Showing papers in "Autophagy in 2011"
TL;DR: The mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria is reviewed and the emerging picture of selectivity affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is addressed.
Abstract: Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated substrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.
1,534 citations
TL;DR: The laboratories recently discovered that AMPK associates with, and directly phosphorylates, ULK1 on several sites and this modification is required for ULK 1 activation after glucose deprivation, and these studies have revealed a molecular mechanism ofULK1 regulation by nutrient signals via the actions of AMPK and mTORC1.
Abstract: The serine/threonine kinase ULK1 is a mammalian homolog of Atg1, part of the Atg1 kinase complex, which is the most upstream component of the core autophagy machinery conserved from yeast to mammals. In budding yeast, activity of the Atg1 kinase complex is inhibited by TORC1 (target of rapamycin complex 1), but how the counterpart ULK1 complex in mammalian cells is regulated has been unknown. Our laboratories recently discovered that AMPK associates with, and directly phosphorylates, ULK1 on several sites and this modification is required for ULK1 activation after glucose deprivation. In contrast, when nutrients are plentiful, the mTORC1 complex phosphorylates ULK1, preventing its association and activation by AMPK. These studies have revealed a molecular mechanism of ULK1 regulation by nutrient signals via the actions of AMPK and mTORC1.
505 citations
TL;DR: Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection.
Abstract: The term microautophagy was first used in 1966 by de Duve and Wattiaux1 and subsequently applied, over the following two decades, to processes described in mammalian cells and involving the presence of lysosome-like organelles having multiple vesicles trapped in their lumen (“multivesicular lysosomes”). Concurrently, many studies suggested a view of microautophagy where the lysosomal membrane was either invaginated or projected arm-like protrusions to sequester cytosolic constituents into intralysosomal vesicles. Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection. The lack of specific approaches to directly detect microautophagy in mammalian systems, aside from electron microscopy, is the major current limitation to addressing its physiological role(s) and possible contribution to particular disease states. In this review we co...
490 citations
TL;DR: This review compares the molecular process of mitophagy with other types of specific autophagic pathways and discusses its biological importance.
Abstract: Mitochondrial dysfunction has severe cellular consequences and is linked with neurodegenerative diseases and aging. Maintaining a healthy population of mitochondria is thus essential for proper cellular homeostasis. Several strategies have evolved to prevent and limit mitochondria damage, and macroautophagy plays a role in degrading superfluous or severely damaged mitochondria. Selective removal of mitochondria by autophagy (termed mitophagy) has been extensively studied recently in both yeast and mammalian cells. In this review, we summarize our current knowledge of mitophagy. We also compare the molecular process of mitophagy with other types of specific autophagic pathways and discuss its biological importance.
375 citations
TL;DR: It is shown that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes and indicates a potential new target in the regulation of apoptosis.
Abstract: Caffeine is one of the most frequently ingested neuroactive compounds. All known mechanisms of apoptosis induced by caffeine act through cell cycle modulation or p53 induction. It is currently unknown whether caffeine-induced apoptosis is associated with other cell death mechanisms, such as autophagy. Herein we show that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, through the use of western blotting, electron microscopy and immunocytochemistry techniques. Phosphorylated p70 ribosomal protein S6 kinase (Thr389), S6 ribosomal protein (Ser235/236), 4E-BP1 (Thr37/46) and Akt (Ser473) were significantly decreased by caffeine. In contrast, ERK1/2 (Thr202/204) was increased by caffeine, suggesting an inhibition of the Akt/mTOR/p70S6K pathway and activation of the ERK1/2 pathway. Although insulin treatment phosphorylated Akt (Ser473) and led to autophagy suppression, the effect of insulin treatment was completely abolished by caffeine addition. Caffeine-induced autophagy was not completely blocked by inhibition of ERK1/2 by U0126. Caffeine induced reduction of mitochondrial membrane potentials and apoptosis in a dose-dependent manner, which was further attenuated by the inhibition of autophagy with 3-methyladenine or Atg7 siRNA knockdown. Furthermore, there was a reduced number of early apoptotic cells (annexin V positive, propidium iodide negative) among autophagy-deficient mouse embryonic fibroblasts treated with caffeine than in their wild-type counterparts. These results support previous studies on the use of caffeine in the treatment of human tumors and indicate a potential new target in the regulation of apoptosis.
