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1.  Novel members of the cdc2-related kinase family in Drosophila: cdk4/6, cdk5, PFTAIRE, and PITSLRE kinase. 
Molecular Biology of the Cell  1996;7(11):1759-1769.
In addition to the previously identified Drosophila cdc2 and cdc2c genes, we have identified four additional cdc2-related genes with low stringency and polymerase chain reaction approaches. Sequence comparisons suggest that the four putative kinases represent the Drosophila homologues of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologues is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4/6 (47% identity). However, like vertebrate cdk4 and cdk6, Drosophila cdk4/6 binds also to a D-type cyclin according to the results of two-hybrid experiments in yeast. Northern blot analysis indicated that the four Drosophila kinases are expressed throughout embryogenesis. Expression in early embryogenesis appeared to be ubiquitous according to in situ hybridization. Abundant expression already at the start of embryogenesis and long before neuron differentiation was also observed in the case of cdk5 protein, which has been described as predominantly neuron specific in mice. Sequence conservation and expression pattern, therefore, suggest that all of these kinases perform important cellular functions.
PMCID: PMC276024  PMID: 8930898
Post-mitotic neurons are typically terminally differentiated and in a quiescent status. However, in Alzheimer disease (AD), many neurons display ectopic re-expression of cell cycle-related proteins. Cyclin-dependent kinase 11 (CDK11) mRNA produces a 110-kDa protein (CDK11p110) throughout the cell cycle, a 58-kDa protein (CDK11p58) that is specifically translated from an internal ribosome entry site and expressed only in the G2/M phase of the cell cycle, and a 46-kDa protein (CDK11p46) that is considered to be apoptosis specific. CDK11 is required for sister chromatid cohesion and the completion of mitosis. In this study, we found that the expression patterns of CDK11 vary such that cytoplasmic CDK11 is increased in AD cellular processes, compared to a pronounced nuclear expression pattern in most controls. We also investigated the effect of amyloid precursor protein (APP) on CDK11 expression in vitro by using M17 cells overexpressing wild-type APP and APP Swedish mutant phenotype and found increased CDK11 expression compared to empty vector. In addition, amyloid-β25-35 resulted in increased CDK11 in M17 cells. These data suggest that CDK11 may play a vital role in cell cycle re-entry in AD neurons in an APP-dependent manner, thus presenting an intriguing novel function of the APP signaling pathway in AD.
PMCID: PMC3153952  PMID: 21461981
Alzheimer disease; APP; CDK11; M17 cells
3.  Agent-based modeling of autophagy reveals emergent regulatory behavior of spatio-temporal autophagy dynamics 
Autophagy is a vesicle-mediated pathway for lysosomal degradation, essential under basal and stressed conditions. Various cellular components, including specific proteins, protein aggregates, organelles and intracellular pathogens, are targets for autophagic degradation. Thereby, autophagy controls numerous vital physiological and pathophysiological functions, including cell signaling, differentiation, turnover of cellular components and pathogen defense. Moreover, autophagy enables the cell to recycle cellular components to metabolic substrates, thereby permitting prolonged survival under low nutrient conditions. Due to the multi-faceted roles for autophagy in maintaining cellular and organismal homeostasis and responding to diverse stresses, malfunction of autophagy contributes to both chronic and acute pathologies.
We applied a systems biology approach to improve the understanding of this complex cellular process of autophagy. All autophagy pathway vesicle activities, i.e. creation, movement, fusion and degradation, are highly dynamic, temporally and spatially, and under various forms of regulation. We therefore developed an agent-based model (ABM) to represent individual components of the autophagy pathway, subcellular vesicle dynamics and metabolic feedback with the cellular environment, thereby providing a framework to investigate spatio-temporal aspects of autophagy regulation and dynamic behavior. The rules defining our ABM were derived from literature and from high-resolution images of autophagy markers under basal and activated conditions. Key model parameters were fit with an iterative method using a genetic algorithm and a predefined fitness function. From this approach, we found that accurate prediction of spatio-temporal behavior required increasing model complexity by implementing functional integration of autophagy with the cellular nutrient state. The resulting model is able to reproduce short-term autophagic flux measurements (up to 3 hours) under basal and activated autophagy conditions, and to measure the degree of cell-to-cell variability. Moreover, we experimentally confirmed two model predictions, namely (i) peri-nuclear concentration of autophagosomes and (ii) inhibitory lysosomal feedback on mTOR signaling.
