自噬在非酒精性脂肪性肝病中的变化及作用

自噬是一种溶酶体依赖性降解途径,已有大量研究报道,自噬参与了非酒精性脂肪性肝病(NAFLD)的发生及发展.在NAFLD早期,自噬增强,并可以通过抑制引起NAFLD的“二次打击”延缓NAFLD的进展.在NAFLD晚期,由于自噬相关基因(Atg)7降解、哺乳动物雷帕霉素靶蛋白通路过度激活、高胰岛素血症、自噬-溶酶体蛋白水解功能减弱、自噬体膜及溶酶体膜脂质构成改变、肝细胞内钙离子水平增加引起自噬减弱,加重了NAFLD.     

Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration

Autophagy is a regulated lysosomal degradation process that involves autophagosome formation and transport. Although recent evidence indicates that basal levels of autophagy protect against neurodegeneration, the exact mechanism whereby this occurs is not known. By using conditional knockout mutant mice, we report that neuronal autophagy is particularly important for the maintenance of local homeostasis of axon terminals and protection against axonal degeneration. We show that specific ablation of an essential autophagy gene, Atg7, in Purkinje cells initially causes cell-autonomous, progressive dystrophy (manifested by axonal swellings) and degeneration of the axon terminals. Consistent with suppression of autophagy, no autophagosomes are observed in these dystrophic swellings, which is in contrast to accumulation of autophagosomes in the axonal dystrophic swellings under pathological conditions. Axonal dystrophy of mutant Purkinje cells proceeds with little sign of dendritic or spine atrophy, indicating that axon terminals are much more vulnerable to autophagy impairment than dendrites. This early pathological event in the axons is followed by cell-autonomous Purkinje cell death and mouse behavioral deficits. Furthermore, ultrastructural analyses of mutant Purkinje cells reveal an accumulation of aberrant membrane structures in the axonal dystrophic swellings. Finally, we observe double-membrane vacuole-like structures in wild-type Purkinje cell axons, whereas these structures are abolished in mutant Purkinje cell axons. Thus, we conclude that the autophagy protein Atg7 is required for membrane trafficking and turnover in the axons. Our study implicates impairment of axonal autophagy as a possible mechanism for axonopathy associated with neurodegeneration.     

Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase

Autophagy mediates the cellular response to nutrient deprivation, protein aggregation, and pathogen invasion in human. Dysfunction of autophagy has been implicated in multiple human diseases including cancer. The identification of novel autophagy factors in mammalian cells will provide critical mechanistic insights into how this complicated cellular pathway responds to a broad range of challenges. Here, we report the cloning of an autophagy-specific protein that we called Barkor (Beclin 1-associated autophagy-related key regulator) through direct interaction with Beclin 1 in the human phosphatidylinositol 3-kinase class III complex. Barkor shares 18% sequence identity and 32% sequence similarity with yeast Atg14. Elimination of Barkor expression by RNA interference compromises starvation- and rapamycin-induced LC3 lipidation and autophagosome formation. Overexpression of Barkor leads to autophagy activation and increased number and enlarged volume of autophagosomes. Tellingly, Barkor is also required for suppression of the autophagy-mediated intracellular survival of Salmonella typhimurium in mammalian cells. Mechanistically, Barkor competes with UV radiation resistance associated gene product (UVRAG) for interaction with Beclin 1, and the complex formation of Barkor and Beclin1 is required for their localizations to autophagosomes. Therefore, we define a regulatory signaling pathway mediated by Barkor that positively controls autophagy through Beclin 1 and represents a potential target for drug development in the treatment of human diseases implicated in autophagic dysfunction.     

Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication

Autophagy is a cellular degradation process involving an intracellular membrane trafficking pathway that recycles cellular components or eliminates intracellular microbes in lysosomes. Many pathogens subvert autophagy to enhance their replication, but the mechanisms these pathogens use to initiate the autophagy process have not been elucidated. This study identifies rotavirus as a pathogen that encodes a viroporin, nonstructural protein 4, which releases endoplasmic reticulum calcium into the cytoplasm, thereby activating a calcium/calmodulin-dependent kinase kinase-β and 5′ adenosine monophosphate-activated protein kinase-dependent signaling pathway to initiate autophagy. Rotavirus hijacks this membrane trafficking pathway to transport viral proteins from the endoplasmic reticulum to sites of viral replication to produce infectious virus. This process requires PI3K activity and autophagy-initiation proteins Atg3 and Atg5, and it is abrogated by chelating cytoplasmic calcium or inhibiting calcium/calmodulin-dependent kinase kinase-β. Although the early stages of autophagy are initiated, rotavirus infection also blocks autophagy maturation. These studies identify a unique mechanism of virus-mediated, calcium-activated signaling that initiates autophagy and hijacks this membrane trafficking pathway to transport viral proteins to sites of viral assembly.Viruses are obligate intracellular parasites that, due to their limited coding capacity, have evolved strategies that usurp cellular processes to facilitate their own propagation. Macroautophagy (hereafter referred to as autophagy) is a cellular catabolic process used to maintain homeostasis by delivering cytoplasmic material to lysosomes for degradation via an intracellular membrane trafficking pathway (1). Autophagy also has intracellular antimicrobial properties and plays a role in the initiation of innate and adaptive immune responses to viral and bacterial infections. Numerous pathogens, including a number of DNA and RNA viruses, have been shown to evade or subvert autophagy (2); however, for most of these viruses, the mechanisms used to initiate autophagy and subvert the normal autophagy process have not been elucidated.The formation of autophagy membranes is complex and not completely understood, but the autophagy (Atg) proteins comprise the core molecular machinery involved in this dynamic membrane rearrangement (3). Autophagy, which is repressed by the mammalian target of rapamycin (mTOR), can be activated by nutrient deprivation; growth factor depletion; or cellular stress, such as hypoxia, energy depletion, endoplasmic reticulum (ER) stress, high temperature, or high cell density conditions (4). Following nutrient deprivation, mTOR is inhibited and a complex composed of Atg13/ULK1/FIP200/Atg101 forms to initiate nucleation of an isolation membrane, or phagophore (5). The phagophore elongates and subsequently encloses cytoplasmic components, forming a double-membrane vacuole, the autophagosome. The elongation phase requires two ubiquitin-like conjugation reactions to form the Atg5/Atg12/Atg16 complex and to conjugate phosphatidylethanolamine (PE) onto microtubule-associated protein light chain 3 (LC3). The lipid tail of LC3 is inserted into the forming autophagosome. Finally, autophagosomes are transported in a dynein-dependent manner on microtubules to lysosomes, where they fuse to form autolysosomes, and the engulfed material is degraded by lysosomal enzymes.Many important pathogens, including RNA viruses [picornaviruses (poliovirus, coxsackievirus, rhinovirus, and hepatitis A), coronaviruses (severe acute respiratory syndrome), and flaviviruses (hepatitis C virus, yellow fever virus, dengue virus, and West Nile virus)] and some DNA viruses (hepatitis B virus and parvovirus) induce the accumulation of autophagosomes or autolysosomes (6–10). It has been proposed for picornaviruses that these dramatically remodeled autophagic intracellular membranes serve as a structural platform for viral replication and assembly. However, the mechanism of autophagy induction for most of these viruses is unknown.Rotavirus is the causative agent of severe gastroenteritis and vomiting in young children and animals worldwide (11). We previously reported that the rotavirus nonstructural protein 4 (NSP4), expressed alone or during virus infection, colocalizes with the endogenous autophagy marker protein LC3 in membranes that surround viroplasms, sites of viral replication and particle assembly, but the functional relevance of autophagy in rotavirus infection and the mechanism of autophagy induction remained unknown (12). The current study investigated whether autophagy is required for rotavirus replication and the mechanism used by rotavirus and NSP4 to initiate autophagy. We report an example of a virus-encoded viroporin that mediates the initiation of autophagy. We discovered that the rotavirus-encoded viroporin NSP4 releases calcium from the ER into the cytoplasm, activating calcium/calmodulin-dependent kinase kinase-β (CaMKK-β) signaling to initiate autophagy. The current study provides insight into a unique mechanism through which rotavirus initiates autophagy and hijacks this membrane trafficking pathway to transport viral proteins from the ER to sites of virus replication for assembly of infectious virus.     

