RESUMO
Autophagy plays multiple roles in host cells challenged with extracellular pathogens. Here, we aimed to explore whether autophagy inhibition could prevent bacterial infections. We first confirmed widely distinct patterns of autophagy responses in host cells infected with Staphylococcus aureus, as compared with Salmonella Only infection with Staphylococcus produced strong accumulation of lipidated autophagy-related protein LC3B (LC3B-II). Infection with virulent Staphylococcus strains induced formation of p62-positive aggregates, suggestive of accumulated ubiquitinated targets. During Salmonella infection, bacteria remain enclosed by lysosomal-associated membrane protein 2 (LAMP2)-positive lysosomes, whereas virulent Staphylococcus apparently exited from enlarged lysosomes and invaded the cytoplasm. Surprisingly, Staphylococcus appeared to escape from the lysosome without generation of membrane-damage signals as detected by galectin-3 recruitment. In contrast, Salmonella infection produced high levels of lysosomal damage, consistent with a downstream antibacterial xenophagy response. Finally, we studied the Unc-51-like autophagy-activating kinase 1 (ULK1) regulatory complex, including the essential subunit autophagy-related protein 13 (ATG13). Infection of cells with either Staphylococcus or Salmonella led to recruitment of ATG13 to sites of cytosolic bacterial cells to promote autophagosome formation. Of note, genetic targeting of ATG13 suppressed autophagy and the ability of Staphylococcus to infect and kill host cells. Two different ULK1 inhibitors also prevented Staphylococcus intracellular replication and host cell death. Interestingly, inhibition of the ULK1 pathway had the opposite effect on Salmonella, sensitizing cells to the infection. Our results suggest that ULK1 inhibitors may offer a potential strategy to impede cellular infection by S. aureus.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Staphylococcus/patogenicidade , Autofagossomos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Morte Celular/efeitos dos fármacos , Citoplasma/metabolismo , Citoplasma/microbiologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Lisossomos/metabolismo , Lisossomos/microbiologia , Salmonella/patogenicidadeRESUMO
It has long been recognised that cancer cells critically depend on reprogrammed patterns of metabolism that can enable robust and abnormally high levels of cell proliferation. As mitochondria form hubs of cellular metabolic activity, it is reasonable to propose that pathways within these organelles can form targets that can be manipulated to compromise the ability of cancer cells to cause disease. However, mitochondria are highly multi-functional, and the full range of mechanistic inter-connections are still being unravelled to enable the full potential of targeting mitochondria in cancer therapeutics. Here, we aim to highlight the potential of modulating mitochondrial dynamics to target key metabolic or apoptotic pathways in cancer cells. Distinct roles have been demonstrated for mitochondrial fission and fusion in different cancer contexts. Targeting of factors mediating mitochondrial dynamics may be directly related to impairment of oxidative phosphorylation, which is essential to sustain cancer cell growth and can also alter sensitivity to chemotherapeutic compounds. This area is still lacking a unified model, although further investigation will more comprehensively map the underlying molecular mechanisms to enable better rational therapeutic strategies based on these pathways.
Assuntos
Dinâmica Mitocondrial , Neoplasias , Humanos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proliferação de CélulasRESUMO
In this study, we examined the activation of non-canonical nuclear factor Kappa B (NFκB) signalling in U2OS cells, a cellular metastatic bone cancer model. Whilst Lymphotoxin α1ß2 (LTα1ß2) stimulated the expected slow, delayed, sustained activation of serine 866/870 p100 phosphorylation and increased cellular expression of p52 NFκB, we found that canonical agonists, Interleukin-1ß (IL-1ß) and also Tumour necrosis factor-α (TNFα) generated a rapid transient increase in pp100, which was maximal by 15-30 min. This rapid phosphorylation was also observed in other cells types, such as DU145 and HCAECs suggesting the phenomenon is universal. IKKα deletion using CRISPR/Cas9 revealed an IKKα-dependent mechanism for serine 866/870 and additionally serine 872 p100 phosphorylation for both IL-1ß and LTα1ß2. In contrast, knockdown of IKKß using siRNA or pharmacological inhibition of IKKß activity was without effect on p100 phosphorylation. Pre-incubation of cells with the NFκB inducing-kinase (NIK) inhibitor, CW15337, had no effect on IL-1ß induced phosphorylation of p100 however, the response to LTα1ß2 was virtually abolished. Surprisingly IL-1ß also stimulated p52 nuclear translocation as early as 60 min, this response and the concomitant p65 translocation was partially reduced by IKKα deletion. Furthermore, p52 nuclear translocation was unaffected by CW15337. In contrast, the response to LTα1ß2 was essentially abolished by both IKKα deletion and CW15337. Taken together, these finding reveal novel forms of NFκB non-canonical signalling stimulated by ligands that activate the canonical NFκB pathway strongly such as IL-1ß.
