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1.
Proc Natl Acad Sci U S A ; 117(35): 21391-21402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817423

RESUMO

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.


Assuntos
Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Escherichia coli , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32513711

RESUMO

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
3.
Nat Rev Mol Cell Biol ; 21(8): 439-458, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32372019

RESUMO

Autophagosomes are double-membrane vesicles newly formed during autophagy to engulf a wide range of intracellular material and transport this autophagic cargo to lysosomes (or vacuoles in yeasts and plants) for subsequent degradation. Autophagosome biogenesis responds to a plethora of signals and involves unique and dynamic membrane processes. Autophagy is an important cellular mechanism allowing the cell to meet various demands, and its disruption compromises homeostasis and leads to various diseases, including metabolic disorders, neurodegeneration and cancer. Thus, not surprisingly, the elucidation of the molecular mechanisms governing autophagosome biogenesis has attracted considerable interest. Key molecules and organelles involved in autophagosome biogenesis, including autophagy-related (ATG) proteins and the endoplasmic reticulum, have been discovered, and their roles and relationships have been investigated intensely. However, several fundamental questions, such as what supplies membranes/lipids to build the autophagosome and how the membrane nucleates, expands, bends into a spherical shape and finally closes, have proven difficult to address. Nonetheless, owing to recent studies with new approaches and technologies, we have begun to unveil the mechanisms underlying these processes on a molecular level. We now know that autophagosome biogenesis is a highly complex process, in which multiple proteins and lipids from various membrane sources, supported by the formation of membrane contact sites, cooperate with biophysical phenomena, including membrane shaping and liquid-liquid phase separation, to ensure seamless segregation of the autophagic cargo. Together, these studies pave the way to obtaining a holistic view of autophagosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Autofagossomos/fisiologia , Animais , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Lisossomos/metabolismo , Macroautofagia , Transporte Proteico
4.
Brain ; 143(3): 783-799, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32185393

RESUMO

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/fisiologia , Demência Frontotemporal/genética , Predisposição Genética para Doença/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Axônios/patologia , Encéfalo/metabolismo , Proteínas de Ligação a DNA , Enzima Desubiquitinante CYLD/metabolismo , Enzimas Desubiquitinantes/metabolismo , Demência Frontotemporal/metabolismo , Camundongos , Mutação de Sentido Incorreto/genética , NF-kappa B/antagonistas & inibidores , Cultura Primária de Células , Transfecção
5.
Neuron ; 105(6): 961-973, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32191859

RESUMO

Autophagy is a key cellular degradative pathway, important for neuronal homeostasis and function. Disruption of autophagy is associated with neuronal dysfunction and neurodegeneration. Autophagy is compartmentalized in neurons, with specific stages of the pathway occurring in distinct subcellular compartments. Coordination of these stages drives progression of autophagy and enables clearance of substrates. Yet, we are only now learning how these distributed processes are integrated across the neuron. In this review, we focus on the cell biological course of autophagy in neurons, from biogenesis at the synapse to degradation in the soma. We describe how the steps of autophagy are distributed across neuronal subcellular compartments, how local machinery regulates autophagy, and the impact of coordinated regulation on neuronal physiology and disease. We also discuss how recent advances in our understanding of neuronal autophagic mechanisms have reframed how we think about the role of local regulation of autophagy in all tissues.


Assuntos
Autofagossomos/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Humanos
6.
FASEB J ; 34(1): 161-179, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914609