Assembly and dynamics of the autophagy-initiating Atg1 complex

The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17–Atg31–Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen–deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼100-nM affinity. The Atg1–Atg13 complex in turn binds as a unit to the Atg17–Atg31–Atg29 scaffold with ∼10-μM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.The engulfment of cytosolic contents by autophagy is an ancient mechanism for cell survival and homeostasis (1, 2). This process is conserved throughout the Eukarya. Autophagy consists of the surrounding of cellular material in a double-membrane structure known as the phagophore (1), which matures into the autophagosome and fuses with the lysosome. The small-molecule metabolites generated by lysosomal degradation replenish energy stores and biosynthetic precursors. Autophagy, or its dysfunction, has roles in neurodegenerative disease, cancer, infection, inflammation, and aging (3). Despite its central importance in human health and disease, current knowledge of autophagosome biogenesis at the structural and molecular mechanistic level is limited (4). Our laboratory and many others have therefore embarked on a protein-by-protein effort to dissect the structures and interactions responsible for the remarkable process of autophagosome biogenesis.In yeast, autophagosome biogenesis commences at a single locus known as the phagophore assembly site (PAS). The autophagosome is nucleated, at least in part, from a cluster of a small number of vesicles with radii of 15–30 nm that contain the integral membrane protein autophagy-related 9 (Atg9) (5–7). The Atg1 complex, consisting of the subunits Atg1, Atg13, Atg17, Atg29, and Atg31, is thought to have a central role in autophagy initiation at the PAS. Atg1 is a protein kinase, yet the Atg1 complex is thought to have essential roles very early in autophagy that are independent of its kinase activity (8). These probably include organizing the vesicle cluster that goes on to form the phagophore (9). The kinase activity of Atg1 is also essential, in part because it phosphorylates Atg9 (10). In human cells, the Unc51-like kinase 1 (ULK1) and ULK2 complexes are largely conserved and thought to serve similar functions (11). The subunits Atg17, Atg29, and Atg31 appear to be capable of assembling at the PAS constitutively. They thus appear to serve as a preexisting scaffold for the recruitment of Atg1 and Atg13 upon activation.The crystal structure of the Atg17–Atg31–Atg29 complex showed that it dimerizes into a structurally unique double crescent (9, 12). Dimerization occurs via the C terminus of Atg17, and is required for formation of the PAS and for autophagy (9). Autophagy initiation also requires the recruitment of Atg1 and Atg13 to the PAS downstream of Atg17–Atg31–Atg29 (13). Atg1 consists of an N-terminal protein kinase domain, a predicted flexible linker, and a C-terminal early autophagy targeting/tethering (EAT) domain. Atg13 consists of an N-terminal HORMA domain (14) and a very long predicted unstructured central and C-terminal region. The presence of extensive regions of presumed intrinsic disorder in the Atg1 and Atg13 subunits has slowed progress in understanding the structure and assembly of the complete pentameric Atg1 complex. Given the essential role of the Atg1 complex in autophagy initiation, we set out to probe its dynamics using hydrogen–deuterium exchange (HDX) coupled to mass spectrometry (MS). In HDX-MS, the intrinsic exchange rate of amide protons is used to measure protein dynamics (15–17). Highly ordered regions of proteins exchange protons slowly, whereas dynamic regions exchange them rapidly.The translocation of the Atg1 and Atg13 subunits to the PAS upon TORC1 inactivation is a critical event in early autophagy and has been the topic of intensive investigation and debate. Dephosphorylated Atg13 is thought to act as a bridge between Atg1 and Atg17, triggering the assembly of the subunits into a dimer of pentamers. Mutation of the eight identified Atg13 phosphorylation sites to Ala induces Atg1 complex formation in yeast in the absence of autophagy induction (18). Recently, a constitutive interaction between Atg1 and Atg13 was observed (19), consistent with findings in human (20–22) and Drosophila (23) cells. The newer report suggests that the Atg1–Atg13 complex is constitutive and is regulated primarily at the level of its conformation, rather than its assembly. In this work, we probe the dynamics and stability of the Atg1 EAT domain and find that much of it is mobile in the absence of Atg13. We also find that the affinity of Atg1 and Atg13 for one another is very high, on the order of ∼100 nM. By contrast, the affinity of the preassembled Atg1–Atg13 and Atg17–Atg31–Atg29 complexes for one another is two orders of magnitude weaker. These findings have implications for models of Atg1 recruitment to the PAS and so for the mechanism of autophagy initiation.     

