effects of runners' sera on a human bone marrow-derived mesenchymal stem (hBM-MSC) cell line. PCR array analyses of PRE RUN versus POST RUN M-CPC total RNAs put in evidence several genes which appeared to be modulated by physical activity. Our results showed that physical exercise promotes differentiation. Osteogenesis-related genes as RUNX2, MSX1, and SPP1 appeared to be upregulated after the run; data showed also increased levels of BMP2 and BMP6 expressions. SOX9, COL2A1, and COMP gene enhanced expression suggested the induction of chondrocytic differentiation as well. The expression of telomerase-associated genes and of two autophagy-related genes, ATG3 and ULK1, was also affected and correlated positively with MSC differentiation. These data highlight an attractive cellular scenario, outlining the role of autophagic response to physical exercise and suggesting new insights into the benefits of physical exercise in counteracting chronic degenerative conditions.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Exercício Físico/fisiologia , Células-Tronco Mesenquimais/fisiologia , Corrida/fisiologia , Fatores de Transcrição SOX9/metabolismo , Adipogenia , Adulto , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Diferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteogênese , RNA Mensageiro/genética , Fatores de Transcrição SOX9/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para Cima
10.
Adv Exp Med Biol ; 1206: 41-65, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31776979

RESUMO

ATG is involved in multiple processes of autophagosome formation, including the initial phase of autophagy. The mammalian autophagy complex-ULK1 complex is composed of ULK1, FIP200, ATG13 and ATG101, and the yeast autophagy initiation complex-ATG1 complex is composed of ATG1, ATG13, ATG17, ATG29 and ATG31. After this complex is activated, it binds and phosphorylates ATG9 on the vesicles. Then PI3KC3-C1 (yeast: ATG34: ATG15: ATG6: ATG14 or mammal animal: ATG34: ATG15: BECN1: ATG14L) is recruited to the PAS. Further, ATG12-ATG5-ATG16 complex is localized on PAS (Yeast) or localized on the outer surface of the membrane (mammal) and makes binding of ATG8 (LC3) with PE to form ATG8-PE complex, promoting autophagic membrane elongation, closure and formation autophagosome and autophagosome lysosome.


Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Regulação da Expressão Gênica , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae
11.
Medicine (Baltimore) ; 98(40): e17313, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31577725

RESUMO

To investigate the impact of carriage of single nucleotide polymorphisms (SNPs) of the Toll-like receptor-4 (TLR4) and of autophagy-related gene 16-like-1 (ATG16L1) in preterm delivery (PTD).A prospective cohort of 145 pregnant women was studied. Women were prospectively followed-up until delivery. Genotyping for rs4986790 (Asp299Gly transition) and rs4986791 (Thr399Ile transition) of TLR4 and for rs2241880 of ATG16L1 was done by PCR-restriction fragment length polymorphism. The primary study endpoint was the impact of carriage of minor alleles of TLR4 on early PTD before gestational week 32. Associations with human chorionic gonadotrophin (hCG) were also analyzed. Peripheral blood mononuclear cells were isolated from 15 healthy women and stimulated for cytokine production.No difference in clinical characteristics was observed between women delivering full term and preterm. The frequency of early PTD was 25% among women carrying minor alleles of TLR4 and 6.8% among women carrying major alleles (P: .032). Odds ratios for PTD were 3.85 among women carrying the GG genotype of rs2241880 and major alleles of TLR4 and 0.26 among carriers of GG genotype and minor alleles of TLR4 (P: .030). The co-presence of GG genotype of rs2241880 and hCG above 70 U/L was an independent variable for PTD. Stimulated production of interleukin-6 was greater among women with GG genotypes of rs2241880.Minor alleles of SNPs of TLR4 predispose to early PTD. The GG genotype of rs2241880 of ATG16L1 is associated with PTD when hCG is supra-elevated.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Nascimento Prematuro/genética , Receptor 4 Toll-Like/genética , Adulto , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Autofagia/fisiologia , Gonadotropina Coriônica/sangue , Citocinas/biossíntese , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Idade Gestacional , Humanos , Polimorfismo de Nucleotídeo Único , Gravidez , Estudos Prospectivos
12.
Mol Cell ; 76(2): 268-285, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31585693