Assuntos
Quinase I-kappa B , Interleucina-1beta , NF-kappa B , Transdução de Sinais , Humanos , Linhagem Celular Tumoral , Quinase I-kappa B/metabolismo , Interleucina-1beta/metabolismo , NF-kappa B/metabolismoRESUMO
The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate.
Assuntos
Aminoácidos , Dinâmica Mitocondrial , Aminas/metabolismo , Aminoácidos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Purinas/metabolismoRESUMO
Hematopoietic stem cells (HSCs) maintain balanced blood cell production in a process called hematopoiesis. As humans age, their HSCs acquire mutations that allow some HSCs to disproportionately contribute to normal blood production. This process, known as age-related clonal hematopoiesis, predisposes certain individuals to cancer, cardiovascular and pulmonary pathologies. There is a growing body of evidence suggesting that factors outside cells, such as extracellular vesicles (EVs), contribute to the disruption of stem cell homeostasis during aging. We have characterized blood EVs from humans and determined that they are remarkably consistent with respect to size, concentration, and total protein content, across healthy subjects aged 20-85 years. When analyzing EV protein composition from mass spectroscopy data, our machine-learning-based algorithms are able to distinguish EV proteins based on age and suggest that different cell types dominantly produce EVs released into the blood, which change over time. Importantly, our data show blood EVs from middle and older age groups (>40 years) significantly stimulate HSCs in contrast to untreated and EVs sourced from young subjects. Our study establishes for the first time that although EV particle size, concentration, and total protein content remain relatively consistent over an adult lifespan in humans, EV content evolves during aging and potentially influences HSC regulation.
Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.
Assuntos
Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Biogênese de Organelas , Proteínas Serina-Treonina Quinases/metabolismo , Aminoácidos/deficiência , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Metoxi-Hidroxifenilglicol/análogos & derivados , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Proteínas Nucleares/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Fura-2/metabolismo , Proteínas de Fluorescência Verde , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Bicamadas Lipídicas , Proteínas Luminescentes/metabolismo , Metoxi-Hidroxifenilglicol/farmacologia , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Aminoácidos/metabolismo , Animais , Autofagossomos/enzimologia , Autofagia/fisiologia , Linhagem Celular Tumoral , Glucose/deficiência , Glucose/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Lisossomos/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Inanição/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
There has been long-standing interest in targeting pro-survival autophagy as a combinational cancer therapeutic strategy. Clinical trials are in progress testing chloroquine (CQ) or its derivatives in combination with chemo- or radiotherapy for solid and haematological cancers. Although CQ has shown efficacy in preclinical models, its mechanism of action remains equivocal. Here, we tested how effectively CQ sensitises metastatic breast cancer cells to further stress conditions such as ionising irradiation, doxorubicin, PI3K-Akt inhibition and serum withdrawal. Contrary to the conventional model, the cytotoxic effects of CQ were found to be autophagy-independent, as genetic targeting of ATG7 or the ULK1/2 complex could not sensitise cells, like CQ, to serum depletion. Interestingly, although CQ combined with serum starvation was robustly cytotoxic, further glucose starvation under these conditions led to a full rescue of cell viability. Inhibition of hexokinase using 2-deoxyglucose (2DG) similarly led to CQ resistance. As this form of cell death did not resemble classical caspase-dependent apoptosis, we hypothesised that CQ-mediated cytotoxicity was primarily via a lysosome-dependent mechanism. Indeed, CQ treatment led to marked lysosomal swelling and recruitment of Galectin3 to sites of membrane damage. Strikingly, glucose starvation or 2DG prevented CQ from inducing lysosomal damage and subsequent cell death. Importantly, we found that the related compound, amodiaquine, was more potent than CQ for cell killing and not susceptible to interference from glucose starvation. Taken together, our data indicate that CQ effectively targets the lysosome to sensitise towards cell death but is prone to a glucose-dependent resistance mechanism, thus providing rationale for the related compound amodiaquine (currently used in humans) as a better therapeutic option for cancer.