Melatonin modulates the autophagic response in acute liver failure induced by the rabbit hemorrhagic disease virus

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin‐1, ubiquitin‐like autophagy‐related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3‐I to autophagosome‐associated LC3‐II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome‐associated membrane protein (LAMP)‐1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase‐3 activity, and immunoglobulin‐heavy‐chain‐binding protein (BiP) and CCAAT/enhancer‐binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.     

Age-related changes in the function of autophagy in rat kidneys

Autophagy is a highly regulated intracellular process for the degradation of cytoplasmic components, especially protein aggregates and damaged organelles. It is essential for maintaining healthy cells. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. In this study, we investigated the effects of age on autophagy in the kidneys of 3-, 12-, and 24-month-old Fischer 344 rats. The results revealed that autophagy-related gene (Atg)7 was significantly downregulated in kidneys of increasing age. The protein expression level of the autophagy marker light chain 3/Atg8 exhibited a marked decline in aged kidneys. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the function of autophagy and proteasomal degradation, increased in older kidneys. The level of 8-hydroxydeoxyguanosine, a marker of mitochondrial DNA oxidative damage, was also increased in older kidneys. Analysis by transmission electron microscope demonstrated swelling and disintegration of cristae in the mitochondria of aged kidneys. These results suggest that autophagic function decreases with age in the kidneys of Fischer 344 rats, and autophagy may mediate the process of kidney aging, leading to the accumulation of damaged mitochondria.     

Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.Autophagy is the major lysosome/vacuole-dependent cellular degradative pathway. During autophagy, cytoplasmic constituents including proteins, lipids, and even entire organelles are surrounded by the phagophore, the initial sequestering compartment. The phagophore then expands to form double-membrane vesicles, termed autophagosomes. The completed autophagosomes fuse with lysosomes/vacuoles allowing access of the cargo to the degradative enzymes within this organelle; the resulting breakdown products are released back into the cytosol as building blocks or catabolic substrates (1–3). Autophagy is not only critical for survival during nutrient deprivation but is also involved in various human pathophysiologies, including cancer and neurodegeneration (4).On autophagy induction, AuTophaGy-related (Atg) proteins accumulate at the phagophore assembly site (PAS) and initiate autophagosome formation. Among the 36 known Atg proteins, Atg1 is the only kinase, and it plays a particularly important role in autophagy induction by controlling the movement of other Atg proteins including Atg9 and Atg23 (5) and in the proper organization of the PAS (6, 7). Atg1 interacts with several proteins, including direct binding to Atg13 (which interacts with Atg17) and Atg11; the kinase activity of Atg1 is regulated in part by its binding to, and/or interaction with, some of these components (8, 9).Atg17 constitutively forms a stable protein complex with Atg29 and Atg31 in both growing and nitrogen starvation conditions (10), and Atg31 directly interacts with Atg17 and Atg29 to bridge these two proteins (11). When autophagy is initiated, the Atg17-Atg31-Atg29 complex is first targeted to the PAS and recruits other Atg proteins, including Atg1 and Atg13, highlighting the significance of the ternary complex (12, 13). Along these lines, a single deletion of the ATG17, ATG29, or ATG31 genes results in a dramatic decrease in autophagy activity (14–16).In this study, we examined the role of posttranslational modification of Atg29. We found that Atg29 is a phosphoprotein and that phosphorylation on the C-terminal domain is critical for autophagy activity; the N terminus of Atg29 contains the functional domain, whereas the C terminus plays a regulatory role. We continued and extended our study to include a structural and functional analysis of the Atg17-Atg31-Atg29 complex. Single-particle electron microscopy (EM) reveals that the recombinant Atg17-Atg31-Atg29 complex is present as an elongated S-shaped dimerized structure, with Atg17 forming the backbone. We further demonstrate that Atg29 has the characteristics of an intrinsically disordered protein (IDP), suggesting that the C-terminal half is flexible and capable of altering its conformation on binding to one or more interacting proteins. Finally, we determined that Atg11 is necessary and sufficient to recruit this complex to the PAS and that phosphorylation of Atg29 is required for its interaction with Atg11 and proper PAS localization.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection

Autophagy, a cytoplasmic catabolic process, plays a critical role in defense against intracellular infection. In turn, evasion or inhibition of autophagy has emerged as an important virulence factor for intracellular pathogens. However, Anaplasma phagocytophilum, the obligatory intracellular bacterium that causes human granulocytic anaplasmosis, replicates in the membrane-bound compartment resembling early autophagosome. Here, we found that Anaplasma translocated substrate 1 (Ats-1), a type IV secretion effector, binds Beclin 1, a subunit of the class III PI3K and Atg14L, and it nucleates autophagosomes with markers of omegasomes, double FYVE-containing protein 1, Atg14L, and LC3. Ats-1 autophagy induction did not activate the starvation signaling pathway of mammalian target of rapamycin. These autophagy proteins were also localized to the Anaplasma inclusion. Ectopically expressed Ats-1 targeted the Anaplasma inclusions and enhanced infection, whereas host cytoplasmic delivery of anti–Ats-1 or Beclin 1 depletion by siRNA suppressed the infection; beclin 1 heterozygous-deficient mice were resistant to Anaplasma infection. Furthermore, Anaplasma growth arrest by the class III PI3K inhibitor 3-methyladenine was alleviated by essential amino acid supplementation. Thus, Anaplasma actively induces autophagy by secreting Ats-1 that hijacks the Beclin 1-Atg14L autophagy initiation pathway likely to acquire host nutrients for its growth.     

Autophagy Inhibits Intercellular Transport of Citrus Leaf Blotch Virus by Targeting Viral Movement Protein

Autophagy is an evolutionarily conserved cellular-degradation mechanism implicated in antiviral defense in plants. Studies have shown that autophagy suppresses virus accumulation in cells; however, it has not been reported to specifically inhibit viral spread in plants. This study demonstrated that infection with citrus leaf blotch virus (CLBV; genus Citrivirus, family Betaflexiviridae) activated autophagy in Nicotiana benthamiana plants as indicated by the increase of autophagosome formation. Impairment of autophagy through silencing of N. benthamiana autophagy-related gene 5 (NbATG5) and NbATG7 enhanced cell-to-cell and systemic movement of CLBV; however, it did not affect CLBV accumulation when the systemic infection had been fully established. Treatment using an autophagy inhibitor or silencing of NbATG5 and NbATG7 revealed that transiently expressed movement protein (MP), but not coat protein, of CLBV was targeted by selective autophagy for degradation. Moreover, we identified that CLBV MP directly interacted with NbATG8C1 and NbATG8i, the isoforms of autophagy-related protein 8 (ATG8), which are key factors that usually bind cargo receptors for selective autophagy. Our results present a novel example in which autophagy specifically targets a viral MP to limit the intercellular spread of the virus in plants.     