RESUMO

The clearance of surplus, broken, or dangerous components is key for maintaining cellular homeostasis. The failure to remove protein aggregates, damaged organelles, or intracellular pathogens leads to diseases, including neurodegeneration, cancer, and infectious diseases. Autophagy is the evolutionarily conserved pathway that sequesters cytoplasmic components in specialized vesicles, autophagosomes, which transport the cargo to the degradative compartments (vacuoles or lysosomes). Research during the past few decades has elucidated how autophagosomes engulf their substrates selectively. This type of autophagy involves a growing number of selective autophagy receptors (SARs) (e.g., Atg19 in yeasts, p62/SQSTM1 in mammals), which bind to the cargo and simultaneously engage components of the core autophagic machinery via direct interaction with the ubiquitin-like proteins (UBLs) of the Atg8/LC3/GABARAP family and adaptors, Atg11 (in yeasts) or FIP200 (in mammals). In this Review, we critically discuss the biology of the SARs with special emphasis on their interactions with UBLs.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas Fúngicas/metabolismo , Transdução de Sinais , Leveduras/metabolismo , Animais , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/genética , Sítios de Ligação , Proteínas Fúngicas/genética , Humanos , Ligantes , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ubiquitinação , Ubiquitinas/metabolismo , Leveduras/genética
13.
Proc Natl Acad Sci U S A ; 116(43): 21508-21513, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31591221

RESUMO

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.


Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases/química , Regulação Alostérica , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Modelos Moleculares , Domínios Proteicos , Transativadores/química , Transativadores/genética , Transativadores/metabolismo
14.
PLoS One ; 14(10): e0223895, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31603923

RESUMO

Significant phenotypic overlaps exist between autophagy and acidogenesis in Aspergillus niger. The possible role of autophagy in the acidogenic growth and metabolism of this fungus was therefore examined and the movement of cytosolic EGFP to vacuoles served to monitor this phenomenon. An autophagy response to typical as well as a metabolic inhibitor-induced nitrogen starvation was observed in A. niger mycelia. The vacuolar re-localization of cytosolic EGFP was not observed upon nitrogen starvation in the A. niger Δatg1 strain. The acidogenic growth of the fungus consisted of a brief log phase followed by an extended autophagy-like state throughout the idiophase of fermentation. Mycelia in the idiophase were highly vacuolated and EGFP was localized to the vacuoles but no autolysis was observed. Both autophagy and acidogenesis are compromised in Δatg1 and Δatg8 strains of A. niger. The acidogenic growth of the fungus thus appears to mimic a condition of nutrient limitation and is associated with an extended autophagy-like state. This crucial role of autophagy in acidogenic A. niger physiology could be of value in improving citric acid fermentation.


Assuntos
Aspergillus niger/crescimento & desenvolvimento , Proteínas Relacionadas à Autofagia/genética , Ácido Cítrico/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Autofagia , Metabolismo dos Carboidratos , Citosol/metabolismo , Fermentação , Proteínas Fúngicas/genética , Mutação , Nitrogênio/metabolismo , Vacúolos/metabolismo
15.
PLoS Biol ; 17(10): e3000508, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31593566

RESUMO

CDGSH iron-sulfur domain-containing protein 2 (Cisd2) is pivotal to mitochondrial integrity and intracellular Ca2+ homeostasis. In the heart of Cisd2 knockout mice, Cisd2 deficiency causes intercalated disc defects and leads to degeneration of the mitochondria and sarcomeres, thereby impairing its electromechanical functioning. Furthermore, Cisd2 deficiency disrupts Ca2+ homeostasis via dysregulation of sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a) activity, resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay cardiac aging and attenuate age-related structural defects and functional decline. In addition, it results in a younger cardiac transcriptome pattern during old age. Our findings indicate that Cisd2 plays an essential role in cardiac aging and in the heart's electromechanical functioning. They highlight Cisd2 as a novel drug target when developing therapies to delay cardiac aging and ameliorate age-related cardiac dysfunction.