RESUMO

Fas-apoptotic inhibitory molecule 2 (FAIM2) is a member of the transmembrane BAX inhibitor motif-containing (TMBIM) family. TMBIM family is comprised of six anti-apoptotic proteins that suppress cell death by regulating endoplasmic reticulum Ca2+ homeostasis. Recent studies have implicated two TMBIM proteins, GRINA and BAX Inhibitor-1, in mediating cytoprotection via autophagy. However, whether FAIM2 plays a role in autophagy has been unknown. Here we show that FAIM2 localizes to the lysosomes at basal state and facilitates autophagy through interaction with microtubule-associated protein 1 light chain 3 proteins in human neuroblastoma SH-SY5Y cells. FAIM2 overexpression increased autophagy flux, while autophagy flux was impaired in shRNA-mediated knockdown (shFAIM2) cells, and the impairment was more evident in the presence of rapamycin. In shFAIM2 cells, autophagosome maturation through fusion with lysosomes was impaired, leading to accumulation of autophagosomes. A functional LC3-interacting region motif within FAIM2 was essential for the interaction with LC3 and rescue of autophagy flux in shFAIM2 cells while LC3-binding property of FAIM2 was dispensable for the anti-apoptotic function in response to Fas receptor-mediated apoptosis. Suppression of autophagosome maturation was also observed in a null mutant of Caenorhabditis elegans lacking xbx-6, the ortholog of FAIM2. Our study suggests that FAIM2 is a novel regulator of autophagy mediating autophagosome maturation through the interaction with LC3.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Autofagia/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Imunossupressores/farmacologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Plasmídeos , Transporte Proteico , Sirolimo/farmacologia
7.
J Mol Biol ; 432(8): 2605-2621, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-31931011

RESUMO

Autophagy is a major degradation pathway where double-membrane vesicles called autophagosomes deliver cytoplasmic content to the lysosome. Increasing evidence suggests that autophagy dysfunction contributes to the pathogenesis of neurodegenerative diseases. In addition, misfolded proteins that accumulate in these diseases and constitute a common pathological hallmark are substrates for autophagic degradation. Astrocytes, a major type of glial cells, are emerging as a critical component in most neurodegenerative diseases. This review will summarize the recent efforts to investigate the role that autophagy plays in astrocytes in the context of neurodegenerative diseases. While the field has mostly focused on the implications of autophagy in neurons, autophagy may also be involved in the clearance of disease-related proteins in astrocytes as well as in maintaining astrocyte function, which could impact the cell autonomous and non-cell autonomous contribution of astrocytes to neurodegeneration.


Assuntos
Astrócitos/patologia , Autofagossomos/fisiologia , Autofagia , Doenças Neurodegenerativas/patologia , Animais , Humanos
8.
J Mol Biol ; 432(8): 2462-2482, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-31682838

RESUMO

Macroautophagy is a conserved catabolic process observed in all eukaryotic cells, during which selected cellular components are transported to and broken down within lysosomes. The process starts with the capture of unnecessary material into autophagosomes, which is followed by autophagosome-lysosome fusion to generate autolysosomes that degrade the cargo. In the past quarter-century, our knowledge about autophagosome formation almost exponentially increased, while the later steps were much less studied. This fortunately changed in the past few years, with more and more publications focusing on the fate of the completed autophagosome. In this review, we aspire to summarize the current knowledge about the molecular mechanisms of autophagosome-lysosome fusion.


Assuntos
Autofagossomos/fisiologia , Autofagia , Lisossomos/fisiologia , Doenças Neurodegenerativas/patologia , Animais , Humanos , Proteínas SNARE/metabolismo
9.
J Mol Biol ; 432(8): 2449-2461, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-31705882

RESUMO

We review current knowledge of the process of autophagosome formation with special emphasis on the very early steps: turning on the autophagy pathway, assembling the autophagy machinery, and building the autophagosome. The pathway is remarkably well coordinated spatially and temporally, and it shows broad conservation across species and cell types, including neurons. In addition, although much current knowledge derives mostly from settings of nonselective autophagy, recent work also indicates that selective autophagy, and more specifically mitophagy, shows similar dynamics. Having an understanding of this remarkable process may help the design of novel therapeutics for neurodegeneration and other pathologies.


Assuntos
Autofagossomos/fisiologia , Autofagia , Mitofagia , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Animais , Humanos
10.
Cells ; 8(12)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861136

RESUMO

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy.


