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1.
Plant Physiol ; 193(1): 304-321, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37195145

RESUMO

As a fundamental metabolic pathway, autophagy plays important roles in plant growth and development, particularly under stress conditions. A set of autophagy-related (ATG) proteins is recruited for the formation of a double-membrane autophagosome. Among them, the essential roles of ATG2, ATG18, and ATG9 have been well established in plant autophagy via genetic analysis; however, the underlying molecular mechanism for ATG2 in plant autophagosome formation remains poorly understood. In this study, we focused on the specific role of ATG2 in the trafficking of ATG18a and ATG9 during autophagy in Arabidopsis (Arabidopsis thaliana). Under normal conditions, YFP-ATG18a proteins are partially localized on late endosomes and translocated to ATG8e-labeled autophagosomes upon autophagic induction. Real-time imaging analysis revealed sequential recruitment of ATG18a on the phagophore membrane, showing that ATG18a specifically decorated the closing edges and finally disassociated from the completed autophagosome. However, in the absence of ATG2, most of the YFP-ATG18a proteins are arrested on autophagosomal membranes. Ultrastructural and 3D tomography analysis showed that unclosed autophagosome structures are accumulated in the atg2 mutant, displaying direct connections with the endoplasmic reticulum membrane and vesicular structures. Dynamic analysis of ATG9 vesicles suggested that ATG2 depletion also affects the association between ATG9 vesicles and the autophagosomal membrane. Furthermore, using interaction and recruitment analysis, we mapped the interaction relationship between ATG2 and ATG18a, implying a possible role of ATG18a in recruiting ATG2 and ATG9 to the membrane. Our findings unveil a specific role of ATG2 in coordinating ATG18a and ATG9 trafficking to mediate autophagosome closure in Arabidopsis.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Saccharomyces cerevisiae , Autofagossomos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/metabolismo , Autofagia/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Arabidopsis/metabolismo
2.
Zhen Ci Yan Jiu ; 47(4): 298-304, 2022 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-35486008

RESUMO

OBJECTIVE: To observe the effect of electroacupuncture (EA) on learning-memory ability, ultrastructural changes of hippocampal CA1 neurons, reactive oxygen species (ROS) level, Nod-like receptor protein 3 (NLRP3) and auto-phagy-related proteins expression in the hippocampus of vascular dementia (VD) rats, so as to reveal its partial mechanisms in treating VD. METHODS: Male SD rats were randomly divided into sham operation, model, and EA groups (n=10 rats in each group). The VD model was established by permanent ligation of bilateral common carotid arteries. Rats of the EA group were treated with EA at "Baihui" (GV20), "Dazhui" (GV14) and bilateral "Shenshu" (BL23) for 30 min, once a day for 4 weeks. Morris water maze was used to evaluate the learning and memory ability of rats before modeling, 4 weeks after modeling and after intervention. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of hippocampal CA1 neurons. The level of ROS in hippocampus was detected by DCFH-DA fluorescence probe. The expressions of NLRP3, autophagy-related protein Beclin1 and microtubule-associated protein 1 light chain 3 (LC3) were measured by Western blot. RESULTS: In comparison with the sham operation group, the average escape latency of rats in the model group was prolonged (P<0.01), and the times of crossing the original platform were reduced (P<0.05), the level of ROS, the expression levels of LC3-Ⅱ/LC3-Ⅰ ratio, Beclin1 and NLRP3 proteins in hippocampus were increased (P<0.01, P<0.05) in the model group. After EA intervention, the average escape latency of rats was significantly shortened (P<0.01), and the times of crossing the original platform were increased (P<0.05), the level of ROS, the expression levels of LC3-Ⅱ/LC3-Ⅰ ratio, Beclin1 and NLRP3 proteins in hippocampus were decreased (P<0.01, P<0.05) in the EA group compared with those of the model group. Outcomes of TEM showed that CA1 neurons in the hippocampus were damaged, chromatin aggregation, mitochondria pyknosis, cristae structure disorder, rough endoplasmic reticulum expanded and degranulated, the number of free ribosomes decreased, and autophagy could be seen in the model group, which were milder in the EA group. CONCLUSION: EA at GV20, GV14 and BL23 can improve the learning and memory abilities of VD rats, alleviate the ultrastructural damage of neurons in hippocampal CA1 area, and repair the damaged neurons. The mechanism may be related to the reduction of ROS level, LC3-Ⅱ/LC3-Ⅰ ratio, NLRP3 and Beclin1 protein expression, the decrease of neuronal autophagy, inhibition of activation of NLRP3 inflammasome and alleviation of central inflammatory response.