Assuntos
Cloroquina/farmacologia , Glucose/metabolismo , Lisossomos/metabolismo , Autofagia , Linhagem Celular Tumoral , HumanosRESUMO
Autophagy plays a critical role in cell metabolism by degrading and recycling internal components when challenged with limited nutrients. This fundamental and conserved mechanism is based on a membrane trafficking pathway in which nascent autophagosomes engulf cytoplasmic cargo to form vesicles that transport their content to the lysosome for degradation. Based on this simple scheme, autophagy modulates cellular metabolism and cytoplasmic quality control to influence an unexpectedly wide range of normal mammalian physiology and pathophysiology. In this review, we summarise recent advancements in three broad areas of autophagy regulation. We discuss current models on how autophagosomes are initiated from endogenous membranes. We detail how the uncoordinated 51-like kinase (ULK) complex becomes activated downstream of mechanistic target of rapamycin complex 1 (MTORC1). Finally, we summarise the upstream signalling mechanisms that can sense amino acid availability leading to activation of MTORC1.
RESUMO
Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1-ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1-ULK1 signalling module.
Assuntos
Autofagia , Transdução de Sinais , Animais , Homeostase , HumanosRESUMO
Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that functions at a constitutive level in cells, which may become further activated by stressors such as nutrient starvation or protein aggregation. Autophagy has multiple beneficial roles for maintaining normal cellular homeostasis and these roles are related to the implications of autophagy in disease mechanisms including neurodegeneration and cancer. We previously searched for novel autophagy regulators and identified Rho-kinase 1 (ROCK1) as a candidate. Here, we show that activated ROCK1 inhibits autophagy in human embryonic kidney 293 cells. Conversely, ROCK inhibitory compounds enhanced the autophagy response to amino acid starvation or rapamycin treatment. Inhibition of ROCK during the starvation period led to a more rapid response with the production of larger early autophagosomes that matured into enlarged late degradative autolysosomes. Despite the production of enlarged LC3-positive early autophagosomes, membrane precursors containing WD-repeat protein interacting with phosphoinositides 1 (WIPI1) and mammalian Atg9 were not affected by ROCK inhibition, suggesting that phagophore elongation had been unusually extended. However, the enlarged autophagosomes were enriched in ULK1 which was essential to allow progression of autophagy flux. Our results demonstrate a novel role for ROCK in the control of autophagosome size and degradative capacity.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Sirolimo/farmacologia , Quinases Associadas a rho/metabolismo , Aminoácidos/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte Vesicular , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that occurs constitutively in cells, but can also be induced by stressors such as nutrient starvation or protein aggregation. Autophagy has been implicated in multiple disease mechanisms including neurodegeneration and cancer, with both tumor suppressive and oncogenic roles. Uncoordinated 51-like kinase 1 (ULK1) is a critical autophagy protein near the apex of the hierarchal regulatory pathway that receives signals from the master nutrient sensors MTOR and AMP-activated protein kinase (AMPK). In mammals, ULK1 has a close homolog, ULK2, although their functional distinctions have been unclear. Here, we show that ULK1 and ULK2 both function to support autophagy activation following nutrient starvation. Increased autophagy following amino acid or glucose starvation was disrupted only upon combined loss of ULK1 and ULK2 in mouse embryonic fibroblasts. Generation of PtdIns3P and recruitment of WIPI2 or ZFYVE1/DFCP1 to the phagophore following amino acid starvation was blocked by combined Ulk1/2 double knockout. Autophagy activation following glucose starvation did not involve recruitment of either WIPI1 or WIPI2 to forming autophagosomes. Consistent with a PtdIns3P-independent mechanism, glucose-dependent autophagy was resistant to wortmannin. Our findings support functional redundancy between ULK1 and ULK2 for nutrient-dependent activation of autophagy and furthermore highlight the differential pathways that respond to amino acid and glucose deprivation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Aminoácidos/metabolismo , Androstadienos/farmacologia , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Linhagem Celular , Feminino , Glucose/metabolismo , Metabolismo dos Lipídeos , Camundongos , Camundongos Knockout , Neoplasias/metabolismo , Fenótipo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transfecção , WortmaninaRESUMO
The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical roles during the activation of autophagy. Previous studies have demonstrated that the conserved C-terminal domain (CTD) of ULK1 controls the regulatory function and localization of the protein. Here, we explored the role of kinase activity and intramolecular interactions to further understand ULK function. We demonstrate that the dominant-negative activity of kinase-dead mutants requires a 7-residue motif within the CTD. Our data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation and conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains other distinct regions that direct membrane association and interaction with the putative human homologue of Atg13, which we have here characterized. Atg13 is required for autophagy and Atg9 trafficking during autophagy. However, Atg13 does not bind the 7-residue dominant-negative motif in the CTD of ULK proteins nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting that in mammalian cells, the CTD may interact with additional autophagy proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Sequência Conservada , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Linhagem Celular , Membrana Celular/metabolismo , Ativação Enzimática , Genes Dominantes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits (beta', beta, or alpha) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes.