362 citations
TL;DR: The findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.
Abstract: Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1(G93A) mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.
335 citations
TL;DR: Functional studies revealed that quercetin activated autophagy by modulation of Akt-mTOR signaling and hypoxia-induced factor 1α (HIF-1α) signaling, and a xenograft model provided additional evidence for occurrence of quercETin-induced apoptosis and Autophagy in vivo.
Abstract: Quercetin, a dietary antioxidant present in fruits and vegetables, is a promising cancer chemopreventive agent that inhibits tumor promotion by inducing cell cycle arrest and promoting apoptotic cell death. In this study, we examined the biological activities of quercetin against gastric cancer. Our studies demonstrated that exposure of gastric cancer cells AGS and MKN28 to quercetin resulted in pronounced pro-apoptotic effect through activating the mitochondria pathway. Meanwhile, treatment with quercetin induced appearance of autophagic vacuoles, formation of acidic vesicular organelles (AVOs), conversion of LC3-I to LC3-II, recruitment of LC3-II to the autophagosomes as well as activation of autophagy genes, suggesting that quercetin initiates the autophagic progression in gastric cancer cells. Furthermore, either administration of autophagic inhibitor chloroquine or selective ablation of atg5 or beclin 1 using small interfering RNA (siRNA) could augment quercetin-induced apoptotic cell death, suggesting that autophagy plays a protective role against quercetin-induced apoptosis. Moreover, functional studies revealed that quercetin activated autophagy by modulation of Akt-mTOR signaling and hypoxia-induced factor 1α (HIF-1α) signaling. Finally, a xenograft model provided additional evidence for occurrence of quercetin-induced apoptosis and autophagy in vivo. Together, our studies provided new insights regarding the biological and anti-proliferative activities of quercetin against gastric cancer, and may contribute to rational utility and pharmacological study of quercetin in future anti-cancer research.
323 citations
TL;DR: This work proposes to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: cell death occurs without the involvement of apoptosis.
Abstract: The concept of autophagic cell death was first established based on observations of increased autophagic markers in dying cells. The major limitation of such a morphology-based definition of autophagic cell death is that it fails to establish the functional role of autophagy in the cell death process, and thus contributes to the confusion in the literature regarding the role of autophagy in cell death and cell survival. Here we propose to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: (i) cell death occurs without the involvement of apoptosis; (ii) there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (iii) suppression of autophagy via both pharmacological inhibitors and genetic approaches is able to rescue or prevent cell death. In light of this new definition, we will discuss some of the common problems and difficulties in the study of autophagic cell death and also revisit some well-reported cases of autophagic cell death, aiming to achieve a better understanding of whether autophagy is a real killer, an accomplice or just an innocent bystander in the course of cell death. At present, the physiological relevance of autophagic cell death is mainly observed in lower eukaryotes and invertebrates such as Dictyostelium discoideum and Drosophila melanogaster. We believe that such a clear definition of autophagic cell death will help us study and understand the physiological or pathological relevance of autophagic cell death in mammals.
316 citations
TL;DR: Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP, however, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed.
Abstract: Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earle's balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors,...
310 citations
TL;DR: It is shown that AtNBR1 is an autophagy substrate degraded in the vacuole dependent on the polymerization property of the PB1 domain and of expression of AtATG7, which is similar to mammalian NBR1 and p62.
Abstract: (Macro)autophagy encompasses both an unselective, bulk degradation of cytoplasmic contents as well as selective autophagy of damaged organelles, intracellular microbes, protein aggregates, cellular structures and specific soluble proteins. Selective autophagy is mediated by autophagic adapters, like p62/SQSTM1 and NBR1. p62 and NBR1 are themselves selective autophagy substrates, but they also act as cargo receptors for degradation of other substrates. Surprisingly, we found that homologs of NBR1 are distributed throughout the eukaryotic kingdom, while p62 is confined to the metazoans. As a representative of all organisms having only an NBR1 homolog we studied Arabidopsis thaliana NBR1 (AtNBR1) in more detail. AtNBR1 is more similar to mammalian NBR1 than to p62 in domain architecture and amino acid sequence. However, similar to p62, AtNBR1 homo-polymerizes via the PB1 domain. Hence, AtNBR1 has hybrid properties of mammalian NBR1 and p62. AtNBR1 has 2 UBA domains, but only the C-terminal UBA domain bound ubiquitin. AtNBR1 bound AtATG8 through a conserved LIR (LC3-interacting region) motif and required co-expression of AtATG8 or human GABARAPL2 to be recognized as an autophagic substrate in HeLa cells. To monitor the autophagic sequestration of AtNBR1 in Arabidopsis we made transgenic plants expressing AtNBR1 fused to a pH-sensitive fluorescent tag, a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive yellow fluorescent proteins. This strategy allowed us to show that AtNBR1 is an autophagy substrate degraded in the vacuole dependent on the polymerization property of the PB1 domain and of expression of AtATG7. A functional LIR was required for vacuolar import.
290 citations
TL;DR: Findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells, indicating that the combination of Autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.
Abstract: Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both ...
University of Michigan1, University of Massachusetts Medical School2, University of Toronto3, University of Pittsburgh4, University of Paris-Sud5, Yeshiva University6, University of California, San Francisco7, University of New Mexico8, Weizmann Institute of Science9, University of Helsinki10, University of Texas MD Anderson Cancer Center11, Virginia Commonwealth University12, University of Paris13, University of Texas Southwestern Medical Center14, Yale University15, Tokyo Medical and Dental University16, University of Cambridge17, University of Oslo18, University of Colorado Denver19, University of Göttingen20, Cancer Research UK21
TL;DR: A comprehensive glossary of autophagy-related terms is developed to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit Autophagy.
Abstract: The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers--even those who work in the field--to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.
TL;DR: The literature that implicates a role for autophagy in the pathophysiology of type 1 and type 2 diabetes mellitus as it applies to β-cell dysfunction, and more broadly to organ systems involved in complications of diabetes including the cardiovascular, renal and nervous systems is reviewed.
Abstract: An emerging body of evidence supports a role for autophagy in the pathophysiology of type 1 and type 2 diabetes mellitus. Persistent high concentrations of glucose lead to imbalances in the antioxidant capacity within the cell resulting in oxidative stress-mediated injury in both disorders. An anticipated consequence of impaired autophagy is the accumulation of dysfunctional organelles such as mitochondria within the cell. Mitochondria are the primary site of the production of reactive oxygen species (ROS), and an imbalance in ROS production relative to the cytoprotective action of autophagy may lead to the accumulation of ROS. Impaired mitochondrial function associated with increased ROS levels have been proposed as mechanisms contributing to insulin resistance. In this article we review and interpret the literature that implicates a role for autophagy in the pathophysiology of type 1 and type 2 diabetes mellitus as it applies to β-cell dysfunction, and more broadly to organ systems involved in complications of diabetes including the cardiovascular, renal and nervous systems.
TL;DR: The impact that electron microscopy has had on autophagy research since the discovery of this self-degradation process in the mid-1950s is summarized.
Abstract: Autophagy was first discovered by transmission electron microscopy more than 50 years ago. For decades, electron microscopy was the only way to reliably detect autophagic compartments in cells because no specific protein markers were known. In the 1970s, however, the introduction of biochemical methods enabled quantitative studies of autophagic-lysosomal degradation, and in the 1980s specific biochemical assays for autophagic sequestration became available. Since the identification of autophagy-related genes in the 1990s, combined fluorescence microscopy, biochemical and genetic methods have taken the leading role in autophagy research. However, electron microscopy is still needed to confirm and verify results obtained by other methods, and also to produce novel knowledge that would not be achievable by any other experimental approach. Confocal microscopy, with its ever-improving resolution, is probably the best-suited morphological approach to investigate the dynamic aspects of autophagy. However, for analyzing the ultrastructural details of the many novel organelles and mechanisms involved in specific subtypes of autophagy, the electron microscope is still indispensable. This review will summarize the impact that electron microscopy has had on autophagy research since the discovery of this self-degradation process in the mid-1950s. Astonishingly, some of the "novel" concepts and principles of autophagy, presented in the recent studies, were already proposed several decades ago by the pioneering, accurate and passionate work of virtuoso electron microscopists.
