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1.
Nat Commun ; 12(1): 2431, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893273

RESUMO

Insulin resistance and lower muscle quality (strength divided by mass) are hallmarks of type 2 diabetes (T2D). Here, we explore whether alterations in muscle stem cells (myoblasts) from individuals with T2D contribute to these phenotypes. We identify VPS39 as an important regulator of myoblast differentiation and muscle glucose uptake, and VPS39 is downregulated in myoblasts and myotubes from individuals with T2D. We discover a pathway connecting VPS39-deficiency in human myoblasts to impaired autophagy, abnormal epigenetic reprogramming, dysregulation of myogenic regulators, and perturbed differentiation. VPS39 knockdown in human myoblasts has profound effects on autophagic flux, insulin signaling, epigenetic enzymes, DNA methylation and expression of myogenic regulators, and gene sets related to the cell cycle, muscle structure and apoptosis. These data mimic what is observed in myoblasts from individuals with T2D. Furthermore, the muscle of Vps39+/- mice display reduced glucose uptake and altered expression of genes regulating autophagy, epigenetic programming, and myogenesis. Overall, VPS39-deficiency contributes to impaired muscle differentiation and reduced glucose uptake. VPS39 thereby offers a therapeutic target for T2D.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Diferenciação Celular/genética , Diabetes Mellitus Tipo 2/genética , Epigenômica/métodos , Mioblastos/metabolismo , Células-Tronco/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Proteínas Relacionadas à Autofagia/deficiência , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Epigênese Genética/genética , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Desenvolvimento Muscular/genética , Proteínas de Transporte Vesicular/deficiência
2.
Mol Cell ; 81(9): 2031-2040.e8, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33909989

RESUMO

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagossomos/patologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A/patogenicidade , Macrolídeos/farmacologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Monensin/farmacologia , Fagocitose , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transdução de Sinais
3.
Molecules ; 26(8)2021 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-33920352

RESUMO

The pathological finding of amyloid-ß (Aß) aggregates is thought to be a leading cause of untreated Alzheimer's disease (AD). In this study, we isolated 2-butoxytetrahydrofuran (2-BTHF), a small cyclic ether, from Holothuria scabra and demonstrated its therapeutic potential against AD through the attenuation of Aß aggregation in a transgenic Caenorhabditis elegans model. Our results revealed that amongst the five H. scabra isolated compounds, 2-BTHF was shown to be the most effective in suppressing worm paralysis caused by Aß toxicity and in expressing strong neuroprotection in CL4176 and CL2355 strains, respectively. An immunoblot analysis showed that CL4176 and CL2006 treated with 2-BTHF showed no effect on the level of Aß monomers but significantly reduced the toxic oligomeric form and the amount of 1,4-bis(3-carboxy-hydroxy-phenylethenyl)-benzene (X-34)-positive fibril deposits. This concurrently occurred with a reduction of reactive oxygen species (ROS) in the treated CL4176 worms. Mechanistically, heat shock factor 1 (HSF-1) (at residues histidine 63 (HIS63) and glutamine 72 (GLN72)) was shown to be 2-BTHF's potential target that might contribute to an increased expression of autophagy-related genes required for the breakdown of the Aß aggregate, thus attenuating its toxicity. In conclusion, 2-BTHF from H. scabra could protect C. elegans from Aß toxicity by suppressing its aggregation via an HSF-1-regulated autophagic pathway and has been implicated as a potential drug for AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Furanos/farmacologia , Holothuria/química , Fármacos Neuroprotetores/farmacologia , Paralisia/prevenção & controle , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Sítios de Ligação , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Furanos/química , Furanos/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Paralisia/genética , Paralisia/metabolismo , Paralisia/patologia , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
BMC Cancer ; 21(1): 411, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33858375