Assuntos
Autofagia/fisiologia , Complexo I de Proteína do Envoltório/metabolismo , Endossomos/metabolismo , Fagossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Complexo I de Proteína do Envoltório/genética , Complexo de Golgi/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Sequestossoma-1 , Transferrina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Autophagy is a vital response to nutrient starvation. Here, we screened a kinase-specific siRNA library using an autophagy assay in human embryonic kidney 293 cells that measures lipidation of the marker protein GFP-LC3 following amino acid starvation. This screen identified ULK1 in addition to other novel candidates that could be confirmed with multiple siRNAs. Knockdown of ULK1, but not the related kinase ULK2, inhibited the autophagic response. Also, ULK1 knockdown inhibited rapamycin-induced autophagy consistent with a role downstream of mTOR. Overexpression of ULK1 inhibited autophagy and this inhibition was independent of its kinase activity. Deletion of the PDZ domain-binding Val-Tyr-Ala motif at the ULK1 C terminus generated a more potent dominant-negative protein. Further deletions revealed that the minimal ULK1 dominant-negative region could be mapped to residues 1-351. Full-length ULK1 localized to cytoplasmic structures, some of which were GFP-LC3-positive, and this localization required the conserved C-terminal domain. In contrast, ULK1-(1-351) was diffuse in the cytoplasm. These experiments reveal at least two domains in ULK1 which likely function via unique sets of effectors to regulate autophagy.
Assuntos
Autofagia/fisiologia , Citoplasma/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mapeamento de Peptídeos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , RNA Interferente Pequeno/genética , Ratos , Deleção de Sequência , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
Nutrient deprivation of eukaryotic cells provokes a variety of stress responses, including autophagy. Autophagy is carried out by autophagosomes which sequester cytosolic components and organelles for degradation after fusion with protease-containing endosomes. To determine the role of microtubules in autophagy, we used nocodazole and vinblastine to disrupt microtubules and independently measured formation and fusion of autophagsosomes in primary rat hepatocytes. By measuring the translocation of GFP-LC3, an autophagosomal marker, to autophagosomes and the lipidation of GFP-LC3, we quantified the rate and magnitude of autophagosome formation. Starvation increased both the rate of autophagosome formation over the basal level and the total number of autophagosomes per cell. Maximal autophagosome formation required an intact microtubule network. Fusion of autophagosomes with endosomes, assayed by acquisition of protease-inhibitor sensitivity as well as overlap with LysoTracker Red-positive endosomes, required intact microtubules. Live-cell imaging demonstrated that autophagosomes were motile structures, and their movement also required microtubules. Interestingly, vinblastine stimulated autophagosome formation more than twofold before any discernable change in the microtubule network was observed. Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.