Assuntos
Demência Vascular , Eletroacupuntura , Animais , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/análise , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Demência Vascular/genética , Demência Vascular/metabolismo , Demência Vascular/terapia , Hipocampo/metabolismo , Masculino , Memória , Proteína 3 que Contém Domínio de Pirina da Família NLR/análise , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas NLR , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/análise
3.
J Neurochem ; 159(1): 145-155, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34129687

RESUMO

Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Mutação/fisiologia , Estresse Oxidativo/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Esclerose Lateral Amiotrófica/patologia , Proteínas Relacionadas à Autofagia/análise , Linhagem Celular , Humanos , Imagem Óptica/métodos
4.
J Pharm Pharmacol ; 73(7): 986-995, 2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-33877365

RESUMO

OBJECTIVES: To explore the potential molecular mechanism underlying the effect of green tea extract (TE), rich in tea polyphenols (TPs), on improving alcohol-induced liver injury. METHODS: Mice were intragastrically treated with 50% (v/v) alcohol administration (15 ml/kg BW) with or without three doses of TE (50, 120 and 300 mg TPs/kg BW) daily for 4 weeks, and biological changes were tested. KEY FINDINGS: The TE improved the functional and histological situations in the liver of the mice accepted alcohol administration, including enzymes for alcohol metabolism, oxidative stress and lipid accumulation. Interestingly, the TE increased the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2), with the decreasing expression of kelch-like ECH-associated protein 1 (Keap1), indicating the association between the effect of TE with Nrf2-mediated antioxidant signalling. Moreover, the TE restored the activity of autophagy, showing as lifted Beclin-1 expression, LC3B-II/LC3B-I ratio, and decreased p62 expression. Importantly, all these effects were dose-dependent. CONCLUSIONS: These findings provide a new notion for the first time that the TE preventing against alcohol-induced liver injury is closely related to accelerated metabolism of alcohol and relieved oxidative stress, which is associated with Nrf2 signalling activation and autophagy restoration, thus the reduction of lipid accumulation in liver.


Assuntos
Autofagia/efeitos dos fármacos , Hepatopatias Alcoólicas , Fator 2 Relacionado a NF-E2/metabolismo , Chá , Animais , Antioxidantes/farmacologia , Proteínas Relacionadas à Autofagia/análise , Proteína Beclina-1/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/prevenção & controle , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
5.
Virchows Arch ; 478(3): 497-506, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32851507

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a disease with a dismal prognosis. Currently, the causing agent(s) are poorly understood. Recent data suggest that senescence and autophagy might play a role in its development, as well as changes in metabolism due to hypoxic conditions. In this study, the expression of senescence markers in 23 cases of usual interstitial pneumonia (UIP)/IPF and UIP/chronic autoimmune diseases (UIP/AuD) was investigated. The status of autophagy was evaluated with respect to either antiinflammatory or antihypoxia function. Formalin-fixed paraffin-embedded tissues of UIP were selected for immunohistochemistry with antibodies for p21, p16, and ß-galactosidase (senescence); for LC3, SIRT1, MAP1S, and pAMKα (autophagy); and for LDH and GLUT1 (metabolism). Epithelial cells in cystic remodeled areas of UIP stained for p16 and p21, p16 being more specific compared with p21. Myofibroblasts were negative in all cases. An upregulation of all four autophagy markers was seen not only in epithelia within remodeled areas and proliferating myofibroblasts, but also in bronchial epithelia and pneumocytes. Upregulated autophagy points to a compensatory mechanism for hypoxia; therefore, LDH and GLUT1 were investigated. Their expression was present in epithelia within cystic remodeling and in myofibroblasts. The cells within the remodeled areas stained for cytokeratin 5, but coexpressed TTF1, confirming their origin from basal cells of bronchioles. Within this population, senescent cells arise. Our results indicated that autophagy in UIP very likely helps cells to survive in hypoxic condition. By phagocytosis of cellular debris, they supplement their need for nutrition, and by upregulating LDH and GLUT1, they compensate for local hypoxia.


Assuntos
Doenças Autoimunes/patologia , Autofagia , Senescência Celular , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Proteínas Relacionadas à Autofagia/análise , Biomarcadores/análise , Proteínas de Ciclo Celular/análise , Hipóxia Celular , Proliferação de Células , Metabolismo Energético , Células Epiteliais/química , Células Epiteliais/patologia , Feminino , Transportador de Glucose Tipo 1/análise , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Imuno-Histoquímica , L-Lactato Desidrogenase/análise , Pulmão/química , Masculino , Pessoa de Meia-Idade , Miofibroblastos/química , Miofibroblastos/patologia , Fagocitose
6.
Autophagy ; 16(12): 2276-2281, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33249989

RESUMO

In less than eleven months, the world was brought to a halt by the COVID-19 outbreak. With hospitals becoming overwhelmed, one of the highest priorities concerned critical care triage to ration the scarce resources of intensive care units. Which patient should be treated first? Based on what clinical and biological criteria? A global joint effort rapidly led to sequencing the genomes of tens of thousands of COVID-19 patients to determine the patients' genetic signature that causes them to be at risk of suddenly developing severe disease. In this commentary, we would like to consider some points concerning the use of a multifactorial risk score for COVID-19 severity. This score includes macroautophagy (hereafter referred to as autophagy), a critical host process that controls all steps harnessed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Abbreviation list: ATG5: autophagy related 5; BECN1: beclin 1; COVID-19: coronavirus infectious disease-2019; EGR1: early growth response 1; ER: endoplasmic reticulum; DMVs: double-membrane vesicles; IBV: infectious bronchitis virus; MAP1LC3: microtubule associated protein 1 light chain 3; LC3-I: proteolytically processed, non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; MEFs: mouse embryonic fibroblasts; MERS-CoV: Middle East respiratory syndrome-coronavirus; MHV: mouse hepatitis virus; NSP: non-structural protein; PEDV: porcine epidemic diarrhea virus; PLP2-TM: membrane-associated papain-like protease 2; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TGEV: transmissible gastroenteritis virus.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , COVID-19/diagnóstico , COVID-19/terapia , Transcriptoma , Animais , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/análise , Biomarcadores/análise , Biomarcadores/metabolismo , COVID-19/genética , COVID-19/patologia , Predisposição Genética para Doença , Humanos , Vírus da Bronquite Infecciosa/fisiologia , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Técnicas de Diagnóstico Molecular/métodos , Prognóstico , Projetos de Pesquisa , Fatores de Risco , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Transcriptoma/fisiologia
7.
Biosci Rep ; 40(6)2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32458987

