Ral GTPase and the exocyst regulate autophagy in a tissue‐specific manner

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress‐induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation‐induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context‐dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch.     

The cyclin-dependent kinase PITSLRE/CDK11 is required for successful autophagy

(Macro)autophagy is a membrane-trafficking process that serves to sequester cellular constituents in organelles termed autophagosomes, which target their degradation in the lysosome. Autophagy operates at basal levels in all cells where it serves as a homeostatic mechanism to maintain cellular integrity. The levels and cargoes of autophagy can, however, change in response to a variety of stimuli, and perturbations in autophagy are known to be involved in the aetiology of various human diseases. Autophagy must therefore be tightly controlled. We report here that the Drosophila cyclin-dependent kinase PITSLRE is a modulator of autophagy. Loss of the human PITSLRE orthologue, CDK11, initially appears to induce autophagy, but at later time points CDK11 is critically required for autophagic flux and cargo digestion. Since PITSLRE/CDK11 regulates autophagy in both Drosophila and human cells, this kinase represents a novel phylogenetically conserved component of the autophagy machinery.     

HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.     

The cyclin-dependent kinase PITSLRE/CDK11 is required for successful autophagy

(Macro)autophagy is a membrane-trafficking process that serves to sequester cellular constituents in organelles termed autophagosomes, which target their degradation in the lysosome. Autophagy operates at basal levels in all cells where it serves as a homeostatic mechanism to maintain cellular integrity. The levels and cargoes of autophagy can, however, change in response to a variety of stimuli, and perturbations in autophagy are known to be involved in the aetiology of various human diseases. Autophagy must therefore be tightly controlled. We report here that the Drosophila cyclin-dependent kinase PITSLRE is a modulator of autophagy. Loss of the human PITSLRE orthologue, CDK11, initially appears to induce autophagy, but at later time points CDK11 is critically required for autophagic flux and cargo digestion. Since PITSLRE/CDK11 regulates autophagy in both Drosophila and human cells, this kinase represents a novel phylogenetically conserved component of the autophagy machinery.     

Autophagy and the ubiquitin-proteasome system: collaborators in neuroprotection

Protein degradation is an essential cellular function that, when dysregulated or impaired, can lead to a wide variety of disease states. The two major intracellular protein degradation systems are the ubiquitin-proteasome system (UPS) and autophagy, a catabolic process that involves delivery of cellular components to the lysosome for degradation. While the UPS has garnered much attention as it relates to neurodegenerative disease, important links between autophagy and neurodegeneration have also become evident. Furthermore, recent studies have revealed interaction between the UPS and autophagy, suggesting a coordinated and complementary relationship between these degradation systems that becomes critical in times of cellular stress. Here we describe autophagy and review evidence implicating this system as an important player in the pathogenesis of neurodegenerative disease. We discuss the role of autophagy in neurodegeneration and review its neuroprotective functions as revealed by experimental manipulation in disease models. Finally, we explore potential parallels and connections between autophagy and the UPS, highlighting their collaborative roles in protecting against neurodegenerative disease.     

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Autophagy is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. It involves the rearrangement of subcellular membranes to sequester cargo for delivery to the lysosome where the sequestered material is degraded and recycled. For many decades, it has been known that autophagy occurs in a wide range of eukaryotic organisms and in multiple different cell types during starvation, cellular and tissue remodeling, and cell death. However, until recently, the functions of autophagy in normal development were largely unknown. The identification of a set of evolutionarily conserved genes that are essential for autophagy has opened up new frontiers for deciphering the role of autophagy in diverse biological processes. In this review, we summarize our current knowledge about the molecular machinery of autophagy and the role of the autophagic machinery in eukaryotic development.     

Inhibitive effects of Photofrin on cellular autophagy

Depending on the circumstances, autophagy can be either a protective or damaging cellular process. The role of autophagy in photodynamic therapy (PDT), a photo‐chemotherapy that utilizes light to activate a photosensitizer drug to achieve localized cellular damage, has been explored in recent years. It has been reported that autophagy in PDT is significantly influenced by the treatment protocol. In this work, the role of Photofrin, a well‐established clinical photosensitizer, in regulating cellular autophagy was investigated. The effects of Photofrin on cellular autophagy induced by conventional starvation or rapamycin techniques were studied. By fluorescence imaging, Western blotting and cell viability assays, it was found that Photofrin can effectively inhibit cellular autophagy induced by starvation or rapamycin. This autophagy blocking is independent of the photosensitizing property of the drug. With Baf‐A1, a well‐established agent that inhibits autophagosome from fusing with lysosome, we also found that, the observed phenomenon is not due to accelerated degradation of existing autophagosomes, thus proving that the drug Photofrin alone, without light excitation, can truly block autophagy. J. Cell. Physiol. 224: 414–422, 2010. © 2010 Wiley‐Liss, Inc.     

Autophagy in protists

Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles, and defense against parasitic invaders. During the last 10-20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.     