RESUMO

BACKGROUND: Little data is available on prognostic biomarkers and effective treatment options for Kidney Renal Papillary Cell Carcinoma (KIRP) patients, to find potential prognostic biomarkers and new targets was an urgent mission for KIRP therapy. METHODS: The differentially expressed autophagy-related genes (DEARGs) were screened out according to the RNA sequencing data in The Cancer Genome Atlas database, then identified survival-related DEARGs to establish a prognostic model for survival predicting of KIRP patients. Then we verified the robustness and validity of the prognostic risk model through clinicopathological data. At last, we evaluate the prognostic value of genes that formed the prognostic risk model individually. RESULTS: We analyzed the expression of 232 autophagy-related genes (ARGs) in 289 KIRP and 32 non-tumor tissue cases, and 40 mRNAs were screened out as DEARGs. The functional and pathway enrichment analysis was done and protein-protein interaction network was constructed for all DEARGs. To sift candidate DEARGs associated with KIRP patients' survival and create an autophagy-related risk prognostic model, univariate and multivariate Cox regression analysis were did separately. Eventually 3 desirable independent prognostic DEARGs (P4HB, NRG1, and BIRC5) were picked out and used for construct the autophagy-related risk model. The accuracy of the prognostic risk model for survival prediction was assessed by Kaplan-Meier plotter, receiver-operator characteristic curve, and clinicopathological correlational analyses. The prognostic value of above 3 genes was verified individually by survival analysis and expression analysis on mRNA and protein level. CONCLUSIONS: The autophagy-related prognostic model is accurate and applicable, it can predict OS independently for KIRP patients. Three independent prognostic DEARGs can benefit for facilitate personalized target treatment too.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Biomarcadores Tumorais , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Autofagia/genética , Carcinoma de Células Renais/mortalidade , Perfilação da Expressão Gênica , Humanos , Neoplasias Renais/mortalidade , Prognóstico , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Curva ROC , Reprodutibilidade dos Testes , Transcriptoma
5.
Mol Cell ; 81(9): 2013-2030.e9, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33773106

RESUMO

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/metabolismo , Metabolismo dos Lipídeos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Inteligência Artificial , Autofagossomos/genética , Autofagossomos/ultraestrutura , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Cisteína Endopeptidases/genética , Células HEK293 , Células HeLa , Humanos , Imageamento Tridimensional , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Mitofagia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transporte Proteico , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
6.
Mol Med Rep ; 23(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33760149

RESUMO

Endometriosis (EM) is a multifactorial and debilitating chronic benign gynecological disease, but the pathogenesis of the disease is not completely understood. Dysregulated expression of microRNAs (miRNA/miR) is associated with the etiology of EM due to their role in regulating endometrial stromal cell proliferation and invasion. The present study aimed to identify the functions and mechanisms underlying miR­143­3p in EM. To explore the role of miR­143­3p in EM, functional miRNAs were analyzed via bioinformatics analysis. miR­143­3p expression levels in endometriotic stromal cells (ESCs) and normal endometrial stromal cells (NESCs) were measured via reverse transcription­quantitative PCR. The role of miR­143­3p in regulating ESC proliferation and invasion was assessed by performing Cell Counting Kit­8 and Transwell assays, respectively. miR­143­3p expression was significantly upregulated in ESCs compared with NESCs. Functionally, miR­143­3p overexpression inhibited ESC proliferation and invasion, whereas miR­143­3p knockdown promoted ESC proliferation and invasion. Moreover, miR­143­3p inhibited autophagy activation in ESCs, as indicated by decreased green puncta, which represented autophagic vacuoles, decreased microtubule associated protein 1 light chain 3α expression and increased p62 expression in the miR­143­4p mimic group compared with the control group. Moreover, compared with the control group, miR­143­3p overexpression significantly decreased the expression levels of autophagy­related 2B (ATG2B), a newly identified target gene of miR­143­3p, in ESCs. ATG2B overexpression reversed miR­143­3p overexpression­mediated inhibition of ESC proliferation and invasion. Collectively, the results of the present study suggested that miR­143­3p inhibited EM progression, thus providing a novel target for the development of therapeutic agents against EM.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Endometriose/genética , MicroRNAs/genética , Proteínas de Transporte Vesicular/genética , Adulto , Movimento Celular/genética , Proliferação de Células/genética , Endometriose/patologia , Endométrio/metabolismo , Endométrio/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica/genética , Humanos , Pessoa de Meia-Idade , Células Estromais/metabolismo , Células Estromais/patologia
7.
Mol Cell ; 81(6): 1337-1354.e8, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33545068

RESUMO

Autophagy deficiency in fed conditions leads to the formation of protein inclusions highlighting the contribution of this lysosomal delivery route to cellular proteostasis. Selective autophagy pathways exist that clear accumulated and aggregated ubiquitinated proteins. Receptors for this type of autophagy (aggrephagy) include p62, NBR1, TOLLIP, and OPTN, which possess LC3-interacting regions and ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins and ubiquitinated substrates. However, the identity of aggrephagy substrates and the redundancy of aggrephagy and related UBD-containing receptors remains elusive. Here, we combined proximity labeling and organelle enrichment with quantitative proteomics to systematically map the autophagic degradome targeted by UBD-containing receptors under basal and proteostasis-challenging conditions in human cell lines. We identified various autophagy substrates, some of which were differentially engulfed by autophagosomal and endosomal membranes via p62 and TOLLIP, respectively. Overall, this resource will allow dissection of the proteostasis contribution of autophagy to numerous individual proteins.