TL;DR: In mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis, and this results are the first to show that liver-specific autophagic proteolysis plays a role in blood glucose regulation.
Abstract: Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.
TL;DR: It is shown that physical exercise activates autophagy in skeletal muscles, and these findings indicate that proper activation of Autophagy is important for muscle homeostasis during physical activity.
Abstract: Autophagy is a catabolic process that provides the degradation of altered/damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagic flux is fundamental for the homeostasis of skeletal muscles in physiological conditions and in response to stress. Defective as well as excessive autophagy is detrimental for muscle health and has a pathogenic role in several forms of muscle diseases. Recently, we found that defective activation of the autophagic machinery plays a key role in the pathogenesis of muscular dystrophies linked to collagen VI. Impairment of the autophagic flux in collagen VI null (Col6a1–/–) mice causes accumulation of dysfunctional mitochondria and altered sarcoplasmic reticulum, leading to apoptosis and degeneration of muscle fibers. Here we show that physical exercise activates autophagy in skeletal muscles. Notably, physical training exacerbated the dystrophic phenotype of Col6a1–/– mice, where autophagy flux is compromised. Autophagy was n...
TL;DR: Observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes.
Abstract: We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD) Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU Disruption of lysosomal activity with bafilomycin A1 or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines
TL;DR: It is demonstrated that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy, and activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.
Abstract: Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.
TL;DR: It is reported that Ulk1/2 in turn phosphorylates all three subunits of AMPK and thereby negatively regulates its activity and is proposed thatUlk1 is not only involved in the induction of autophagy, but also in terminating signaling events that trigger autophagic induction.
Abstract: Unc-51-like kinase 1 (Ulk1) plays a central role in autophagy induction. It forms a stable complex with Atg13 and focal adhesion kinase (FAK) family interacting protein of 200 kDa (FIP 200). This complex is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) in a nutrient-dependent way. AMP-activated protein kinase (AMPK), which is activated by LKB1/Strad/Mo25 upon high AMP levels, stimulates autophagy by inhibiting mTORC1. Recently, it has been described that AMPK and Ulk1 interact and that the latter is phosphorylated by AMPK. This phosphorylation leads to the direct activation of Ulk1 by AMPK bypassing mTOR-inhibition. Here we report that Ulk1/2 in turn phosphorylates all three subunits of AMPK and thereby negatively regulates its activity. Thus, we propose that Ulk1 is not only involved in the induction of autophagy, but also in terminating signaling events that trigger autophagy. In our model, phosphorylation of AMPK by Ulk1 represents a negative feedback circuit.
TL;DR: These findings reveal an important role of transcription in the regulation of starvation-induced autophagy, and link lysosomal biogenesis to autophagic process.
Abstract: Autophagy is a fundamental catabolic process that plays a central role in health and disease. An efficient autophagic process relies on the cooperation of two distinct types of organelles: the autophagosome and the lysosome. We have identified a gene network that regulates the biogenesis and function of both organelles. Our findings reveal an important role of transcription in the regulation of starvation-induced autophagy, and link lysosomal biogenesis to autophagy.
TL;DR: The results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.
Abstract: In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.
TL;DR: It is demonstrated that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro, and a new mechanism whereby ULK 1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor is proposed, which is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORc1 inhibition when nutrient supplies are limiting.
Abstract: Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.
TL;DR: It is shown for the first time that in wild-type macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection, and it is demonstrated that if efficiently activated, autophagy can control B. Cepacia infection and ameliorate the associated inflammation.
Abstract: Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.
TL;DR: It is concluded that p62 and NDP52 act cooperatively to drive efficient antibacterial autophagy by targeting the protein complexes they coordinate to distinct microdomains associated with bacteria.
Abstract: Autophagy is an innate immune defense against bacterial invasion. Recent studies show that two adaptor proteins, p62 and NDP52, are required for autophagy of the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium). However, it is not known why two different adaptors are required to target the same bacterial cargo to autophagy. Here we show that both adaptors are recruited to bacteria with similar kinetics, that they are recruited to bacteria independently of each other, and that depletion of either adaptor leads to impairment of antibacterial autophagy. Depletion of both adaptors does not synergistically impair autophagy, indicating they act in the same pathway. Remarkably, we observed that these adaptors do not colocalize, but rather form non-overlapping microdomains surrounding bacteria. We conclude that p62 and NDP52 act cooperatively to drive efficient antibacterial autophagy by targeting the protein complexes they coordinate to distinct micro-domains associated with bacteria.