RESUMO

Pressure ulcers (PUs) are a common clinical issue lacking effective treatment and validated pharmacological therapy in hospital settings. Ischemia-reperfusion injury of deep tissue, especially muscle, plays a vital role in the formation and development of the overwhelming majority of PUs. However, muscular protein expression study in PUs has not been reported. Herein, we aimed to investigate the muscular proteins profiles in PUs and to explore the pathological mechanism of PUs. The iTRAQ LC-MS/MS was conducted to detect the protein profiles in clinical muscle samples of PUs. The GO and KEGG pathways analyses were performed for annotation of differentially expressed proteins. Protein-protein interaction (PPI) network was constructed by STRING online database, and hub proteins were validated by the immunoblotting. Based on proteomics results, we found a number of proteins that were differentially expressed in PU muscle samples compared with the normal and identified unique proteins expression patterns between these two groups, suggesting that they might involve in pathological process of the disease. Importantly, cathepsin B and D, as well as other autophagy-lysosome and apoptosis associated proteins were identified. Further experiments characterize the expression of these proteins and their regulation in the process of apoptosis and autophagy. These findings may provide novel insights into the mechanisms of lysosome-associated pathways involved in the initiation of PUs. This is the first study linking proteomics to PUs muscle tissues, which indicated cathepsin B and D might be key drug target for PUs.


Assuntos
Cromatografia Líquida , Proteínas Musculares/análise , Músculo Esquelético/química , Úlcera por Pressão/metabolismo , Proteoma , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Proteínas Reguladoras de Apoptose/análise , Proteínas Relacionadas à Autofagia/análise , Biomarcadores/análise , Estudos de Casos e Controles , Catepsina B/análise , Catepsina D/análise , Biologia Computacional , Humanos , Músculo Esquelético/patologia , Úlcera por Pressão/patologia , Mapas de Interação de Proteínas
8.
Methods Mol Biol ; 1880: 231-242, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610701

RESUMO

Correlative live-cell imaging and super-resolution microscopy of autophagy was developed to combine the temporal resolution of time-lapse fluorescence microscopy with the spatial resolution of super-resolution microscopy. HEK293 cells that express recombinant proteins of interest fused to fluorescent tags are imaged live to capture the formation of autophagosomes, fixed on stage to "snap-freeze" these structures, stained with appropriate antibodies, relocated, and imaged at super resolution by direct stochastic optical reconstruction microscopy. This chapter provides an easy-to-follow protocol along with practical tips and background information to help set up and perform an experiment.


Assuntos
Autofagia , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Técnicas de Cultura de Células/métodos , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Transfecção/métodos
9.
Methods Mol Biol ; 1880: 491-510, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610717

RESUMO

Three-dimensional (3D) models are acquiring importance in cancer research due to their ability to mimic multiple features of the tumor microenvironment more accurately than standard monolayer two-dimensional (2D) cultures. Several groups, including our laboratory, are now accumulating evidence that autophagy in solid tumors is also better represented in 3D than in 2D. Here we detail how we generate 3D models, both in vitro multicellular spheroids generated from cell lines and ex vivo tumor fragment spheroids generated from tumor samples, and how autophagy can be measured in 3D cultures.


Assuntos
Autofagia/fisiologia , Técnicas de Cultura de Células/métodos , Mesotelioma/patologia , Esferoides Celulares/patologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Técnicas de Cultura de Células/instrumentação , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismo
11.
Methods Mol Biol ; 1880: 691-701, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610732

RESUMO

Selective autophagy enables degradation of specific cargo such as protein aggregates or organelles and thus plays an essential role in the regulation of cellular homeostasis. Cargo specificity is achieved on the level of autophagy receptors that concurrently bind the cargo and the autophagosomal membrane. Recent studies have demonstrated that selective autophagy is tightly regulated by posttranslational modifications of autophagy receptors, in particular protein phosphorylation. Phosphorylation of autophagy receptors by different kinases, including Tank-binding kinase (TBK1), can increase their affinity toward the cargo or autophagosomes and thereby regulate the specificity and activity of selective autophagy depending on the cellular condition.Here, we report an approach for quantitative analysis of phosphorylation sites on autophagy receptors using mass spectrometry-based proteomics. In this protocol, GFP-tagged autophagy receptors are purified based on the high-affinity binding between GFP and GFP-Trap agarose. Interaction partners and background binders are subsequently removed by washes under denaturing conditions to obtain a pure fraction of the bait protein, thereby reducing the complexity of the analyzed sample. The bait protein is then digested on-bead, and peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The described approach permits systematic identification and quantification of phosphorylation sites on autophagy receptors and other autophagic components. In addition to phosphorylation, this protocol is suitable for investigating other posttranslational modifications, including protein ubiquitylation.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/análise , Autofagia/fisiologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Células HEK293 , Humanos , Isótopos de Nitrogênio/química , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteômica/instrumentação , Espectrometria de Massas em Tandem/instrumentação
12.
Methods Mol Biol ; 1882: 197-206, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30378056