Impaired autophagic function in rat islets with aging

Type 2 diabetes is characterized by a deficit in β-cell function and mass, and its incidence increases with age. Autophagy is a highly regulated intracellular process for degrading cytoplasmic components, particularly protein aggregates and damaged organelles. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. Autophagy may be necessary to maintain structure, mass, and function of pancreatic β-cells. In this study, we investigated the effects of age on β-cell function and autophagy in pancreatic islets of 4-month-old (young), 14-month-old (adult), and 24-month-old (old) male Wistar rats. We found that islet β-cell function decreased gradually with age. Protein expression of the autophagy markers LC3/Atg8 and Atg7 exhibited a marked decline in aged islets. The expression of Lamp-2, a good indicator of autophagic degradation rate, was significantly reduced in the islets of old rats, suggesting that autophagic degradation is decreased in the islets of aged rats. However, protein expression of beclin-1/Atg6, which plays an important role in the induction and formation of the pre-autophagosome structure by associating with a multimeric complex of autophagy regulatory proteins (Atg14, Vps34/class 3 PI3 kinase, and Vps15), was most prominent in the islets of adult rats, and was higher in 24-month-old islets than in 4-month-old islets. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the functions of autophagy and proteasomal degradation, were increased in aging islets. 8-Hydroxydeoxyguanosine, a marker of mitochondrial and nuclear DNA oxidative damage, exhibited strong immunostaining in old islets. Analysis by electron microscopy demonstrated swelling and disintegration of cristae in the mitochondria of aged islets. These results suggest that β-cell and autophagic function in islets decline simultaneously with increasing age in Wistar rats, and that impaired autophagy in the islets of older rats may cause accumulation of misfolded and aggregated proteins and reduce the removal of abnormal mitochondria in β-cells, leading to reduced β-cell function. Dysfunctional autophagy in islets during the aging process may be an important mechanism leading to the development of type 2 diabetes.     

Autophagy in the heart and liver during normal aging and calorie restriction

Autophagy is a highly regulated intracellular process for the degradation of cellular constituents and essential for the maintenance of a healthy cell. We evaluated the effects of age and life-long calorie restriction on autophagy in heart and liver of young (6 months) and old (26 months) Fisher 344 rats. We observed that the occurrence of autophagic vacuoles was higher in heart than liver. The occurrence of autophagic vacuoles was not affected by age in either tissue, but was increased with calorie restriction in heart but not in liver. Next, we examined the expression of proteins involved in the formation and maturation of autophagosomes (beclin-1, LC3, Atg7, Atg9) or associated with autolysosomes and lysosomes (LAMP-1; cathepsin D). In hearts of both ad libitum-fed and calorie-restricted rats, we observed an increase in expression of beclin-1 and procathepsin D, but not mature cathepsin D, and a decrease in expression of LAMP-1 because of aging. In hearts, calorie restriction stimulated the expression of Atg7 and Atg9 and the lipidation of Atg8 (elevated LC3-II/I ratios) in aged rats. In hearts of ad libitum-fed rats, expression of Atg7 and lipidation of Atg8 were unaffected by age, while the cellular levels of Atg9 were lower in aged animals. Furthermore, we observed that the age- and diet-dependent expression levels of those proteins differed between heart and liver. In conclusion, autophagy in heart and liver did not decrease with age in ad libitum-fed rats, but was enhanced by calorie restriction in the heart. Thus, calorie restriction may mediate some of its beneficial effects by stimulating autophagy in the heart, indicating the potential for cardioprotective therapies.     