Assuntos
Autofagossomos , Autofagia , Mapas de Interação de Proteínas , Proteólise , Proteostase , Ubiquitinação , Autofagossomos/genética , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteômica
8.
Eur J Pharmacol ; 897: 173920, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33571535

RESUMO

Hepatic ischemia-reperfusion (HIR) injury is a common pathophysiological process in many clinical settings. This study was designed to compare the protective role of octreotide (somatostatin analogue, OCT) and melatonin (N-acetyl-5-methoxytryptamine, MLT) through the modulation of autophagy against HIR injury in rats. Male albino rats were divided into sham, HIR, OCT at three doses (50, 75, and 100 µg/kg), MLT, MLT + OCT75, compound C (AMPK inhibitor, CC), and CC + OCT75 groups. Ischemia was induced for 30 min followed by 24 h reperfusion. Biochemical, histopathological, immunohistochemical, lipid peroxidation, ELISA, qPCR, and western blot techniques were performed in our study. Liver autophagy was restored by OCT at doses (50 or 75 µg/kg) as indicated by elevating the expressions of Beclin-1, ATG7, and LC3 accompanied by the reduction of p62 expression through induction of AMPK/S317-ULK1 and inhibition of PI3K/AKT/mTOR/S757-ULK1 signaling pathways. As well, OCT maintained the integrity of the Keap1-Nrf2 system for the normal hepatic functions via controlling the Keap1 turnover through autophagy in a p62-dependent manner, resulting in upholding a series of anti-oxidant and anti-inflammatory cascades. These effects were abolished by compound C. On the other hand, MLT showed a decrease in the autophagy markers via inhibiting AMPK/pS317-ULK1 and activating PI3K/AKT/mTOR/pS757-ULK1 pathways. Autophagy inhibition with MLT markedly reversed the hepatoprotective effects of OCT75 after HIR injury. Finally, our results proved for the first time that OCT75 was more effective than MLT as it was sufficient to induce protective autophagy in our HIR model, which led to the induction of Nrf2-dependent AMPK/autophagy pathways.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Hepatopatias/prevenção & controle , Fígado/efeitos dos fármacos , Melatonina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Octreotida/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Modelos Animais de Doenças , Fígado/enzimologia , Fígado/patologia , Hepatopatias/enzimologia , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Fator 2 Relacionado a NF-E2/genética , Fosfatidilinositol 3-Quinase/genética , Ratos , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/genética
9.
Int Immunopharmacol ; 93: 107378, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33529915

RESUMO

OBJECTIVE: This study was performed to test whether ATG16L1 rs2241880, rs6758317 and ATG16L2 rs11235604 polymorphisms were associated with RA and further examine the genetic interaction between ATG16L1 and ATG16L2 in RA among a Chinese population. METHODS: A total of 594 RA patients and 604 healthy controls were included, and the genetic polymorphisms were genotyped based on HI-SNP technology. RESULTS: Significant associations of ATG16L1 rs2241880 polymorphism with RA (T/T versus C/T + C/C, OR = 1.32, 95% CI 1.04-1.67, P = 0.02), cyclic citrullinated peptide (CCP)-positive RA (genotype comparison, P = 5.38 × 10-3; T/T versus C/T + C/C, OR = 1.45, 95% CI 1.12-1.87, P = 4.86 × 10-3) and rheumatoid factor (RF)-positive RA (genotype comparison, P = 0.03; T versus C, OR = 1.23, 95% CI 1.01-1.49, P = 0.04; T/T versus C/T + C/C, OR = 1.44, 95% CI 1.10-1.88, P = 7.62 × 10-3) were found. Significant genetic interaction between ATG16L1 rs2241880 and ATG16L2 rs11235604 was associated RA (P = 0.03), and significant genetic interaction between ATG16L1 rs6758317 and ATG16L2 rs11235604 was associated with RA (P = 7.57 × 10-3), CCP-positive RA (P = 0.01) and RF-positive RA (P = 0.01). Consistently, stratification analysis found that significant associations of RA with ATG16L1 rs2241880, rs6758317 polymorphisms were only detected among individuals carrying C/T genotype of the ATG16L2 rs11235604 polymorphism. CONCLUSIONS: Our results indicated that ATG16L1 rs2241880 polymorphism was associated with RA in Chinese population, and provided evidence for genetic interaction between ATG16L1 and ATG16L2 in determing the development of RA, highlighting the involvement of autophagy in the pathogenesis of RA.