Agent-based modeling represents a novel approach to investigate autophagy dynamics, function and dysfunction with high biological realism. Our model accurately recapitulates short-term behavior and cell-to-cell variability under basal and activated conditions of autophagy. Further, this approach also allows investigation of long-term behaviors emerging from biologically-relevant alterations to vesicle trafficking and metabolic state.
Electronic supplementary material
The online version of this article (doi:10.1186/s12964-014-0056-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4172826  PMID: 25214434
Agent-based modeling; Autophagy; Autophagic flux; Cell-to-cell variability; Systems biology; Computational biology; mTOR; Lysosomes
4.  Network organization of the human autophagy system 
Nature  2010;466(7302):68-76.
Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. While conserved protein kinase, lipid kinase, and ubiquitin-like (UBL) protein conjugation sub-networks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here, we report a proteomic analysis of the autophagy interaction network (AIN) in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 4 09 candidate interacting proteins with extensive connectivity among sub-networks. Many new AIN components have roles in vesicle trafficking, protein or lipid phosphorylation, and protein ubiquitination, and affect autophagosome number or flux when depleted by RNAi. The six ATG8 orthologs in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions (LIR) in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.
PMCID: PMC2901998  PMID: 20562859
5.  Host Cell Autophagy Modulates Early Stages of Adenovirus Infections in Airway Epithelial Cells 
Journal of Virology  2013;87(4):2307-2319.
Human adenoviruses typically cause mild infections in the upper or lower respiratory tract, gastrointestinal tract, or ocular epithelium. However, adenoviruses may be life-threatening in patients with impaired immunity and some serotypes cause epidemic outbreaks. Attachment to host cell receptors activates cell signaling and virus uptake by endocytosis. At present, it is unclear how vital cellular homeostatic mechanisms affect these early steps in the adenovirus life cycle. Autophagy is a lysosomal degradation pathway for recycling intracellular components that is upregulated during periods of cell stress. Autophagic cargo is sequestered in double-membrane structures called autophagosomes that fuse with endosomes to form amphisomes which then deliver their content to lysosomes. Autophagy is an important adaptive response in airway epithelial cells targeted by many common adenovirus serotypes. Using two established tissue culture models, we demonstrate here that adaptive autophagy enhances expression of the early region 1 adenovirus protein, induction of mitogen-activated protein kinase signaling, and production of new viral progeny in airway epithelial cells infected with adenovirus type 2. We have also discovered that adenovirus infections are tightly regulated by endosome maturation, a process characterized by abrupt exchange of Rab5 and Rab7 GTPases, associated with early and late endosomes, respectively. Moreover, endosome maturation appears to control a pool of early endosomes capable of fusing with autophagosomes which enhance adenovirus infection. Many viruses have evolved mechanisms to induce autophagy in order to aid their own replication. Our studies reveal a novel role for host cell autophagy that could have a significant impact on the outcome of respiratory infections.
PMCID: PMC3571477  PMID: 23236070
6.  The incredible ULKs 
Macroautophagy (commonly abbreviated as autophagy) is an evolutionary conserved lysosome-directed vesicular trafficking pathway in eukaryotic cells that mediates the lysosomal degradation of intracellular components. The cytoplasmic cargo is initially enclosed by a specific double membrane vesicle, termed the autophagosome. By this means, autophagy either helps to remove damaged organelles, long-lived proteins and protein aggregates, or serves as a recycling mechanism for molecular building blocks. Autophagy was once invented by unicellular organisms to compensate the fluctuating external supply of nutrients. In higher eukaryotes, it is strongly enhanced under various stress conditions, such as nutrient and growth factor deprivation or DNA damage. The serine/threonine kinase Atg1 was the first identified autophagy-related gene (ATG) product in yeast. The corresponding nematode homolog UNC-51, however, has additional neuronal functions. Vertebrate genomes finally encode five closely related kinases, of which UNC-51-like kinase 1 (Ulk1) and Ulk2 are both involved in the regulation of autophagy and further neuron-specific vesicular trafficking processes. This review will mainly focus on the vertebrate Ulk1/2-Atg13-FIP200 protein complex, its function in autophagy initiation, its evolutionary descent from the yeast Atg1-Atg13-Atg17 complex, as well as the additional non-autophagic functions of its components. Since the rapid nutrient- and stress-dependent cellular responses are mainly mediated by serine/threonine phosphorylation, it will summarize our current knowledge about the relevant upstream signaling pathways and the altering phosphorylation status within this complex during autophagy induction.