Assuntos
Autofagia/fisiologia , Sintaxina 16/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Lisossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Sintaxina 16/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismo , Rede trans-Golgi/fisiologia
11.
Cancer Res ; 79(20): 5245-5259, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31395606

RESUMO

Cyclin-dependent kinase 4 (CDK4) is well-known for its role in regulating the cell cycle, however, its role in cancer metabolism, especially mTOR signaling, is undefined. In this study, we established a connection between CDK4 and lysosomes, an emerging metabolic organelle crucial for mTORC1 activation. On the one hand, CDK4 phosphorylated the tumor suppressor folliculin (FLCN), regulating mTORC1 recruitment to the lysosomal surface in response to amino acids. On the other hand, CDK4 directly regulated lysosomal function and was essential for lysosomal degradation, ultimately regulating mTORC1 activity. Pharmacologic inhibition or genetic inactivation of CDK4, other than retaining FLCN at the lysosomal surface, led to the accumulation of undigested material inside lysosomes, which impaired the autophagic flux and induced cancer cell senescence in vitro and in xenograft models. Importantly, the use of CDK4 inhibitors in therapy is known to cause senescence but not cell death. To overcome this phenomenon and based on our findings, we increased the autophagic flux in cancer cells by using an AMPK activator in combination with a CDK4 inhibitor. The cotreatment induced autophagy (AMPK activation) and impaired lysosomal function (CDK4 inhibition), resulting in cell death and tumor regression. Altogether, we uncovered a previously unknown role for CDK4 in lysosomal biology and propose a novel therapeutic strategy to target cancer cells. SIGNIFICANCE: These findings uncover a novel function of CDK4 in lysosomal biology, which promotes cancer progression by activating mTORC1; targeting this function offers a new therapeutic strategy for cancer treatment.


Assuntos
Quinase 4 Dependente de Ciclina/fisiologia , Lisossomos/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Neoplasias/fisiologia , Adenilato Quinase/metabolismo , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Autofagossomos/fisiologia , Autofagia/fisiologia , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Senescência Celular/fisiologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Sinergismo Farmacológico , Feminino , Técnicas de Inativação de Genes , Humanos , Insulina/fisiologia , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Proto-Oncogênicas/metabolismo , Pironas/farmacologia , Pironas/uso terapêutico , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nat Commun ; 10(1): 3436, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366886

RESUMO

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.


Assuntos
Autofagia/fisiologia , Doenças do Desenvolvimento Ósseo/genética , Calcificação Fisiológica/fisiologia , Efrina-B2/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Autofagossomos/fisiologia , Autofagia/genética , Doenças do Desenvolvimento Ósseo/patologia , Remodelação Óssea/fisiologia , Linhagem Celular , Efrina-B2/genética , Camundongos , Camundongos Endogâmicos C57BL , Osteócitos/metabolismo , Osteócitos/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína rhoA de Ligação ao GTP
13.
Sci Rep ; 9(1): 10762, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341174

RESUMO

Recent clinical trials suggest that early nutritional support might block the induction of autophagy in critically ill patients leading to the development of organ failure. However, the regulation of autophagy, especially by nutrients, in critical illness is largely unclear. The autophagy flux (AF) in relation to critical illness and nutrition was investigated by using an in vitro model of human primary myotubes incubated with serum from critically ill patients (ICU). AF was calculated as the difference of p62 expression in the presence and absence of chloroquine (50 µM, 6 h), in primary myotubes incubated for 24 h with serum from healthy volunteers (n = 10) and ICU patients (n = 93). We observed 3 different phenotypes in AF, non-altered (ICU non-responder group), increased (ICU inducer group) or blocked (ICU blocker group). This block was not associate with a change in amino acids serum levels and was located at the accumulation of autophagosomes. The increase in the AF was associated with lower serum levels of non-essential amino acids. Thus, early nutrition during critical illness might not block autophagy but could attenuate the beneficial effect of starvation on reactivation of the autophagy process. This could be of clinical importance in the individual patients in whom this process is inhibited by the critical illness insult.