Assuntos
Artrite Reumatoide/genética , Grupo com Ancestrais do Continente Asiático/genética , Proteínas Relacionadas à Autofagia/genética , Adulto , Idoso , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
10.
EMBO J ; 40(6): e105543, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33586810

RESUMO

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Vírus da Influenza A/patogenicidade , Proteínas Associadas aos Microtúbulos/metabolismo , Infecções por Orthomyxoviridae/genética , Deleção de Sequência , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Animais , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Embrião de Galinha , Citocinas/metabolismo , Cães , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Domínios Proteicos , Replicação Viral
11.
Gene ; 782: 145537, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33636294

RESUMO

Detection of TCGA data revealed that WIPI1 is highly expressed in osteosarcoma cells. So we explore the mechanisms of WIPI1 affecting the proliferation of osteosarcoma cells through Affymetrix microarray analysis. Functional analysis of differentially expressed genes shows that the classical signaling pathways affecting tumor formation and development have changed significantly. By fitting analysis, it is speculated that the WIPI1 may function in the direction of osteosarcoma by regulating the expression of multiple cell cycle-related genes such as CDKN1A, CDK4 and CCND1. Therefore, the key genes are selected for RT-PCR and Western-blot verification. Combined with flow and other means, WIPI1 may affect the cell cycle and the osteosarcoma by regulating the expression of CDKN1A, CDK4 and CCND1. To verify the results, the effect of WIPI1 on cell proliferation was quantified by MTT, cell counts and nude mouse tumorigenicity assay. The results showed that WIPI1 promotes osteosarcoma cell proliferation.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Neoplasias Ósseas/genética , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Proteínas de Membrana/genética , Osteossarcoma/genética , Animais , Proteínas Relacionadas à Autofagia/fisiologia , Neoplasias Ósseas/patologia , Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Proteínas de Membrana/fisiologia , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/patologia , Software , Transcriptoma
12.
J Exp Bot ; 72(9): 3340-3351, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33587749

RESUMO

Autophagy is a highly conserved degradative pathway that ensures cellular homeostasis through the removal of damaged or useless intracellular components including proteins, membranes, or even entire organelles. A main hallmark of autophagy is the biogenesis of autophagosomes, double-membrane vesicles that engulf and transport to the vacuole the material to be degraded and recycled. The formation of autophagosomes responds to integrated signals produced as a consequence of metabolic reactions or different types of stress and is mediated by the coordinated action of core autophagy-related (ATG) proteins. ATG4 is a key Cys-protease with a dual function in both ATG8 lipidation and free ATG8 recycling whose balance is crucial for proper biogenesis of the autophagosome. ATG4 is conserved in the green lineage, and its regulation by different post-translational modifications has been reported in the model systems Chlamydomonas reinhardtii and Arabidopsis. In this review, we discuss the major role of ATG4 in the integration of stress and redox signals that regulate autophagy in algae and plants.


Assuntos
Proteínas Relacionadas à Autofagia , Proteínas Associadas aos Microtúbulos , Peptídeo Hidrolases , Arabidopsis , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Chlamydomonas reinhardtii , Proteínas Associadas aos Microtúbulos/metabolismo , Oxirredução
13.
J Mol Biol ; 433(5): 166809, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33484718

RESUMO

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12-Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12-Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113-131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64-99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.