Assuntos
Endossomos/metabolismo , Microtúbulos/metabolismo , Fagossomos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Células Cultivadas , Meios de Cultura , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/fisiologia , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Nocodazol/farmacologia , Fagossomos/efeitos dos fármacos , Fagossomos/ultraestrutura , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Vimblastina/farmacologiaRESUMO
Autophagy, fundamentally a lysosomal degradation pathway, functions in cells during normal growth and certain pathological conditions, including starvation, to maintain homeostasis. Autophagosomes are formed through a mechanism that is not well understood, despite the identification of many genes required for autophagy. We have studied the mammalian homologue of Atg9p, a multi-spanning transmembrane protein essential in yeast for autophagy, to gain a better understanding of the function of this ubiquitious protein. We show that both the N- and C-termini of mammalian Atg9 (mAtg9) are cytosolic, and predict that mAtg9 spans the membrane six times. We find that mAtg9 is located in the trans-Golgi network and late endosomes and colocalizes with TGN46, the cation-independent mannose-6-phosphate receptor, Rab7 and Rab9. Amino acid starvation or rapamycin treatment, which upregulates autophagy, causes a redistribution of mAtg9 from the TGN to peripheral, endosomal membranes, which are positive for the autophagosomal marker GFP-LC3. siRNA-mediated depletion of the putative mammalian homologue of Atg1p, ULK1, inhibits this starvation-induced redistribution. The redistribution of mAtg9 also requires PI 3-kinase activity, and is reversed after restoration of amino acids. We speculate that starvation-induced autophagy, which requires mAtg9, may rely on an alteration of the steady-state trafficking of mAtg9, in a Atg1-dependent manner.
Assuntos
Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Rede trans-Golgi/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Rede trans-Golgi/ultraestruturaRESUMO
To investigate the role of Raf-1 in v-Ha-ras transformation, we have isolated and characterized a number of Raf-1 mutants that display increased transforming activity in Rat2 fibroblasts. A dipeptide deletion (Delta144-145) in the cysteine-rich domain (CRD) of conserved region (CR) 1 increased the interaction between Raf-1 and v-Ha-ras effector loop mutants in the yeast two-hybrid system, supporting the proposal that the CRD serves as a secondary ras-binding domain. Many activating mutations were located in CR2. Two representative CR2 mutants (Delta250-258 and S257L) displayed increased interaction with v-Ha-ras effector loop mutants and with mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 in the two-hybrid system. One novel mutation in CR3 was recovered; G361S affected the third glycine of the GXGXXG protein kinase motif involved in ATP binding. Expression of G361S Raf-1 in Rat2 fibroblasts activated MEK and ERK. The CR1, CR2, and CR3 activating mutations, when combined in cis, cooperated in transforming Rat2 fibroblasts. Conversely, Raf-1 transforming activity was decreased when the S257L or G361S mutation was combined in cis with the R89E substitution, which disrupts ras-Raf interaction. This mutant analysis provides additional information about the distinct functions of individual Raf-1 regions and documents a novel genetic mechanism for activating an oncogenic kinase.
Assuntos
Transformação Celular Neoplásica/genética , Sequência Conservada , Mutação , Proteínas Proto-Oncogênicas c-raf/genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Fibroblastos , Genes ras , Sistema de Sinalização das MAP Quinases/fisiologia , Microscopia de Contraste de Fase , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteínas Recombinantes/metabolismoRESUMO
Huntington's disease is caused by polyglutamine expansion (exp) in huntingtin (Htt). Htt-associated protein-1 (HAP1) was the first identified Htt-binding partner. The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) is an intracellular Ca2+ release channel that plays an important role in neuronal function. Recently, we identified a InsP3R1-HAP1A-Htt ternary complex in the brain and demonstrated that Httexp, but not normal Htt, activates InsP3R1 in bilayers and facilitates InsP3R1-mediated intracellular Ca2+ release in medium spiny striatal neurons [MSN; T.-S. Tang et al. (2003) Neuron, 39, 227-239]. Here we took advantage of mice with targeted disruption of both HAP1 alleles (HAP1 -/-) to investigate the role of HAP1 in functional interactions between Htt and InsP3R1. We determined that: (i) HAP1 is expressed in the MSN; (ii) HAP1A facilitates functional effects of Htt and Htt(exp) on InsP3R1 in planar lipid bilayers; (iii) HAP1 is required for changes in MSN basal Ca2+ levels resulting from Htt or Htt(exp) overexpression; (iv) HAP1 facilitates potentiation of InsP3R1-mediated Ca2+ release by Htt(exp) in mouse MSN. Our present results indicate that HAP1 plays an important role in functional interactions between Htt and InsP3R1.