RESUMO

Macroautophagy is a catabolic process through which redundant, aged, or damaged cellular structures are first enclosed within double-membrane vesicles (called autophagosomes), and thereafter degraded within lysosomes. Macroautophagy provides a primary route for the turnover of macromolecules, membranes and organelles, and as such plays a major role in cell homeostasis. As part of the stress response, autophagy is crucial to determine the cell fate in response to extracellular or intracellular injuries. Autophagy is involved in cancerogenesis and in cancer progression. Here we illustrate the essential methods for monitoring autophagy in pancreatic cancer cells.


Assuntos
Proteínas Relacionadas à Autofagia/análise , Autofagia , Immunoblotting/métodos , Neoplasias Pancreáticas/patologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/metabolismo , Carcinogênese/patologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Progressão da Doença , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes/química , Humanos , Immunoblotting/instrumentação , Lisossomos/patologia , Macrolídeos/farmacologia , Camundongos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Pâncreas/citologia , Pâncreas/patologia
13.
Histol Histopathol ; 33(10): 1075-1087, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29809274

RESUMO

Keloid is a fibro-proliferative skin disorder with tumor-like behavior and dependence on anaerobic glycolysis (the Warburg effect), but its exact pathogenesis is unknown. Although autophagy is widely accepted as a lysosomal pathway for cell survival and cellular homeostasis (specifically upon exposure to stressors such as hypoxia), very few studies have investigated the involvement of autophagy and related glycolytic effectors in keloidogenesis. Here the authors examined the expression and cellular localization of autophagy proteins (LC3, pan-cathepsin), glycolytic markers (LDH, MCT1, MCT4) and the transcription factor HIF isoforms in human keloid samples using immunohistochemical analysis and double-labeling immunofluorescence methods. Based on H&E staining and expression of CD31, keloids were compartmentalized into hypoxic central and normoxic marginal zones. Vimentin-expressing fibroblasts in the central zone exhibited greater autophagy than their marginal-zone counterparts, as evidenced by increased LC3 puncta formation and co-localization with lysosomal pan-cathepsin. LDH (a lactate stimulator), MCT4 (a lactate exporter) and HIF-1 α expression levels were also higher in central-zone fibroblasts. Conversely, HIF-2 α expression was upregulated in fibroblasts and endothelial cells of the peripheral zone, while MCT1 was expressed in both zones. Taken together, these observations suggest that upregulation of autophagy and glycolysis markers in keloid hypoxic-zone fibroblasts may indicate a prosurvival mechanism allowing the extrusion of lactate to marginal-zone fibroblasts via metabolic coupling. The authors believe this is the first report on differential expression of autophagic and glycolytic markers in keloid-zone fibroblasts. The study results indicate that autophagy inhibitors and MCT4 blockers may have therapeutic implications in keloid treatment.


Assuntos
Proteínas Relacionadas à Autofagia/análise , Autofagia , Fibroblastos/química , Glicólise , Queloide/metabolismo , Pele/química , Biomarcadores/análise , Hipóxia Celular , Fibroblastos/patologia , Imunofluorescência , Humanos , Queloide/patologia , Queloide/cirurgia , Transdução de Sinais , Pele/patologia
14.
Toxicol Ind Health ; 34(1): 23-35, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29166827

RESUMO

The exploding popularity of mobile phones and their close proximity to the brain when in use has raised public concern regarding possible adverse effects from exposure to radiofrequency electromagnetic fields (RF-EMF) on the central nervous system. Numerous studies have suggested that RF-EMF emitted by mobile phones can influence neuronal functions in the brain. Currently, there is still very limited information on what biological mechanisms influence neuronal cells of the brain. In the present study, we explored whether autophagy is triggered in the hippocampus or brain stem after RF-EMF exposure. C57BL/6 mice were exposed to 835 MHz RF-EMF with specific absorption rates (SAR) of 4.0 W/kg for 12 weeks; afterward, the hippocampus and brain stem of mice were dissected and analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that several autophagic genes, which play key roles in autophagy regulation, were significantly upregulated only in the hippocampus and not in the brain stem. Expression levels of LC3B-II protein and p62, crucial autophagic regulatory proteins, were significantly changed only in the hippocampus. In parallel, transmission electron microscopy (TEM) revealed an increase in the number of autophagosomes and autolysosomes in the hippocampal neurons of RF-EMF-exposed mice. The present study revealed that autophagy was induced in the hippocampus, not in the brain stem, in 835 MHz RF-EMF with an SAR of 4.0 W/kg for 12 weeks. These results could suggest that among the various adaptation processes to the RF-EMF exposure environment, autophagic degradation is one possible mechanism in specific brain regions.


Assuntos
Autofagia/efeitos da radiação , Tronco Encefálico/efeitos da radiação , Campos Eletromagnéticos/efeitos adversos , Hipocampo/efeitos da radiação , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Perfilação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
15.
Methods Enzymol ; 587: 171-188, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28253954

RESUMO

Posttranslational modifications (PTMs) increase the functional diversity of proteins and play a key role in many cellular processes. Macroautophagy (hereafter simply referred to as autophagy) is an evolutionarily conserved, lysosome-dependent degradation pathway. This process is finely regulated by autophagy-related (ATG) genes widely conserved among eukaryotes from yeast to mammals. Various PTMs of ATG proteins such as phosphorylation, ubiquitination, and acetylation have been theorized to play a critical role in modulating autophagic processes and activity. In this chapter, we introduce several antibody-based tools (e.g., Western blot, Simple Western™, immunofluorescence, and immunoprecipitation) that are widely used to assess the PTMs of ATG proteins in mammalian cells.


Assuntos
Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Western Blotting/métodos , Imunofluorescência/métodos , Imunoprecipitação/métodos , Processamento de Proteína Pós-Traducional , Animais , Células Cultivadas , Humanos , Mamíferos
16.
Methods Enzymol ; 587: 207-225, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28253956

RESUMO

While only one Atg4 is present in yeast, there are four Atg4 homologues in human and in mouse with different substrate specificities and catalytic efficiencies. The molecule Atg4 is a type of cysteine protease, and is known for its crucial roles in cleavage of the Atg8 family proteins before they can be conjugated to phospholipids, and also in cleavage of the conjugated Atg8 molecules from the membrane, a process known as deconjugation. Both processes are required for the maximal efficiency in autophagosome biogenesis. Atg4 could thus be a target for intervention of the autophagy process. It is thus important to measure Atg4 activity to determine and to modulate the autophagy function. Here, we review the catalytic functions and regulatory mechanisms of human Atg4 proteases and discuss the methodology for analyzing Atg4 activity in details.


Assuntos
Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/metabolismo , Biologia Molecular/métodos , Engenharia de Proteínas/métodos , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/isolamento & purificação , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Cisteína Endopeptidases/química , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Conformação Proteica , Proteínas Recombinantes/genética
17.
Methods Enzymol ; 587: 21-42, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28253957

RESUMO

Autophagy relies on the sequential, hierarchical association of proteins with phagophores, and forming autophagosomes to allow completion of the process. Additionally, the trafficking of the unique transmembrane autophagy-related protein ATG9 is vital for autophagy progression. In this chapter, we discuss methods to monitor autophagosome number using confocal microscopy, by following the association of different autophagosomal markers with the phagophore and completed autophagosome. We also discuss methods to monitor the trafficking of ATG9 in mammalian cells under starvation conditions.