Assuntos
Proteína 12 Relacionada à Autofagia/química , Proteína 5 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/química , Proteínas Relacionadas à Autofagia/química , Membrana Celular/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Autofagia/genética , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Sítios de Ligação , Membrana Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Lipossomos/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
14.
BMC Cancer ; 21(1): 58, 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33435917

RESUMO

BACKGROUND: Autophagy-related genes (ARGs) have been confirmed to have an important role in tumorigenesis and tumor microenvironment formation. Nevertheless, a systematic analysis of ARGs and their clinical significance in sarcoma patients is lacking. METHODS: Gene expression files from The Cancer Genome Atlas (TCGA) database and Genotype-Tissue Expression (GTEx) were used to select differentially expressed genes (DEGs). Differentially expressed ARGs (DEARGs) were determined by matching the DEG and HADb gene sets, which were evaluated by functional enrichment analysis. Unsupervised clustering of the identified DEARGs was conducted, and associations with tumor microenvironment (TME), immune checkpoints, and immune cells were analyzed simultaneously. Two prognostic signatures, one for overall survival (OS) and one for disease-free survival (DFS), were established and validated in an independent set. RESULTS: In total, 84 DEARGs and two clusters were identified. TME scores, five immune checkpoints, and several types of immune cells were found to be significantly different between two clusters. Two prognostic signatures incorporating DEARGs showed favorable discrimination and were successfully validated. Two nomograms combining signature and clinical variables were generated. The C-indexes were 0.818 and 0.747 for the OS and DFS nomograms, respectively. CONCLUSION: This comprehensive analyses of the ARG landscape in sarcoma showed novel ARGs related to carcinogenesis and the immune microenvironment. These findings have implications for prognosis and therapeutic responses, which reveal novel potential prognostic biomarkers, promote precision medicine, and provide potential novel targets for immunotherapy.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Nomogramas , Sarcoma/patologia , Transcriptoma , Humanos , Prognóstico , Curva ROC , Sarcoma/genética , Microambiente Tumoral
15.
Nat Commun ; 12(1): 374, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446636

RESUMO

During autophagy the enzyme Atg3 catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region of human Atg3 (hAtg3) communicates information from the N-terminal membrane curvature-sensitive amphipathic helix (AH), which presumably targets the enzyme to the tip of phagophore, to the C-terminally located catalytic core for LC3-PE conjugation. Mutations in the putative communication region greatly reduce or abolish the ability of hAtg3 to catalyze this conjugation in vitro and in vivo, and alter the membrane-bound conformation of the wild-type protein, as reported by NMR. Collectively, our results demonstrate that the N-terminal conserved region of hAtg3 works in concert with its geometry-selective AH to promote LC3-PE conjugation only on the target membrane, and substantiate the concept that highly curved membranes drive spatial regulation of the autophagosome biogenesis during autophagy.


Assuntos
Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membrana Celular/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/genética , Biocatálise , Membrana Celular/genética , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Enzimas de Conjugação de Ubiquitina/genética
16.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33328309

RESUMO

Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins; however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing small interfering RNA (siRNA)-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release, suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that indirect interference with ULK1 through inhibition of the upstream regulator AMP-activated protein kinase (AMPK) impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99), and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure the efficient release of viral particles.IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Citomegalovirus/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Liberação de Vírus , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Capsídeo/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Regulação para Cima , Proteínas da Matriz Viral/metabolismo , Replicação Viral
17.
Eur J Pharmacol ; 892: 173660, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33310181

RESUMO

Chemoresistance has doubled the effort needed to reach an effective treatment for cancer. Now, scientists should consider molecular pathways and mechanisms involved in chemoresistance to overcome cancer. Autophagy is a "self-digestion" mechanism in which potentially toxic and aged organelles and macromolecules are degraded. Increasing evidence has shown that autophagy possesses dual role in cancer cells (onco-suppressor or oncogene). So, it is vital to identify its role in cancer progression and malignancy. MicroRNAs (miRs) are epigenetic factors capable of modulation of autophagy in cancer cells. In the current review, we emphasize on the relationship between miRs and autophagy in cancer chemotherapy. Besides, we discuss upstream mediators of miR/autophagy axis in cancer chemotherapy including long non-coding RNAs, circular RNAs, Nrf2 c-Myc, and HIF-1α. At the final section, we provide a discussion about how anti-tumor compounds affect miR/autophagy axis in ensuring chemosensitivity. These topics are described in this review to show how autophagy inhibition/induction can lead to chemosensitivity/chemoresistance, and miRs are considered as key players in these discussions.