Assuntos
Autofagia/fisiologia , Estado Terminal/terapia , Adulto , Idoso , Aminoácidos/sangue , Autofagossomos/fisiologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/fisiologia , Apoio Nutricional/efeitos adversos , Apoio Nutricional/métodos , Pesquisa Médica Translacional
14.
J Dairy Sci ; 102(9): 8264-8272, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31255277

RESUMO

Staphylococcus aureus is an important pathogen causing chronic and subclinical mastitis of cows. Autophagy is an important regulatory mechanism that participates in the elimination of invading pathogenic organisms. Here, we hypothesize that autophagy is involved in the process of Staph. aureus survival in bovine mammary epithelial cells (BMEC). In this study, we detected the expression of autophagy-related proteins during infection and assessed the effect of autophagosome formation and degradation on the proliferation of intracellular Staph. aureus. Infection with Staph. aureus increased the protein expression of microtubule-associated protein 1 light chain 3-II (MAP1LC3, also called LC3-II) and sequestosome-1 (SQSTM1, also called p62) in BMEC. After infection, the formation of the autophagosomes increased but the autophagosomes and lysosomes could not fuse normally to form autolysosomes. When the formation of the autophagosomes was enhanced or the degradation of the autolysosomes was inhibited, the number of Staph. aureus in the BMEC increased. However, the intracellular proliferation of Staph. aureus was slowed when formation of autophagosomes was inhibited. Therefore, autophagy was induced in BMEC challenged by Staph. aureus but the autophagic flux was obstructed. Inhibiting the formation of autophagosomes in BMEC facilitated the clearance of intracellular Staph. aureus, which may offer a new strategy for the treatment of mastitis in cows.


Assuntos
Autofagossomos/fisiologia , Autofagia/fisiologia , Células Epiteliais/fisiologia , Glândulas Mamárias Animais/citologia , Mastite Bovina/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Bovinos , Contagem de Células , Feminino , Proteína Sequestossoma-1/análise , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia
15.
Exp Cell Res ; 382(1): 111447, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31150612

RESUMO

Heterotypic CICs (cell-in-cell structures) have been found between tumor cells and various immune cells in a variety of cancer tissues. The frequency of CICs has been found to correlate with tumor malignancy in some studies but not in others. Herein, we examined in depth the CICs observed in colon cancer to determine their potential significance in disease progression. Heterotypic CICs were observed by histochemistry between epithelial cells and lymphocytes in an expanded spectrum of colon tissue from colitis to cancer and in vitro studies were performed using the colonic tumor cell line HCT8 and human peripheral blood lymphocytes. Our data revealed that the CICs formed by colonic epithelial cells and infiltrated lymphocytes not only positively correlated with tumor malignancy but also were upregulated by the inflammatory cytokine IL-6. In addition, we observed that colon cancer cells could initiate autophagy for survival after cytotoxic lymphocyte internalization and that IL-6 could also be involved in this process to promote the death of lymphocytes in CIC structures. Furthermore, certain changes were observed in tumor cells after experiencing CICs. Our findings suggest that CICs formed by colon cancer cells and lymphocytes contribute to tumor escape from immune surveillance, which could be facilitated by IL-6, and might represent a previously undescribed pathway for tumor cells to adapt and evade host immune defense.


Assuntos
Autofagia/fisiologia , Formação de Célula em Célula/fisiologia , Neoplasias do Colo/patologia , Interleucina-6/fisiologia , Evasão Tumoral/fisiologia , Adenocarcinoma/patologia , Adenoma/patologia , Autofagossomos/fisiologia , Linhagem Celular Tumoral , Colite Ulcerativa/patologia , Progressão da Doença , Células Epiteliais/patologia , Humanos , Células Matadoras Ativadas por Linfocina/patologia , Linfócitos do Interstício Tumoral/patologia , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Citotóxicos/patologia
16.
Arch Virol ; 164(8): 2005-2013, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102052