TL;DR: The findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
Abstract: Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
TL;DR: It is demonstrated that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53, and compelling evidence for the interplay betweenAutophagy and apoptosis induced by DHA is supported by the findings that Autophagy inhibition suppressed apoptosis and further autophagic induction enhanced apoptosis in response to DHA treatment.
Abstract: Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here we show that DHA increased both the level of microtubule-associated protein 1 light chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.
TL;DR: These studies show that both mTOR and AMPK directly regulate Ulk1 and coordinate the mammalian autophagy initiation, indicating the regulation of autophagic initiation in mammals is different from that in yeast.
Abstract: Ulk1 is a serine/threonine kinase and the mammalian functional homolog of yeast Atg1. It acts at the initiation step of autophagy and forms a complex with mAtg13, FIP200 and Atg101. Assembly of this complex is independent of mTOR signaling, indicating the regulation of autophagy initiation in mammals is different from that in yeast. In a recent study, we reported that Ulk1 can be phosphorylated by mTOR and AMPK kinases. AMPK associates with Ulk1 in nutrient-dependent manner. Rapid dissociation between Ulk1 and AMPK primes cells for fast autophagy induction upon nutrient withdrawal. These studies show that both mTOR and AMPK directly regulate Ulk1 and coordinate the mammalian autophagy initiation.
TL;DR: Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.
Abstract: Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.
TL;DR: It is shown that EPI induced autophagy in human breast cancer MCF-7 cells and the use of clinically applicable autophagic inhibitors might be one of the important strategies for breast cancer therapy.
Abstract: Epirubicin (EPI) is one of the most effective drugs against cancer. But the acquired resistance of cancer cells to EPI is becoming a major obstacle for successful cancer therapy. Recently, some studies have revealed that macroautophagy (here referred to as autophagy) may protect the cancer cell from anticancer drug-induced death, so autophagy might be related to the development of drug resistance to these reagents. However, the relationship between autophagy and drug resistance has yet to be defined. Our study showed that EPI induced autophagy in human breast cancer MCF-7 cells. And the EPI-induced autophagy protected MCF-7 cells from EPI-induced apoptosis. Furthermore, autophagy was elevated in EPI-resistant MCF-7 cells (MCF-7er cells), and inhibition of autophagy restored the sensitivity of MCF-7er cells to EPI. Therefore, autophagy is a prosurvival factor and has a role in the development of EPI-acquired resistance in EPI-treated MCF-7 cells. Also, this finding indicates that the use of clinically appl...
TL;DR: Findings suggest that Bif-1 acts as a critical regulator of Atg9 puncta formation presumably by mediating Golgi fission for autophagosome biogenesis during starvation.
Abstract: Atg9 is a transmembrane protein essential for autophagy which cycles between the Golgi network, late endosomes and LC3-positive autophagosomes in mammalian cells during starvation through a mechanism that is dependent on ULK1 and requires the activity of the class III phosphatidylinositol-3-kinase (PI3KC3). In this study, we demonstrate that the N-BAR-containing protein, Bif-1, is required for Atg9 trafficking and the fission of Golgi membranes during the induction of autophagy. Upon starvation, Atg9-positive membranes undergo continuous tubulation and fragmentation to produce cytoplasmic punctate structures that are positive for Rab5, Atg16L and LC3. Loss of Bif-1 or inhibition of the PI3KC3 complex II suppresses starvation-induced fission of Golgi membranes and peripheral cytoplasmic redistribution of Atg9. Moreover, Bif-1 mutants, which lack the functional regions of the N-BAR domain that are responsible for membrane binding and/or bending activity, fail to restore the fission of Golgi membranes as well as the formation of Atg9 foci and autophagosomes in Bif-1-deficient cells starved of nutrients. Taken together, these findings suggest that Bif-1 acts as a critical regulator of Atg9 puncta formation presumably by mediating Golgi fission for autophagosome biogenesis during starvation.