Assuntos
Autofagossomos/ultraestrutura , Microscopia Confocal/métodos , Animais , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Técnica Indireta de Fluorescência para Anticorpo/métodos , Células HEK293 , Humanos , Mamíferos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Imagem Molecular/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
18.
EMBO J ; 36(10): 1392-1411, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28331029

RESUMO

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P2, but this function appears normal in SynaptojaninRQ knock-in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P2, two lipids found in autophagosomal membranes. Using advanced imaging, we show that SynaptojaninRQ mutants accumulate the PI(3)P/PI(3,5)P2-binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell-derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in SynaptojaninRQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic-specific autophagy defects to Parkinson's disease.


Assuntos
Autofagossomos/metabolismo , Autofagia , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Terminações Pré-Sinápticas/enzimologia , Terminações Pré-Sinápticas/metabolismo , Substituição de Aminoácidos , Animais , Proteínas Relacionadas à Autofagia/análise , Células Cultivadas , Drosophila , Humanos , Proteínas de Membrana/análise , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/patologia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/genética
19.
Methods Enzymol ; 588: 323-365, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28237109

RESUMO

The budding yeast Saccharomyces cerevisiae is a powerful and versatile model organism for studying multiple aspects of the biology of eukaryotic cells, including the molecular principles underlying autophagy. One of the unique advantages of this unicellular system is its amenability to genetic and biochemical approaches, which had a pivotal role in the discovery and characterization of most of the autophagy-related (Atg) proteins, the central players of autophagy. The relevance of investigating autophagy in this cell model lies in the high conservation of this pathway among eukaryotes, i.e., most of the yeast Atg proteins possess one or more mammalian orthologs. In addition to the experimental advantages, a very large collection of reagents keeps S. cerevisiae in a leading position for the study of the molecular mechanism and regulation of autophagy. In this chapter, we describe fluorescence microscopy and biochemical methods that allow to monitor in vivo the assembly the of Atg machinery, a key step of autophagy. These approaches can be very useful to those researchers that would like to assess the progression of the autophagosomal precursor structure formation under various conditions, in the presence of specific Atg protein mutants or in the absence of other factors.


Assuntos
Autofagia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/metabolismo , Microscopia de Fluorescência , Transporte Proteico , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Methods Enzymol ; 588: 445-465, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28237115

RESUMO

Drosophila is an excellent model system for studying autophagy during animal development due to the availability of genetic reagents and opportunity for in vivo cell biological analysis. The regulation and mechanism of autophagy are highly evolutionarily conserved and the role of autophagy has been characterized during various stages of Drosophila development as well as following starvation. Studies in Drosophila have revealed novel insights into the role of distinct components of the autophagy machinery. This chapter describes protocols for examining autophagy during Drosophila development. A crucial step in the induction of autophagy is the incorporation of Atg8a into the autophagosome. This can be measured as autophagic puncta using live fluorescent imaging, immunostaining, or immunoblot analysis of LC3/Atg8a processing. The level of autophagy can also be examined using other specific components of the autophagy pathway as markers detected by immunofluorescent imaging. Based on the distinct morphology of autophagy, it can also be examined by transmission electron microscopy. In addition, one of the advantages of using Drosophila as a model is the ability to undertake genetic analysis of individual components of the autophagy machinery. Current approaches that can be used to monitor autophagy, including the overall flux and individual steps in Drosophila melanogaster, will be discussed.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Animais , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/genética , Dissecação/métodos , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Técnicas de Silenciamento de Genes/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Interferência de RNA
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