RESUMO

We previously found that genetic factors are associated with a familial predisposition for developing liver cirrhosis and hepatocellular carcinoma during chronic hepatitis B virus (HBV) infection. Autophagy has been shown to play a role in HBV replication and the course of disease. More than 190 host genes have been identified that modify the process of autophagy, but which of these genes are involved in chronicity of HBV infection and how this occurs remains unclear. Chronic hepatitis B (CHB) patients were recruited to investigate the expression of autophagy-modulating genes in peripheral blood mononuclear cells (PBMCs). mRNA prepared from PBMCs from members of two families with clustering HBV infection, including 11 CHB patients and nine healthy spouses, was hybridized to high-density oligonucleotide arrays. Immunoblot analysis was used to determine the level of autophagy. Of the 192 autophagy-modulating genes, 18 were found to be differently expressed. Of these, 11 displayed decreased expression in CHB patients, while seven displayed increased expression compared to those in healthy controls. Functional analysis showed that these genes are closely involved in initiation, nucleation, elongation of phagophores, formation of autophagosomes, transportation to lysosomes, and the process of degradation. Western blot analysis revealed inhibited autophagy in PBMCs based on decreased lipidation of LC3II. A differential expression profile of autophagy-modulating genes was observed, and decreased autophagy in PBMCs could be closely associated with chronicity of HBV infection, suggesting a novel strategy for the treatment of patients with chronic HBV infection.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Redes Reguladoras de Genes/genética , Hepatite B Crônica/genética , Leucócitos Mononucleares/fisiologia , Autofagossomos/fisiologia , Análise por Conglomerados , Feminino , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/virologia , Humanos , Lisossomos/fisiologia , Masculino , RNA Mensageiro/genética
17.
J Cell Biol ; 218(6): 1787-1798, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-30952800

RESUMO

During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER-autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.


Assuntos
Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Lipídeos/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Sistemas CRISPR-Cas , Células HEK293 , Humanos , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/genética
18.
Autophagy ; 15(10): 1801-1809, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30939979

RESUMO

Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, Crassostrea gigas. However, the autophagy pathway has never been explored in the hemocytes of C. gigas, which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of C. gigas and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level. Abbreviations: MAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NH4Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy.


Assuntos
Autofagia/fisiologia , Crassostrea , Hemócitos/fisiologia , Animais , Autofagossomos/fisiologia , Autofagossomos/ultraestrutura , Crassostrea/citologia , Crassostrea/metabolismo , Crassostrea/ultraestrutura , Citometria de Fluxo , Hemócitos/citologia , Hemócitos/ultraestrutura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência
19.
J Cell Biol ; 218(6): 1908-1927, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31010855

RESUMO

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.


Assuntos
Autofagossomos/fisiologia , Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Membranas Intracelulares , Lisossomos/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas rab5 de Ligação ao GTP/genética
20.
Autophagy ; 15(10): 1787-1800, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30894053

RESUMO

It has been indicated that the Golgi apparatus contributes to autophagy, but how it is involved in autophagosome formation and maturation is not well understood. Here we show that amino acid starvation causes trans-Golgi derived membrane fragments to colocalize with autophagosomes. Depletion of the Golgi stacking protein GORASP2/GRASP55, but not GORASP1/GRASP65, increases both MAP1LC3 (LC3)-II and SQSTM1/p62 levels. We demonstrate that GORASP2 facilitates autophagosome-lysosome fusion by physically linking autophagosomes and lysosomes through the interactions with LC3 on autophagosomes and LAMP2 on late endosomes/lysosomes. Furthermore, we provide evidence that GORASP2 interacts with BECN1 to facilitate the assembly and membrane association of the phosphatidylinositol 3-kinase (PtdIns3K) UVRAG complex. These findings indicate that GORASP2 plays an important role in autophagosome maturation during amino acid starvation. Abbreviations: ATG14: autophagy related 14; BafA1: bafilomycin A1; BSA: bovine serum albumin; CQ: chloroquine; EBSS: earle's balanced salt solution; EM: electron microscopy; EEA1: early endosome antigen 1; GFP: green fluorescent protein; GORASP1/GRASP65: golgi reassembly stacking protein 1; GORASP2/GRASP55: golgi reassembly stacking protein 2; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PK: protease K; PNS: post-nuclear supernatant; RFP: red fluorescent protein; SD: standard deviation; TGN: trans-Golgi network; UVRAG: UV radiation resistance associated.


Assuntos
Autofagossomos/fisiologia , Proteínas da Matriz do Complexo de Golgi/fisiologia , Lisossomos/fisiologia , Fusão de Membrana/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/genética , Células Cultivadas , Proteínas da Matriz do Complexo de Golgi/genética , Células HeLa , Humanos , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Ratos , Proteínas Supressoras de Tumor/fisiologia
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