Assuntos
Senilidade Prematura/genética , Envelhecimento/fisiologia , Bloqueio Atrioventricular/genética , Proteínas Relacionadas à Autofagia/genética , Coração/fisiopatologia , Proteínas do Tecido Nervoso/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Senilidade Prematura/metabolismo , Senilidade Prematura/fisiopatologia , Animais , Bloqueio Atrioventricular/diagnóstico por imagem , Bloqueio Atrioventricular/metabolismo , Bloqueio Atrioventricular/fisiopatologia , Proteínas Relacionadas à Autofagia/deficiência , Cálcio/metabolismo , Eletrocardiografia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Coração/fisiologia , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Proteínas do Tecido Nervoso/deficiência , Sarcômeros/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transcriptoma
16.
Life Sci ; 237: 116902, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31610195

RESUMO

AIMS: Insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) promotes hepatic stellate cell (HSC) autophagy and activation. However, the underlying mechanism remains unknown. Noncoding RNAs (ncRNAs) including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), have received increasing attention. We aimed to investigate the roles of the lncRNA nuclear enriched abundant transcript 1 (NEAT1), miR-29b, and autophagy related protein 9a (Atg9a), and their relationships with each other during IGFBPrP1-induced HSC autophagy and activation. MAIN METHODS: Levels of NEAT1, miR-29b, Atg9a, and autophagy were detected in adenovirus-mediated IGFBPrP1 (AdIGFBPrP1)-treated mouse liver tissue and immortalized mouse hepatic stellate cell line JS1 transfected with either AdIGFBPrP1 or siIGFBPrP1. In AdIGFBPrP1-treated JS1 cells, autophagy and activation were detected after altering NEAT1, miR-29b, or Atg9a levels. In AdIGFBPrP1-treated JS1 cells, relationships among NEAT1, miR-29b, and Atg9a were explored using dual-luciferase reporter assays, Western blot, qRT-PCR, and immunofluorescence. KEY FINDINGS: IGFBPrP1 increased levels of NEAT1, Atg9a, and autophagy while decreasing the level of miR-29b in mouse liver tissues and mouse HSCs. Moreover, NEAT1 increased HSC autophagy and activation while miR-29b decreased both processes. Atg9a also participated in IGFBPrP1-induced HSC autophagy and activation. Importantly, NEAT1, miR-29b, and Atg9a formed a NEAT1/miR-29b/Atg9a regulatory axis for IGFBPrP1-induced HSC autophagy and activation. SIGNIFICANCE: Our study unveiled the new NEAT1/miR-29b/Atg9a regulatory axis involved in IGFBPrP1-induced mouse HSC autophagy and activation. The study thus provides new insights in the pathogenesis and potential therapeutic strategies of liver fibrosis.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Células Estreladas do Fígado/patologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Cirrose Hepática/patologia , Proteínas de Membrana/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteínas de Transporte Vesicular/metabolismo , Adenoviridae/genética , Animais , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Células Estreladas do Fígado/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte Vesicular/genética
17.
Microb Cell Fact ; 18(1): 177, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615527

RESUMO

BACKGROUND: Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae. RESULTS: The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production. CONCLUSION: These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.