Autophagy-dependent survival is controlled with a unique regulatory network upon various cellular stress events

Although autophagy is a type of programmed cell death, it is also essential for cell survival upon tolerable level of various stress events. For the cell to respond adequately to an external and/or internal stimulus induced by cellular stress, autophagy must be controlled in a highly regulated manner. By using systems biology techniques, here we explore the dynamical features of autophagy induction. We propose that the switch-like characteristic of autophagy induction is achieved by a control network, containing essential feedback loops of four components, so-called autophagy inducer, autophagy controller, mTORC1 and autophagy executor, respectively. We show how an autophagy inducer is capable to turn on autophagy in a cellular stress-specific way. The autophagy controller acts as a molecular switch and not only promotes autophagy but also blocks the permanent hyperactivation of the process via downregulating the autophagy inducer. In this theoretical analysis, we explore in detail the properties of all four proposed controlling elements and their connections. Here we also prove that the kinetic features of this control network can be considered accurate in various stress processes (such as starvation, endoplasmic reticulum stress and oxidative stress), even if the exact components may be different. The robust response of the resulting control network is essential during cellular stress.Subject terms: Biochemistry, Molecular biology     

Autophagy in protists

Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles, and defense against parasitic invaders. During the last 10-20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.     

Autophagy process is associated with anti-neoplastic function

Autophagy is a highly conserved process of cellular degradation, which is present in yeast, plants, and mammals. Under normal physiological conditions, autophagy acts to maintain cellular homeostasis and regulate the turnover of organelles. In response to cellular stresses, autophagy prevents the accumulation of impaired proteins and organelles, which serves to inhibit carcinogenesis. On this basis, it is widely accepted that most tumor suppressors, such as beclin 1 associated proteins, forkhead box class O (FoxO) family proteins, multiple mammalian target of Rapamycin (mTOR) inactivators, and nuclear p53 play a role in inducing autophagy. Here, we focus on how the process of autophagy is associated with anti-neoplastic function.     

PKD at the crossroads of necrosis and autophagy

Reactive oxygen species (ROS) that accumulate under oxidative pressure cause severe damage to cellular components, and induce various cellular responses, including apoptosis, programmed necrosis and autophagy, depending on the cellular setting. Various studies have described ROS-induced autophagy, but only a few direct factors that regulate autophagy under oxidative stress are known to date. We have identified DAPK and PKD as such regulators by demonstrating their role in the process of autophagy in general, and specifically during oxidative stress. PKD acts as a downstream effector of DAPk in the regulation of autophagy. Furthermore, PKD functions within the autophagic network as an activator of VPS34, by associating with and phosphorylating VPS34, leading to its activation. Significantly, PKD is recruited to the autophagosomal membranes, placing it within proximity of its autophagic target.     

PKD at the crossroads of necrosis and autophagy

Reactive oxygen species (ROS) that accumulate under oxidative pressure cause severe damage to cellular components, and induce various cellular responses, including apoptosis, programmed necrosis and autophagy, depending on the cellular setting. Various studies have described ROS-induced autophagy, but only a few direct factors that regulate autophagy under oxidative stress are known to date. We have identified DAPK and PKD as such regulators by demonstrating their role in the process of autophagy in general, and specifically during oxidative stress. PKD acts as a downstream effector of DAPk in the regulation of autophagy. Furthermore, PKD functions within the autophagic network as an activator of VPS34, by associating with and phosphorylating VPS34, leading to its activation. Significantly, PKD is recruited to the autophagosomal membranes, placing it within proximity of its autophagic target.     

Sustained Autophagy Contributes to Measles Virus Infectivity

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.     

Autophagy and senescence: A new insight in selected human diseases

Senescence and autophagy play important roles in homeostasis. Cellular senescence and autophagy commonly cause several degenerative processes, including oxidative stress, DNA damage, telomere shortening, and oncogenic stress; hence, both events are known to be interrelated. Autophagy is well known for its disruptive effect on human diseases, and it is currently proposed to have a direct effect on triggering senescence and quiescence. However, it is yet to be proven whether autophagy has a positive or negative impact on senescence. It is known that elevated levels of autophagy induce cell death, whereas inadequate autophagy can trigger cellular senescence. Both have important roles in human diseases such as aging, renal degeneration, neurodegenerative disorders, and cancer. Therefore, this review aims to highlight the relevance of senescence and autophagy in selected human ailments through a summary of recent findings on the connection and effects of autophagy and senescence in these diseases.     

Bnip3-mediated defects in oxidative phosphorylation promote mitophagy

The Bcl-2 proteins are best known as regulators of the intrinsic mitochondrial pathway of apoptosis. However, recent studies have demonstrated that they can also regulate autophagy. For many years, autophagy was considered to be a nonselective process where the autophagosomes randomly sequestered contents in the cytosol to supply the cells with amino acids and fatty acids during nutrient deprivation. However, it is now clear that autophagy is important for cellular homeostasis under normal conditions, and that it can be a selective process where specific protein aggregates or organelles, such as mitochondria, are targeted for removal by the autophagosomes. Removal of damaged mitochondria is essential for cellular survival, and defects in this process lead to accumulation of dysfunctional mitochondria and cell death. However, the molecular mechanism underlying the selective removal of mitochondria in cells is still poorly understood. A recent study from our laboratory demonstrates that the BH3-only protein Bnip3 is a specific activator of mitochondrial autophagy (mitophagy) and that this process is independent of its role in apoptotic signaling. Here, we discuss how Bnip3-mediated impairment of mitochondrial oxidative phosphorylation facilitates mitochondrial turnover via autophagy in the absence of permeabilization of the mitochondrial membrane and apoptosis.     

WNK1 is an unexpected autophagy inhibitor

Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions.