LC3B-II deacetylation by histone deacetylase 6 is involved in serum-starvation-induced autophagic degradation

Autophagy is a conserved mechanism for controlling the degradation of misfolded proteins and damaged organelles in eukaryotes and can be induced by nutrient withdrawal, including serum starvation. Although differential acetylation of autophagy-related proteins has been reported to be involved in autophagic flux, the regulation of acetylated microtubule-associated protein 1 light chain 3 (LC3) is incompletely understood. In this study, we found that the acetylation levels of phosphotidylethanolamine (PE)-conjugated LC3B (LC3B-II), which is a critical component of double-membrane autophagosome, were profoundly decreased in HeLa cells upon autophagy induction by serum starvation. Pretreatment with lysosomal inhibitor chloroquine did not attenuate such deacetylation. Under normal culture medium, we observed increased levels of acetylated LC3B-II in cells treated with tubacin, a specific inhibitor of histone deacetylase 6 (HDAC6). However, tubacin only partially suppressed serum-starvation-induced LC3B-II deacetylation, suggesting that HDAC6 is not the only deacetylase acting on LC3B-II during serum-starvation-induced autophagy. Interestingly, tubacin-induced increase in LC3B-II acetylation was associated with p62/SQSTM1 accumulation upon serum starvation. HDAC6 knockdown did not influence autophagosome formation but resulted in impaired degradation of p62/SQSTM1 during serum starvation. Collectively, our data indicated that LC3B-II deacetylation, which was partly mediated by HDAC6, is involved in autophagic degradation during serum starvation.     

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.     

Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment

Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the host's autophagy machinery is activated during HBV infection to enhance HBV replication.     

The late stage of autophagy: cellular events and molecular regulation

Autophagy is an intracellular degradation system that delivers cytoplasmic contents to the lysosome for degradation. It is a “self-eating” process and plays a “house-cleaner” role in cells. The complex process consists of several sequential steps—induction, autophagosome formation, fusion of lysosome and autophagosome, degradation, efflux transportation of degradation products, and autophagic lysosome reformation. In this review, the cellular and molecular regulations of late stage of autophagy, including cellular events after fusion step, are summarized.     

HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin

CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous polyglutamine aggregates via a process that appears to involve capture and degradation of aggregates by autophagy. The ubiquitin-proteasome system protects cells against proteotoxicity by degrading soluble monomeric misfolded aggregation-prone proteins but is ineffective against, and impaired by, non-native protein oligomers. Here we show that autophagy is induced in response to impaired ubiquitin proteasome system activity. We show that ATG proteins, molecular determinants of autophagic vacuole formation, and lysosomes are recruited to pericentriolar cytoplasmic inclusion bodies by a process requiring an intact microtubule cytoskeleton and the cytoplasmic deacetylase HDAC6. These data suggest that HDAC6-dependent retrograde transport on microtubules is used by cells to increase the efficiency and selectivity of autophagic degradation.     

Inhibition of histone deacetylase1 induces autophagy

Autophagy is a process where cytoplasmic materials are degraded by lysosomal machinery. Histone deacetylase (HDAC) inhibitors induce autophagy, and HDAC6, one of class II HDAC isotypes, is directly involved in autophagic degradation in the cell. However, it is unclear if class I HDAC isotype such as HDCA1 is involved in this process. To investigate if class I HDAC isotype is involved in autophagy, a specific class I HDAC inhibitor and an siRNA of HDAC1 were used to treat HeLa cells. Autophagic markers were then investigated. Both inhibition and genetic knock-down of HDAC1 in the cells significantly induced autophagic vacuole formation and lysosome function. Moreover, disruption of HDAC1 leads to the conversion of LC3-I to LC3-II. Together, these results demonstrate that HDAC1 could play a role in autophagy and specific inhibition of HDAC1 can induce autophagy.     

A reporter cell system to monitor autophagy based on p62/SQSTM1

Macroautophagy (hereafter referred to as autophagy) is a catabolic pathway to isolate and transport cytosolic components to the lysosome for degradation. Recently, autophagy receptors, like p62/SQSTM1 and NBR1, which physically link autophagic cargo to ATG8/MAP1-LC3/GABARAP family members located on the forming autophagic membranes, have been identified. To identify conditions or compounds that affect autophagy cell systems that efficiently report on autophagic flux are required. Here we describe reporter cell systems based on induced expression of GFP-p62, GFP-NBR1 or GFP-LC3B. The degradation of the fusion proteins was followed after promoter shut off by flow cytometry of live cells. All three fusion proteins were degraded at a basal rate by autophagy. Surprisingly, the basal degradation rate varied for the three reporter fusion proteins. GFP-LC3B was the most stable protein. GFP-NBR1 was most efficiently degraded under basal conditions while degradation of GFP-p62 displayed the strongest response to amino acid starvation. GFP-p62 was found to perform best of the tested reporters. Single cell analysis of autophagic flux by flow cytometry allows estimates of heterogeneous cell populations. The feasibility of this approach was demonstrated using transient overexpression of a dominant negative ULK1 kinase and siRNA-mediated knock-down of LC3B to inhibit autophagic degradation of GFP-p62. The inducible GFP-p62 cell system allows quantification by several approaches and will be useful in screening for compounds or conditions that affect the rate of autophagy. Inducers of autophagy can be identified using rich medium whereas inhibitors are identified under starvation conditions.     

Autophagosome–lysosome fusion in neurons requires INPP5E,a protein associated with Joubert syndrome

Autophagy is a multistep membrane traffic pathway. In contrast to autophagosome formation, the mechanisms underlying autophagosome–lysosome fusion remain largely unknown. Here, we describe a novel autophagy regulator, inositol polyphosphate‐5‐phosphatase E (INPP5E), involved in autophagosome–lysosome fusion process. In neuronal cells, INPP5E knockdown strongly inhibited autophagy by impairing the fusion step. A fraction of INPP5E is localized to lysosomes, and its membrane anchoring and enzymatic activity are necessary for autophagy. INPP5E decreases lysosomal phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P₂), one of the substrates of the phosphatase, that counteracts cortactin‐mediated actin filament stabilization on lysosomes. Lysosomes require actin filaments on their surface for fusing with autophagosomes. INPP5E is one of the genes responsible for Joubert syndrome, a rare brain abnormality, and mutations found in patients with this disease caused defects in autophagy. Taken together, our data reveal a novel role of phosphoinositide on lysosomes and an association between autophagy and neuronal disease.     

Selective removal of dishevelled by autophagy: A role of p62

Autophagy has long been viewed as a process to remove long-lived or misfolded protein aggregates and aging and damaged organelles. Our study identified a previously unknown function of autophagy in suppression of Wnt signaling. A signaling protein, Dishevelled (Dvl), can be degraded via the autophagy pathway upon starvation. In this selective degradation, p62/sequestosome-1 binds to ubiquitinated Dvl proteins and promotes Dvl aggregation. Intriguingly, LC3 can also directly interact with Dvl. The studies on the mechanism for autophagic clearance of Dishevelled led to several interesting findings.     

The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes

PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C.?elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function?causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of?protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for?omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides?a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.     

Absence of TDP‐43 is difficult to digest

It is well established that TDP‐43 accumulates in degenerating neurons in patients with ALS/FTLD, which might affect normal TDP‐43 function. In this issue of The EMBO Journal Xia et al ( 2016 ) show a novel connection between TDP‐43 loss of function and autophagy failure. Using knockdown models of TDP‐43, they observed enhanced autophagosome and lysosome biogenesis through mTORC1 activity inhibition and TFEB activation. Impaired autophagosome–lysosome fusion was also observed, however in an mTORC1‐independent manner. The data identify dysfunctions at multiple stages of the autophagic pathway following TDP‐43 depletion that might represent possible targets of future therapeutic interventions.     

Connexin 31.1 degradation requires the Clathrin‐mediated autophagy in NSCLC cell H1299

Connexins have relative short half‐lives. Connexin 31.1 (Cx31.1) was newly reported to be down‐regulated in non‐small cell lung cancer cell lines, and displayed tumour‐suppressive properties. However, no reports describing how a cell regulates Cx31.1 level were found. In this study, Cx31.1 was suggested to be degraded through both ubiquitin–proteasome system (UPS) and autophagy. Blockage of UPS with MG‐132 increased Cx31.1 level, but could not inhibit the degradation of Cx31.1 completely. In H1299 cells stably expressing Cx31.1, Cx31.1 reduced when autophagy was induced through starvation or Brefeldin A treatment. Knockdown of autophagy‐related protein ATG5 could increase the cellular level of Cx31.1 both under normal growth condition and starvation‐induced autophagy. Colocalization of Cx31.1 and autophagy marker light chain 3 (LC3) was revealed by immunofluorescence analysis. Coimmunoprecipitation and immunofluorescence showed that Cx31.1 might interact with clathrin heavy chain which was newly reported to regulate autophagic lysosome reformation (ALR) and controls lysosome homoeostasis. When clathrin expression was knockdown by siRNA treatment, the level of Cx31.1 increased prominently both under normal growth condition and starvation‐induced autophagy. Under starvation‐induced autophagy, LC3‐II levels were slightly accumulated with siCla. treatment compared to that of siNC, which could be ascribed to that clathrin knockdown impaired the late stage of autophagy, ALR. Taken together, we found autophagy contributed to Cx31.1 degradation, and clathrin might be involved in the autophagy of Cx31.1.     