Assuntos
Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/metabolismo , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais
18.
Acta Biochim Biophys Sin (Shanghai) ; 53(2): 140-148, 2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33382073

RESUMO

Myocardial injury is still a serious condition damaging the public health. Clinically, myocardial injury often leads to cardiac dysfunction and, in severe cases, death. Reperfusion of the ischemic myocardial tissues can minimize acute myocardial infarction (AMI)-induced damage. MicroRNAs are commonly recognized in diverse diseases and are often involved in the development of myocardial ischemia/reperfusion injury. However, the role of miR-431 remains unclear in myocardial injury. In this study, we investigated the underlying mechanisms of miR-431 in the cell apoptosis and autophagy of human cardiomyocytes in hypoxia/reoxygenation (H/R). H/R treatment reduced cell viability, promoted cell apoptotic rate, and down-regulated the expression of miR-431 in human cardiomyocytes. The down-regulation of miR-431 by its inhibitor reduced cell viability and induced cell apoptosis in the human cardiomyocytes. Moreover, miR-431 down-regulated the expression of autophagy-related 3 (ATG3) via targeting the 3'-untranslated region of ATG3. Up-regulated expression of ATG3 by pcDNA3.1-ATG3 reversed the protective role of the overexpression of miR-431 on cell viability and cell apoptosis in H/R-treated human cardiomyocytes. More importantly, H/R treatments promoted autophagy in the human cardiomyocytes, and this effect was greatly alleviated via miR-431-mimic transfection. Our results suggested that miR-431 overexpression attenuated the H/R-induced myocardial damage at least partly through regulating the expression of ATG3.


Assuntos
Apoptose , Proteínas Relacionadas à Autofagia/metabolismo , MicroRNAs/biossíntese , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/genética , Humanos , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/genética , Enzimas de Conjugação de Ubiquitina/genética
19.
Arterioscler Thromb Vasc Biol ; 41(2): e82-e96, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33356389

RESUMO

OBJECTIVE: Atherosclerotic lesions are often characterized by accumulation of OxLDL (oxidized low-density lipoprotein), which is associated with vascular inflammation and lesion vulnerability to rupture. Extracellular AIBP (apolipoprotein A-I binding protein; encoded by APOA1BP gene), when secreted, promotes cholesterol efflux and regulates lipid rafts dynamics, but its role as an intracellular protein in mammalian cells remains unknown. The aim of this work was to determine the function of intracellular AIBP in macrophages exposed to OxLDL and in atherosclerotic lesions. Approach and Results: Using a novel monoclonal antibody against human and mouse AIBP, which are highly homologous, we demonstrated robust AIBP expression in human and mouse atherosclerotic lesions. We observed significantly reduced autophagy in bone marrow-derived macrophages, isolated from Apoa1bp-/- compared with wild-type mice, which were exposed to OxLDL. In atherosclerotic lesions from Apoa1bp-/- mice subjected to Ldlr knockdown and fed a Western diet, autophagy was reduced, whereas apoptosis was increased, when compared with that in wild-type mice. AIBP expression was necessary for efficient control of reactive oxygen species and cell death and for mitochondria quality control in macrophages exposed to OxLDL. Mitochondria-localized AIBP, via its N-terminal domain, associated with E3 ubiquitin-protein ligase PARK2 (Parkin), MFN (mitofusin)1, and MFN2, but not BNIP3 (Bcl2/adenovirus E1B 19-kDa-interacting protein-3), and regulated ubiquitination of MFN1 and MFN2, key components of mitophagy. CONCLUSIONS: These data suggest that intracellular AIBP is a new regulator of autophagy in macrophages. Mitochondria-localized AIBP augments mitophagy and participates in mitochondria quality control, protecting macrophages against cell death in the context of atherosclerosis.


Assuntos
Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Lipoproteínas LDL/toxicidade , Macrófagos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Fosfoproteínas/metabolismo , Racemases e Epimerases/metabolismo , Animais , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apoptose/efeitos dos fármacos , Aterosclerose/genética , Aterosclerose/patologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Modelos Animais de Doenças , Células HEK293 , Células Hep G2 , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfoproteínas/genética , Racemases e Epimerases/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
20.
Front Immunol ; 11: 573646, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33329536

RESUMO

During their lifespan, dendritic cells (DCs) are exposed to different pO2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.


Assuntos
Autofagia/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Hipóxia Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Humanos , Ligantes , Antígeno 96 de Linfócito/agonistas , Transdução de Sinais , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo
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