Protective role of autophagy in palmitate-induced INS-1 beta-cell death

Autophagy, a vacuolar degradative pathway, constitutes a stress adaptation that avoids cell death or elicits the alternative cell-death pathway. This study was undertaken to determine whether autophagy is activated in palmitate (PA)-treated beta-cells and, if activated, what the role of autophagy is in the PA-induced beta-cell death. The enhanced formation of autophagosomes and autolysosomes was observed by exposure of INS-1 beta-cells to 400 microm PA in the presence of 25 mm glucose for 12 h. The formation of green fluorescent protein-LC3-labeled structures (green fluorescent protein-LC3 dots), with the conversion from LC3-I to LC3-II, was also distinct in the PA-treated cells. The phospho-mammalian target of rapamycin level, a typical signal pathway that inhibits activation of autophagy, was gradually decreased by PA treatment. Blockage of the mammalian target of rapamycin signaling pathway by treatment with rapamycin augmented the formation of autophagosomes but reduced PA-induced INS-1 cell death. In contrast, reduction of autophagosome formation by knocking down the ATG5, inhibition of fusion between autophagosome and lysosome by treatment with bafilomycin A1, or inhibition of proteolytic degradation by treatment with E64d/pepstatin A, significantly augmented PA-induced INS-1 cell death. These findings showed that the autophagy system could be activated in PA-treated INS-1 beta-cells, and suggested that the induction of autophagy might play an adaptive and protective role in PA-induced cell death.     

Impaired autophagy: A mechanism of mitochondrial dysfunction in anoxic rat hepatocytes

Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy-related protein 7 (Atg7), Beclin-1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid-free and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusion: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy, and this subsequently leads to MPT-dependent hepatocyte death after A/R.     

The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.     

Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)

The class III phosphatidylinositol 3-kinase (PI3KC3) is crucial for autophagosome biogenesis. It has been long speculated to nucleate the autophagosome membrane, but the biochemical mechanism of such nucleation activity remains unsolved. We recently identified Barkor/Atg14(L) as the targeting factor for PI3KC3 to autophagosome membrane. Here, we show that we have characterized the region of Barkor/Atg14(L) required for autophagosome targeting and identified the BATS [Barkor/Atg14(L) autophagosome targeting sequence] domain at the carboxyl terminus of Barkor. Bioinformatics and mutagenesis analyses revealed that the BATS domain binds to autophagosome membrane via the hydrophobic surface of an intrinsic amphipathic alpha helix. BATS puncta overlap with Atg16 and LC3, and partially with DFCP1, in a stress-inducible manner. Ectopically expressed BATS accumulates on highly curved tubules that likely represent intermediate autophagic structures. PI3KC3 recruitment and autophagy stimulation by Barkor/Atg14(L) require the BATS domain. Furthermore, our biochemical analyses indicate that the BATS domain directly binds to the membrane, and it favors membrane composed of phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 4,5-biphosphate [PtdIns(4,5)P2]. By binding preferentially to curved membranes incorporated with PtdIns(3)P but not PtdIns(4,5)P2, the BATS domain is capable of sensing membrane curvature. Thus, we propose a novel model of PI3KC3 autophagosome membrane nucleation in which its autophagosome-specific adaptor, Barkor, accumulates on highly curved PtdIns(3)P enriched autophagic membrane via its BATS domain to sense and maintain membrane curvature.     

Melatonin attenuates methamphetamine-induced deactivation of the mammalian target of rapamycin signaling to induce autophagy in SK-N-SH cells

Abstract:  Methamphetamine (METH) is a commonly abused drug that damages nerve terminals by causing reactive oxygen species (ROS) formation, apoptosis, and neuronal damage. Autophagy, a type of programmed cell death independent of apoptosis, is negatively regulated by the mammalian target of the rapamycin (mTOR) signaling pathway. It is not known, however, whether autophagy is involved in METH-induced neurotoxicity. Therefore, we investigated the effect of METH on autophagy and its upstream regulator, the mTOR signaling pathway. Using the SK-N-SH dopaminergic cell line, we found that METH induces the expression of LC3-II, a protein associated with the autophagosome membrane, in a dose-dependent manner. Moreover, METH inhibits the phosphorylation of mTOR and the action of its downstream target, the eukaryotic initiation factor (eIF)4E-binding protein, 4EBP1. Melatonin, a major secretory product of pineal, is a potent naturally produced antioxidant that acts through various mechanisms to ameliorate the toxic effects of ROS. We found that a pretreatment with melatonin enhances mTOR activity and 4EBP1 phosphorylation and protects against the formation of LC3-II in SK-N-SH cells exposed to METH. This work demonstrates a novel role for melatonin as a neuroprotective agent against METH.     

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion

The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs’ previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through “PI4P shuttling.” Importantly, PI4KIIα’s role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP₂) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome’s fusion machinery.Macroautophagy (autophagy) is orchestrated by multiple autophagy-related (Atg) proteins (1). Among these, the Atg8 proteins are essential for autophagosome biogenesis and maturation. Mammals have at least six Atg8 orthologs that can be broadly classified into two large subfamilies: LC3s (light-chain 3) and GABARAPs (γ-aminobutyric acid receptor-associated proteins)/GATE-16s (Golgi-associated ATPase enhancer of 16 kDa), hereafter referred to collectively as GABARAPs. GABARAPs were initially identified as trafficking modulators for transmembrane receptors from the Golgi to the plasma membrane (2), and subsequently as Atg8s (1). Functional studies in mammalian cells have placed GABARAPs downstream of LC3 during autophagy (3).The complexity of the mammalian Atg8 protein network was highlighted by a recent screen that revealed a cohort of at least 67 binding partners, a third of which are unique to either the LC3 or GABARAP families (4). Phosphatidylinositol 4-kinase IIα (PI4KIIα), which generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol, was identified as a binding partner for GABARAPs, but not for LC3 (4). PI4KIIα is one of four vertebrate PI4Ks (5), and it has not been previously implicated in autophagy. Here we establish for the first time, to our knowledge, that GABARAPs govern the fusion of autophagosomes with lysosomes (A:L fusion) through PI4KIIα-mediated in situ PI4P generation on autophagosomes. We propose a working model that integrates GABARAPs’ critical roles as trafficking modulators and autophagy effectors through PI4KIIα.     

Autophagy enhances hepatocellular carcinoma progression by activation of mitochondrial β-oxidation

Background

Several types of cancers, including hepatocellular carcinoma (HCC), show resistance to hypoxia and nutrient starvation. Autophagy is a means of providing macromolecules for energy generation under such stressed-conditions. The aim of this study was to clarify the role of autophagy in HCC development under hypoxic conditions.

Methods

The expression of microtubule-associated protein 1 light chain 3 (LC3), which is a key gene involved in autophagosome formation, was evaluated in human HCC using immunohistochemistry and western blot. The relationship between LC3 and hypoxia-induced factor 1α (HIF1α) expression was examined using real-time PCR. In addition, human HCC cell line Huh7 was treated with pharmacological autophagy-inhibitor and inactive mutant of Atg4B (Atg4B^C74A) under hypoxic condition to evaluate the effects of hypoxia-induced autophagy on cell survival, intracellular ATP, and mitochondrial β-oxidation.

Results

LC3 was significantly highly expressed in HCC as compared with noncancerous tissues. LC3 expression, correlated with HIF1α expression, was also significantly correlated with tumor size, and only in the context of large tumors, was an independent predictor of HCC recurrence after surgery. In addition, Huh7 treated with autophagy-inhibitor under hypoxia had lower viability, with low levels of intracellular ATP due to impaired mitochondrial β-oxidation.

Conclusions

Autophagy in HCC works to promote HIF1α-mediated proliferation through the maintenance of intracellular ATP, depending on the activation of mitochondrial β-oxidation. These findings demonstrated the feasibility of anti-autophagic treatment as a potential curative therapy for HCC, and improved understanding of the factors determining adaptive metabolic responses to hypoxic conditions.