PMCID: PMC3330011  PMID: 22413737
Atg1; Atg13; Atg17; UNC-51; EPG-1; Ulk1; Ulk2; FIP200; Atg101; Autophagy; Serine/threonine phosphorylation
7.  NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses 
PLoS Genetics  2013;9(1):e1003196.
Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.
Author Summary
Autophagy is an evolutionarily conserved process that sequestrates and delivers cytoplasmic macromolecules and organelles to the vacuoles or lysosomes for degradation. In plants, autophagy is involved in supplying internal nutrients during starvation and in promoting cell survival during senescence and during biotic and abiotic stresses. Arabidopsis NBR1 is a homolog of mammalian autophagy cargo adaptors P62 and NBR1. Disruption of Arabidopsis NBR1 caused increased sensitivity to a spectrum of abiotic stresses but had no significant effect on plant senescence, responses to carbon starvation, or resistance to a necrotrophic pathogen. NBR1 contains an ubiquitin-binding domain, and the compromised stress tolerance of autophagy mutants was associated with increased accumulation of NBR1 and ubiquitin-positive cellular protein aggregates in the insoluble protein fraction under stress conditions. Based on these results, we propose that NBR1 targets ubiquitinated protein aggregates most likely derived from denatured and otherwise damaged nonnative proteins for autophagic clearance under stress conditions.
PMCID: PMC3547818  PMID: 23341779
8.  Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila 
BMC Cell Biology  2013;14:29.
Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized.
Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (α, β or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1α.
We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1α/sima may contribute to upregulation of autophagy by impaired proteasomal activity.
PMCID: PMC3700814  PMID: 23800266
Autophagy; Drosophila; HIF-1α/sima; Hypoxia; p62/Ref2P; Proteasome
9.  PITSLRE protein kinase activity is associated with apoptosis. 
Minimal ectopic expression of a 58-kDa protein kinase (PITSLRE beta 1), distantly related to members of the cdc2 gene family, induces telophase delay, abnormal chromosome segregation, and decreased growth rates in Chinese hamster ovary cells. Here we show that this decrease in cell growth rate is due to apoptosis. Apoptosis is also induced by ectopic expression of an amino-terminal deletion mutant containing the catalytic and C-terminal domains of PITSLRE beta 1 but not by other mutants lacking histone H1 kinase activity or by other members of the cdc2 gene family. However, unlike the wild-type PITSLRE beta 1 over-expressors, ectopic expression of the N-terminal PITSLRE beta 1 mutant does not result in telophase delay or abnormal chromosome segregation. These results suggested that the function of this protein kinase could be linked to apoptotic signaling. To test this hypothesis, we examined levels of PITSLRE mRNA, steady-state protein, and enzyme activity in human T cells undergoing apoptosis after activation with the anti-Fas monoclonal antibody (MAb). All were substantially elevated shortly after Fas MAb treatment. In addition to new transcription and translation, proteolysis contributed to the increased steady-state levels of a novel 50-kDa PITSLRE protein, as suggested by the diminution of larger PITSLRE isoforms observed in the same cells. Indeed, treatment of the Fas-activated T cells with a serine protease inhibitor prevented apoptotic death and led to the accumulation of larger, less active PITSLRE kinase isoforms but not the enzymatically active 50-kDa PITSLRE isoform. Finally, induction of apoptosis by glucocorticoids in the same cell line, as well as by Fas MAb treatment of another T-cell line, led to a similar induction of 50-kDa PITSLRE protein levels over time. These findings suggest that (i) PITSLRE kinase(s) may lie within apoptotic signaling pathway(s), (ii) serine protease activation may be an early event in Fas-activated apoptosis of human T cells, and (iii) some PITSLRE kinase isoforms may be targets of apoptotic proteases.
PMCID: PMC231901  PMID: 7528324
10.  Sustained Autophagy Contributes to Measles Virus Infectivity 
PLoS Pathogens  2013;9(9):e1003599.
The interplay between autophagy and intracellular pathogens is intricate as autophagy is an essential cellular response to fight against infections, whereas numerous microbes have developed strategies to escape this process or even exploit it to their own benefit. The fine tuned timing and/or selective molecular pathways involved in the induction of autophagy upon infections could be the cornerstone allowing cells to either control intracellular pathogens, or be invaded by them. We report here that measles virus infection induces successive autophagy signallings in permissive cells, via distinct and uncoupled molecular pathways. Immediately upon infection, attenuated measles virus induces a first transient wave of autophagy, via a pathway involving its cellular receptor CD46 and the scaffold protein GOPC. Soon after infection, a new autophagy signalling is initiated which requires viral replication and the expression of the non-structural measles virus protein C. Strikingly, this second autophagy signalling can be sustained overtime within infected cells, independently of the expression of C, but via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Whereas this sustained autophagy signalling leads to the autophagy degradation of cellular contents, viral proteins escape from degradation. Furthermore, this autophagy flux is ultimately exploited by measles virus to limit the death of infected cells and to improve viral particle formation. Whereas CD150 dependent virulent strains of measles virus are unable to induce the early CD46/GOPC dependent autophagy wave, they induce and exploit the late and sustained autophagy. Overall, our work describes distinct molecular pathways for an induction of self-beneficial sustained autophagy by measles virus.
Author Summary
Autophagy is an evolutionarily conserved lysosomal dependent degradative pathway for recycling of long-lived proteins and damaged organelles. Autophagy is also an essential cellular response to fight infection by destroying infectious pathogens trapped within autophagosomes and plays a key role in the induction of both innate and adaptive immune responses. Numerous viruses have evolved strategies to counteract autophagy in order to escape from degradation or/and to inhibit immune signals. The kinetic and molecular pathways involved in the induction of autophagy upon infections might determine if cells would be able to control pathogens or would be invaded by them. We showed that measles virus (MeV) infection induces successive autophagy signallings in cells via distinct molecular pathways. A first autophagy wave is induced by the engagement of the MeV cellular receptor CD46 and the scaffold protein GOPC. A second wave is initiated after viral replication by the expression of the non-structural MeV protein C and is sustained overtime within infected cells thanks to the formation of syncytia. This sustained autophagy is exploited by MeV to limit the death of infected cells and to improve viral particle formation. We describe new molecular pathways by which MeV hijacks autophagy to promote its infectivity.
PMCID: PMC3784470  PMID: 24086130
11.  Autophagy: A Critical Regulator of Cellular Metabolism and Homeostasis 
Molecules and Cells  2013;36(1):7-16.
Autophagy is a dynamic process by which cytosolic material, including organelles, proteins, and pathogens, are sequestered into membrane vesicles called autophagosomes, and then delivered to the lysosome for degradation. By recycling cellular components, this process provides a mechanism for adaptation to starvation. The regulation of autophagy by nutrient signals involves a complex network of proteins that include mammalian target of rapamycin, the class III phosphatidylinositol-3 kinase/Beclin 1 complex, and two ubiquitin-like conjugation systems. Additionally, autophagy, which can be induced by multiple forms of chemical and physical stress, including endoplasmic reticulum stress, and hypoxia, plays an integral role in the mammalian stress response. Recent studies indicate that, in addition to bulk assimilation of cytosol, autophagy may proceed through selective pathways that target distinct cargoes to autophagosomes. The principle homeostatic functions of autophagy include the selective clearance of aggregated protein to preserve proteostasis, and the selective removal of dysfunctional mitochondria (mitophagy). Additionally, autophagy plays a central role in innate and adaptive immunity, with diverse functions such as regulation of inflammatory responses, antigen presentation, and pathogen clearance. Autophagy can preserve cellular function in a wide variety of tissue injury and disease states, however, maladaptive or pro-pathogenic outcomes have also been described. Among the many diseases where autophagy may play a role include proteopathies which involve aberrant accumulation of proteins (e.g., neurodegenerative disorders), infectious diseases, and metabolic disorders such as diabetes and metabolic syndrome. Targeting the autophagy pathway and its regulatory components may eventually lead to the development of therapeutics.
PMCID: PMC3887921  PMID: 23708729
autophagy; innate immunity; metabolism; mitophagy; neurodegeneration; proteostasis
12.  LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy 
Molecular Cell  2012;48(3):329-342.
Autophagy protects cellular homeostasis by capturing cytosolic components and invading pathogens for lysosomal degradation. Autophagy receptors target cargo to autophagy by binding ATG8 on autophagosomal membranes. The expansion of the ATG8 family in higher eukaryotes suggests that specific interactions with autophagy receptors facilitate differential cargo handling. However, selective interactors of ATG8 orthologs are unknown. Here we show that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity since cells lacking either protein cannot protect their cytoplasm against Salmonella. LC3C is required for antibacterial autophagy because in its absence the remaining ATG8 orthologs do not support efficient antibacterial autophagy. Structural analysis revealed that the selectivity of NDP52 for LC3C is conferred by a noncanonical LIR, in which lack of an aromatic residue is balanced by LC3C-specific interactions. Our report illustrates that specificity in the interaction between autophagy receptors and autophagy machinery is of functional importance to execute selective autophagy.
Graphical Abstract
► The autophagy receptor NDP52 binds selectively the ATG8 family member LC3C ► A noncanonical LIR motif in NDP52 provides specificity for LC3C ► LC3C and NDP52 are required for autophagy and restriction of S. Typhimurium ► LC3C required to recruit all ATG8 orthologs into antibacterial autophagosomes
PMCID: PMC3510444  PMID: 23022382
13.  Image-Based Genome-Wide siRNA Screen Identifies Selective Autophagy Factors 
Nature  2011;480(7375):113-117.
Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction1–4. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy, and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation5. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. To identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide siRNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to mRNA processing, interferon signaling, vesicle trafficking, cytoskeletal motor function, and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, Smurf1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, Smurf1-deficient mice display an accumulation of damaged mitochondria in heart, brain, and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines Smurf1 as a newly recognized mediator of both viral autophagy and mitophagy.
PMCID: PMC3229641  PMID: 22020285
14.  The machinery of macroautophagy 
Cell Research  2013;24(1):24-41.
Autophagy is a primarily degradative pathway that takes place in all eukaryotic cells. It is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and through its action in various aspects of immunity including the elimination of invasive microbes and its participation in antigen presentation. The most prevalent form of autophagy is macroautophagy, and during this process, the cell forms a double-membrane sequestering compartment termed the phagophore, which matures into an autophagosome. Following delivery to the vacuole or lysosome, the cargo is degraded and the resulting macromolecules are released back into the cytosol for reuse. The past two decades have resulted in a tremendous increase with regard to the molecular studies of autophagy being carried out in yeast and other eukaryotes. Part of the surge in interest in this topic is due to the connection of autophagy with a wide range of human pathophysiologies including cancer, myopathies, diabetes and neurodegenerative disease. However, there are still many aspects of autophagy that remain unclear, including the process of phagophore formation, the regulatory mechanisms that control its induction and the function of most of the autophagy-related proteins. In this review, we focus on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.
PMCID: PMC3879710  PMID: 24366339
autophagosome; autophagy; degradation; lysosome; phagophore; stress; vacuole
15.  Autophagy Driven by a Master Regulator of Hematopoiesis 
Molecular and Cellular Biology  2012;32(1):226-239.
Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy.
PMCID: PMC3255705  PMID: 22025678
16.  14-3-3 Proteins are Regulators of Autophagy 
Cells  2012;1(4):754-773.