Assuntos
Citosol/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Butileno Glicóis/metabolismo , Etanol/metabolismo , Fermentação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Engenharia Metabólica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Am J Clin Oncol ; 42(10): 767-776, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31517637

RESUMO

OBJECTIVES: Autophagy plays a dual role in tumorigenesis. In the initial stages, it promotes cell survival and suppresses carcinogenesis, whereas in cancer development, it induces cancer cell survival. In this study, we investigate the role of autophagy as a protective or tumor suppressor mechanism in colorectal cancer (CRC) cell lines and evaluate its role as a potential biomarker in human tumor samples. MATERIALS AND METHODS: The data of 68 patients with CRC treated at our Department from January 1 to December 31, 2016 were analyzed. Immunohistochemistry evaluation of p62, LC3B, Beclin-1, and Rab-7 in formalin-fixed paraffin-embedded tissue samples was performed and their expression was correlated with clinicopathologic characteristics, mutation status, and therapeutic approach. The χ was used to test an association among categorical variables. Survival curves were estimated using the Kaplan-Meier method and differences were assessed using the log-rank test. Colo-205, HT29, SW-480, and Caco-2 cell lines were also used so as to test the autophagy markers with oxaliplatin, irinotecan, hydroxychloroquine, and 3-methyladenine. RESULTS: Overexpression of Beclin-1 is associated with poor survival (P=0.001) in patients with CRC treated with chemotherapy, irrespective of the stage and mutational status. Rab-7 is also correlated with progression-free survival (PFS) (P=0.088). Oxaliplatin (10 and 20 µΜ) and irinotecan (10 and 20 µΜ) inhibit autophagy in microsatellite stable (MSS) CRC cell lines. The inhibition of autophagy in MSS CRC cell lines after treatment with oxaliplatin and irinotecan is further identified through monodancylcadaverine staining. Moreover, inhibition of autophagy with molecules such as hydroxychloroquine (20 µΜ) and 3-methyladenine (5 mM) was identified by the accumulation of p62 and LC3B. CONCLUSIONS: Beclin-1 is an independent prognostic factor of overall survival and PFS. Also, Rab-7 is identified as an independent prognostic factor of PFS. Besides, several chemotherapeutic drugs such as oxaliplatin and irinotecan inhibit autophagy in MSS CRC cell lines in a similar way like hydroxychloroquine and 3-methyladenine. Thus, in MSS patients who develop chemoresistance, a combination of other therapies that include an autophagy inhibitor could be more beneficial. Further clinical trials are needed to investigate these therapeutic strategies.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteína Beclina-1/metabolismo , Biomarcadores Tumorais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Terapia Combinada/métodos , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Estudos Retrospectivos , Estatísticas não Paramétricas , Análise de Sobrevida
19.
Fish Shellfish Immunol ; 94: 113-121, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31491526

RESUMO

Autophagy related gene 16 (Atg16), which encodes a core protein for autophagosome formation, participates in autophagy activity, the ubiquitin proteasome system and inflammatory response in mammals. In this study, we cloned and characterized an Atg16 homolog from orange-spotted grouper (Epinephelus coioides) (EcAtg16L1). EcAtg16L1 encodes a 656-amino acid polypeptide, which shares 94.22% and 72.65% homology with large yellow croakers (Larimichthys crocea) and humans (Homo sapiens), respectively. EcAtg16L1 contains a conserved Atg16 domain and a WD-repeat-containing domain. Subcellular localization showed that EcAtg16L1 was distributed in the cytoplasm of grouper cells with a dot-like pattern. EcAtg16L1 overexpression promoted Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by the increase in viral gene transcription and viral coat protein. Furthermore, EcAtg16L1 overexpression negatively regulated interferon (IFN)-related molecules and proinflammatory cytokines, and decreased IFN, IFN-stimulated response element, and nuclear factor κB promoter activities. Taken together, aside from its function in autophagosome formation, EcAtg16L1 also plays role in promoting SGIV and RGNNV replication and the pro-viral effect might involve its down regulation to interferon and inflammatory responses.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas Relacionadas à Autofagia/química , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Nodaviridae/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
20.
Proc Natl Acad Sci U S A ; 116(38): 19187-19192, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31484757

RESUMO

Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening.


Assuntos
Aminopeptidases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Estômatos de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Aminopeptidases/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas Relacionadas à Autofagia/genética , Homeostase , Mutação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais
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