Dysregulation of autophagy by HIV-1 Nef in human astrocytes

Viruses often exploit autophagy, a common cellular process of degradation of damaged proteins, organelles, and pathogens, to avoid destruction. HIV-1 dysregulates this process in several cell types by means of Nef protein. Nef is a small HIV-1 protein which is expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HIV-Associated Neurocognitive Disorders (HAND). In order to explore its effect in the CNS with respect to autophagy, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFA) using an adenovirus vector (Ad-Nef). We observed that Nef expression triggered the accumulation of autophagy markers, ATG8/LC3 and p62 (SQSMT1). Similar results were obtained with Bafilomycin A1, an autophagy inhibitor which blocks the fusion of autophagosome to lysosome. Furthermore co-expression of tandem LC3 vector (mRFP-EGFP-LC3) and Ad-Nef in these cells produced mainly yellow puncta (mRFP+, EGFP+) strongly suggesting that autophagosome fusion to lysosome is blocked in PHFA cells in the presence of Nef. Together these data indicate that HIV-1 Nef mimics Bafilomycin A1 and blocks the last step of autophagy thereby helping HIV-1 virus to avoid autophagic degradation in human astrocytes.     

Getting ready for building: signaling and autophagosome biogenesis

Autophagy is the main cellular catabolic process responsible for degrading organelles and large protein aggregates. It is initiated by the formation of a unique membrane structure, the phagophore, which engulfs part of the cytoplasm and forms a double‐membrane vesicle termed the autophagosome. Fusion of the outer autophagosomal membrane with the lysosome and degradation of the inner membrane contents complete the process. The extent of autophagy must be tightly regulated to avoid destruction of proteins and organelles essential for cell survival. Autophagic activity is thus regulated by external and internal cues, which initiate the formation of well‐defined autophagy‐related protein complexes that mediate autophagosome formation and selective cargo recruitment into these organelles. Autophagosome formation and the signaling pathways that regulate it have recently attracted substantial attention. In this review, we analyze the different signaling pathways that regulate autophagy and discuss recent progress in our understanding of autophagosome biogenesis.     

Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.     

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.     

GFP‐LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP‐LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP‐LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP‐Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin‐positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP‐LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP‐LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP‐LC3, a fusion protein widely used as a marker for autophagic vesicles and pre‐autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley‐Liss, Inc.     

A unique hairpin-type tail-anchored SNARE starts to solve a long-time puzzle

Macroautophagy mediates recycling of intracellular material by a multistep pathway, ultimately leading to the fusion of closed double-membrane structures, called autophagosomes, with the lysosome. This event ensures the degradation of the autophagosome content by lysosomal proteases followed by the release of macromolecules by permeases and, thus, it accomplishes the purpose of macroautophagy (hereafter referred to as autophagy). Because fusion of unclosed autophagosomes (i.e., phagophores) with the lysosome would fail to degrade the autophagic cargo, this critical step has to be tightly controlled. Yet, until recently, little was known about the regulation of this event and the factors orchestrating it. A punctum in this issue highlights the recent paper by Noboru Mizushima and his collaborators that answered the question of how premature fusion of phagophores with the lysosome is prevented prior to completion of autophagosome closure.     

Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus

Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogs A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication-deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualize autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of infectious bronchitis virus (IBV). IBV induced autophagic signaling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV nonstructural protein 6 alone did induce autophagic signaling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.     

NBR1 co-operates with p62 in selective autophagy of ubiquitinated targets

Selective degradation of intracellular targets, such as misfolded proteins and damaged organelles, is an important homeostatic function that autophagy has acquired in addition to its more general role in restoring the nutrient balance during stress and starvation. Although the exact mechanism underlying selection of autophagic substrates is not known, ubiquitination is a candidate signal for autophagic degradation of misfolded and aggregated proteins. p62/SQSTM1 was the first protein shown to bind both target-associated ubiquitin (Ub) and LC3 conjugated to the phagophore membrane, thereby effectively acting as an autophagic receptor for ubiquitinated targets. Importantly, p62 not only mediates selective degradation but also promotes aggregation of ubiquitinated proteins that can be harmful in some cell types. Is p62 the only autophagic receptor for selective autophagy? Looking for proteins that interact with ATG8 family proteins, we identified NBR1 (neighbor of BRCA1 gene 1) as an additional LC3- and Ub-binding protein. NBR1 is degraded by autophagy depending on its LC3-interacting region (LIR) but does not strictly require p62 for this process. Like p62, NBR1 accumulates and aggregates when autophagy is inhibited and is a part of pathological inclusions. We propose that NBR1 together with p62 promotes autophagic degradation of ubiquitinated targets and simultaneously regulates their aggregation when autophagy becomes limited.