14-3-3 proteins are implicated in the regulation of proteins involved in a variety of signaling pathways. 14-3-3-dependent protein regulation occurs through phosphorylation-dependent binding that results, in many cases, in the release of survival signals in cells. Autophagy is a cell digestion process that contributes to overcoming nutrient deprivation and is initiated under stress conditions. However, whether autophagy is a cell survival or cell death mechanism remains under discussion and may depend on context. Nevertheless, autophagy is a cellular process that determines cell fate and is tightly regulated by different signaling pathways, some of which, for example MAPK, PI3K and mTOR, are tightly regulated by 14-3-3 proteins. It is therefore important to understand the role of 14-3-3 protein in modulating the autophagic process. Within this context, direct binding of 14-3-3 to mTOR regulatory proteins, such as TSC2 and PRAS40, connects 14-3-3 with autophagy regulatory processes. In addition, 14-3-3 binding to human vacuolar protein sorting 34 (hVps34), a class III phosphatidylinositol-3-kinase (PI3KC3), indicates the involvement of 14-3-3 proteins in regulating autophagosome formation. hVps34 is involved in vesicle trafficking processes such as autophagy, and its activation is needed for initiation of autophagy. Chromatography and overlay techniques suggest that hVps34 directly interacts with 14-3-3 proteins under physiological conditions, thereby maintaining hVps34 in an inactive state. In contrast, nutrient starvation promotes dissociation of the 14-3-3–hVps34 complex, thereby enhancing hVps34 lipid kinase activity. Thus, 14-3-3 proteins are regulators of autophagy through regulating key components of the autophagic machinery. This review summarizes the role of 14-3-3 protein in the control of target proteins involved in regulating the master switches of autophagy.
PMCID: PMC3901138  PMID: 24710529
14-3-3 proteins; autophagy; cell signaling
17.  Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors 
PLoS Genetics  2013;9(8):e1003715.
Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.
Author Summary
Autophagy is a eukaryotic cellular process that transports cytoplasmic contents into lysosomes/vacuoles for degradation. It has been linked to multiple human diseases, including cancer and neurodegenerative disorders. The molecular machinery of autophagy was first identified and has been best characterized in the budding yeast Saccharomyces cerevisiae, but little is known about the autophagy machinery in another important unicellular model organism, the fission yeast Schizosaccharomyces pombe. In this study, we performed an unbiased and comprehensive screening of the fission yeast autophagy genes by profiling the mating phenotypes of nearly 3000 deletion strains. Following up on the screening results, we systematically characterized both previously known and newly identified fission yeast autophagy factors by examining their localization and the phenotype of their mutants. Our analysis increased the number of experimentally defined fission yeast autophagy factors from 14 to 23, including two novel factors that act in ways different from all previously known autophagy proteins. Together, our data reveal unexpected evolutionary divergence of autophagy mechanisms and establish a new model system for unraveling the molecular details of the autophagy process.
PMCID: PMC3738441  PMID: 23950735
18.  Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex 
PLoS Genetics  2014;10(10):e1004626.
Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.
Author Summary
Beclin 1 was not only the first-described mammalian autophagy protein, but is one of the most widely-characterized players in autophagy regulation. It is implicated in multiple human disease conditions. As a core component of the essential lipid kinase complex (PI3K-III), beclin 1 has largely been characterized to date in the context of autophagy through its recruitment of additional autophagy proteins for the assembly of the PI3K-III complexes involved in the nucleation of the autophagosome. Little is known, however, about how beclin 1 regulates specific functions of PI3K-III in other membrane trafficking pathways. Furthermore, although beclin 1 has been linked to multiple neurodegenerative diseases, the function of beclin 1 in the brain remains uncharacterized. Herein, we used genetic animal models and mutant cell lines to demonstrate that beclin 1 participates in multiple organelle trafficking pathways. Beclin 1 deficiency in neurons causes severe neurodegeneration, concomitant with aberrant late endosome formation and impaired phospholipid localization. Our mechanistic study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy. Our study clarifies the physiological function of beclin 1, which leads for further understanding of its role in tumorigenesis, infectious disease and neurodegenerative disease.
PMCID: PMC4183436  PMID: 25275521
19.  Autophagy termination and lysosome reformation regulated by mTOR 
Nature  2010;465(7300):942-946.
Autophagy is an evolutionarily conserved process to catabolize cytoplasmic proteins and organelles1, 2. During starvation, the target of rapamycin (TOR), a nutrient-responsive kinase, is inhibited, thereby inducing autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of autophagosome cargo in autolysosomes3-5, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly defined. Here we show that mTOR signaling is inhibited during autophagy initiation, but reactivated with prolonged starvation. mTOR reactivation is autophagy-dependent, and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell – a process we identify in multiple animal species. Thus, an evolutionarily-conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.
PMCID: PMC2920749  PMID: 20526321
20.  Autophagy in the Lung 
Autophagy is a cellular process for the disposal of damaged organelles or denatured proteins through a lysosomal degradation pathway. By reducing endogenous macromolecules to their basic components (i.e., amino acids, lipids), autophagy serves a homeostatic function by ensuring cell survival during starvation. Increased autophagy can be found in dying cells, although the relationships between autophagy and programmed cell death remain unclear. To date, few studies have examined the regulation and functional significance of autophagy in human lung disease. The lung, a complex organ that functions primarily in gas exchange, consists of diverse cell types (i.e., endothelial, epithelial, mesenchymal, inflammatory). In lung cells, autophagy may represent a general inducible adaptive response to injury resulting from exposure to stress agents, including hypoxia, oxidants, inflammation, ischemia–reperfusion, endoplasmic reticulum stress, pharmaceuticals, or inhaled xenobiotics (i.e., air pollution, cigarette smoke). In recent studies, we have observed increased autophagy in mouse lungs subjected to chronic cigarette smoke exposure, and in pulmonary epithelial cells exposed to cigarette smoke extract. Knockdown of autophagic proteins inhibited apoptosis in response to cigarette smoke exposure in vitro, suggesting that increased autophagy was associated with epithelial cell death. We have also observed increased morphological and biochemical markers of autophagy in human lung specimens from patients with chronic obstructive pulmonary disease (COPD). We hypothesize that increased autophagy contributes to COPD pathogenesis by promoting epithelial cell death. Further research will examine whether autophagy plays a homeostatic or maladaptive role in COPD and other human lung diseases.
PMCID: PMC3137145  PMID: 20160144
autophagy; apoptosis; pulmonary disease
21.  Beyond autophagy 
Autophagy  2008;4(6):817-820.
Autophagy is a lysosome-directed membrane trafficking event for the degradation of cytoplasmic components, including organelles. The past few years have seen a great advance in our understanding of the cellular machinery of autophagosome biogenesis, the hallmark of autophagy. However, our global understanding of autophagosome maturity remains relatively poor and fragmented. The topological similarity of autophagosome and endosome delivery to lysosomes suggests that autophagic and endosomal maturation may have evolved to share associated machinery to promote the lysosomal delivery of their cargoes. We have recently discovered that UVRAG, originally identified as a Beclin 1-binding autophagy protein, appears to be an important factor in autophagic and endosomal trafficking through its interaction with the class C Vps tethering complex. Given the ability of UVRAG to bind Beclin 1 and the class C Vps complex in a genetically and functionally separable manner, it may serve as an important regulator for the spatial and/or temporal control of diverse cellular trafficking events. As more non-autophagic functions of UVRAG are unveiled, our understanding of seemingly different cellular processes may move a step further.
PMCID: PMC2878715  PMID: 18612260
UVRAG; the class C Vps complex; autophagy; autophagosome maturation; membrane trafficking
22.  Using LC3 to Monitor Autophagy Flux in the Retinal Pigment Epithelium 
Autophagy  2009;5(8):1190-1193.
Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation.1,2 Autophagy begins with the formation of membranes arising in part from the endoplasmic reticulum, that elongate and fuse engulfing cytoplasmic constituents into a classic double-membrane bound nascent autophagosome. These early autophagosomes undergo a stepwise maturation process to form the late autophagosome or amphisome that ultimately fuses with a lysosome. Efficient autophagy is dependent on an equilibrium between the formation and elimination of autophagosomes; thus, a deficit in any part of this pathway will cause autophagic dysfunction. Autophagy plays a role in aging and age-related diseases. 1,2,7 However, few studies of autophagy in retinal disease have been reported.
Recent studies show that autophagy and changes in lysosomal activity are associated with both retinal aging and age-related macular degeneration (AMD).3,4 This article describes methods which employ the target protein LC3 to monitor autophagic flux in retinal pigment epithelial cells. During autophagy, the cytosolic form of LC3 (LC3-I) is processed and recruited to the phagophore where it undergoes site specific proteolysis and lipidation near the C terminus to form LC3-II.5 Monitoring the formation of cellular autophagosome puncta containing LC3 and measuring the ratio of LC3-II to LC3-I provides the ability to monitor autophagy flux in the retina.
PMCID: PMC3704326  PMID: 19855195
autophagy flux; LC3; retinal pigment epithelium; lysosomes; age-related macular degeneration; aging
23.  The molecular machinery of autophagy: unanswered questions 
Journal of cell science  2005;118(Pt 1):7-18.
Autophagy is a process in which cytosol and organelles are sequestered within double-membrane vesicles that deliver the contents to the lysosome/vacuole for degradation and recycling of the resulting macromolecules. It plays an important role in the cellular response to stress, is involved in various developmental pathways and functions in tumor suppression, resistance to pathogens and extension of lifespan. Conversely, autophagy may be associated with certain myopathies and neurodegenerative conditions. Substantial progress has been made in identifying the proteins required for autophagy and in understanding its molecular basis; however, many questions remain. For example, Tor is one of the key regulatory proteins at the induction step that controls the function of a complex including Atg1 kinase, but the target of Atg1 is not known. Although autophagy is generally considered to be nonspecific, there are specific types of autophagy that utilize receptor and adaptor proteins such as Atg11; however, the means by which Atg11 connects the cargo with the sequestering vesicle, the autophagosome, is not understood. Formation of the autophagosome is a complex process and neither the mechanism of vesicle formation nor the donor membrane origin is known. The final breakdown of the sequestered cargo relies on well-characterized lysosomal/vacuolar proteases; the roles of lipases, by contrast, have not been elucidated, and we do not know how the integrity of the lysosome/vacuole membrane is maintained during degradation.
PMCID: PMC1828869  PMID: 15615779
Lysosome; Pexophagy; Protein targeting; Vacuole; Yeast
24.  Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy 
PLoS Biology  2013;11(11):e1001708.
A novel Drosophila model system of chloroquine myopathy reveals how glycogen is targeted to the lysosome and what the significance of this process is for muscle cells.
Several myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8.
Author Summary
Lysosomes are organelles that work as a disposal system for the cell. It is known that lysosomes can degrade glycogen and that defects in this function trigger the accumulation of vesicles containing glycogen in animals that lead to vacuolar myopathies—diseases that result in muscle weakness. However, it remains unclear how and why glycogen is degraded through this system, and what significance it has for the pathology of such diseases. Here, we addressed these questions by establishing a fruitfly model system to study glycogen autophagy in skeletal muscles. By feeding the flies chloroquine (CQ), we induce a vacuolar myopathy associated with massive accumulation of glycogen-filled vesicles, and assay the role of autophagy and glycogen metabolic enzymes in this process. We show that CQ-induced glycogen autophagy is completely dependent on the core conserved autophagy genes and that this autophagy is triggered by nutrient deprivation in a Tor-dependent manner. Interestingly, while glycogen autophagy and enzymatic glycogen breakdown can compensate for each other, concurrent inhibition of both systems blocks glycogen breakdown. Finally, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of glycogen synthase—the main enzyme involved in converting glucose to glycogen—in regulating autophagy through its interaction with the autophagosome.
PMCID: PMC3825659  PMID: 24265594
25.  An Overview of the Molecular Mechanism of Autophagy 
Autophagy is a highly conserved cellular degradation process in which portions of cytosol and organelles are sequestered into a double-membrane vesicle, an autophagosome, and delivered into a degradative organelle, the vacuole/lysosome, for breakdown and eventual recycling of the resulting macromolecules. This process relieves the cell from various stress conditions. Autophagy plays a critical role during cellular development and differentiation, functions in tumor suppression, and may be linked to life span extension. Autophagy also has diverse roles in innate and adaptive immunity, such as resistance to pathogen invasion. Substantial progress has been made in the identification of many autophagy-related (ATG) genes that are essential to drive this cellular process, including both selective and nonselective types of autophagy. Identification of the ATG genes in yeast, and the finding of orthologs in other organisms, reveals the conservation of the autophagic machinery in all eukaryotes. Here, we summarize our current knowledge about the machinery and molecular mechanism of autophagy.
PMCID: PMC2832191  PMID: 19802558

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