Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.     

Human antigen R regulates hypoxia-induced mitophagy in renal tubular cells through PARKIN/BNIP3L expressions

Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we aimed to understand the role of human antigen R (HuR) in hypoxia-induced mitophagy process in the renal tubular cells. Mitophagy marker expressions (PARKIN, p-PARKIN, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. Immunofluorescence studies were performed to analyze mitophagosome, mitolysosome, co-localization of p-PARKIN/TOMM20 and BNIP3L/TOMM20. HuR-mediated regulation of PARKIN/BNIP3L expressions was determined by RNA-immunoprecipitation analysis and RNA stability experiments. Hypoxia induced mitochondrial dysfunction by increased ROS, decline in membrane potential and activated mitophagy through up-regulated PARKIN, PINK1, BNIP3 and BNIP3L expressions. HuR knockdown studies revealed that HuR regulates hypoxia-induced mitophagosome and mitolysosome formation. HuR was significantly bound to PARKIN and BNIP3L mRNA under hypoxia and thereby up-regulated their expressions through mRNA stability. Altogether, our data highlight the importance of HuR in mitophagy regulation through up-regulating PARKIN/BNIP3L expressions in renal tubular cells.     

Transiently expressed ATG16L1 inhibits autophagosome biogenesis and aberrantly targets RAB11-positive recycling endosomes

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12–ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1⁺ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.     

Oxidative stress impairs autophagy through oxidation of ATG3 and ATG7

Dysfunctional macroautophagy/autophagy has been causatively linked to aging and the pathogenesis of many diseases, which are also broadly characterized by dysregulated cellular redox. As the autophagy-related (ATG) conjugation systems that mediate autophagosome maturation are cysteine dependent, their oxidation may account for loss in this catabolic process under conditions of oxidative stress. During active autophagy, LC3 is transferred from the catalytic thiol of ATG7 to the active site thiol of ATG3, where it is conjugated to phosphatidylethanolamine. In our recent study, we show LC3 is bound to the catalytic thiols of inactive ATG3 and ATG7 through a stable thioester, which becomes transient upon autophagy stimulation. Transient interaction with LC3 exposes the catalytic thiols on ATG3 and ATG7, which under pro-oxidizing conditions undergo inhibitory oxidation. This process was found to be upregulated in aged mouse tissue and therefore may account, at least in part, for impaired autophagy observed during aging.     

Crosstalk between lysine methylation and phosphorylation of ATG16L1 dictates the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes

Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12–ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.     

Hsa_circ_0092276 promotes doxorubicin resistance in breast cancer cells by regulating autophagy via miR-348/ATG7 axis

Previous study has confirmed that hsa_circ_0092276 is highly expressed in doxorubicin (DOX)-resistant breast cancer cells, indicating that hsa_circ_0092276 may be involved in regulating the chemotherapy resistance of breast cancer. Here we attempted to investigate the biological role of hsa_circ_0092276 in breast cancer. We first constructed DOX-resistant breast cancer cells (MCF-7/DOX and MDA-MB-468/DOX). The 50% inhibiting concentration of MCF-7/DOX and MDA-MB-468/DOX cells was significantly higher than that of their parental breast cancer cells, MCF-7 and MDA-MB-46. MCF-7/DOX and MDA-MB-468/DOX cells also exhibited an up-regulation of drug resistance-related protein MDR1. Compared with MCF-7 and MDA-MB-46 cells, hsa_circ_0092276 was highly expressed in MCF-7/DOX and MDA-MB-468/DOX cells. Hsa_circ_0092276 overexpression enhanced proliferation and the expression of LC3-II/LC3-I and Beclin-1, and repressed apoptosis of breast cancer cells. The effect of hsa_circ_0092276 up-regulation on breast cancer cells was abolished by 3-methyladenine (autophagy inhibitor). Hsa_circ_0092276 modulated autophagy-related gene 7 (ATG7) expression via sponging miR-384. Hsa_circ_0092276 up-regulation promoted autophagy and proliferation, and repressed apoptosis of breast cancer cells, which was abolished by miR-384 overexpression or ATG7 knockdown. In addition, LV-circ_0092276 transfected MCF-7 cell transplantation promoted autophagy and tumor growth of breast cancer in mice. In conclusion, our data demonstrate that hsa_circ_0092276 promotes autophagy and DOX resistance in breast cancer by regulating miR-348/ATG7 axis. Thus, this article highlights a novel competing endogenous RNA circuitry involved in DOX resistance in breast cancer.     

SNX18 tubulates recycling endosomes for autophagosome biogenesis

The role of membrane remodeling and phosphoinositide-binding proteins in autophagy remains elusive. PX domain proteins bind phosphoinositides and participate in membrane remodeling and trafficking events and we therefore hypothesized that one or several PX domain proteins are involved in autophagy. Indeed, the PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation using an image-based siRNA screen. We show that SNX18 interacts with ATG16L1 and LC3, and functions downstream of ATG14 and the class III PtdIns3K complex in autophagosome formation. SNX18 facilitates recruitment of ATG16L1 to perinuclear recycling endosomes, and its overexpression leads to tubulation of ATG16L1- and LC3-positive membranes. We propose that SNX18 promotes LC3 lipidation and tubulation of recycling endosomes to provide membrane for phagophore expansion.     

SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin‐2

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.     

Structure of the WD40‐domain of human ATG16L1

Autophagy‐related protein ATG16L1 is a component of the mammalian ATG12～ATG5/ATG16L1 complex, which acts as E3‐ligase to catalyze lipidation of LC3 during autophagosome biogenesis. The N‐terminal part of ATG16L1 comprises the ATG5‐binding site and coiled‐coil dimerization domain, both also present in yeast ATG16 and essential for bulk and starvation induced autophagy. While absent in yeast ATG16, mammalian ATG16L1 further contains a predicted C‐terminal WD40‐domain, which has been shown to be involved in mediating interaction with diverse factors in the context of alternative functions of autophagy, such as inflammatory control and xenophagy. In this work, we provide detailed information on the domain boundaries of the WD40‐domain of human ATG16L1 and present its crystal structure at a resolution of 1.55 Å.     

The Ca channel TRPML3 specifically interacts with the mammalian ATG8 homologue GATE16 to regulate autophagy

TRPML3 is a Ca²⁺ permeable cation channel expressed in multiple intracellular compartments. Although TRPML3 is implicated in autophagy, how TRPML3 can regulate autophagy is not understood. To search interacting proteins with TRPML3 in autophagy, we performed split-ubiquitin membrane yeast two-hybrid (MY2H) screening with TRPML3-loop as a bait and identified GATE16, a mammalian ATG8 homologue. GST pull-down assay revealed that TRPML3 and TRPML3-loop specifically bind to GATE16, not to LC3B. Co-immunoprecipitation (co-IP) experiments showed that TRPML3 and TRPML3-loop pull down only the lipidated form of GATE16, indicating that the interaction occurs exclusively at the organellar membrane. The interaction of TRPML3 with GATE16 and GATE16-positive vesicle formation were increased in starvation induced autophagy, suggesting that the interaction facilitates the function of GATE16 in autophagosome formation. However, GATE16 was not required for TRPML3 trafficking to autophagosomes. Experiments using dominant-negative (DN) TRPML3(D458K) showed that GATE16 is localized not only in autophagosomes but also in extra-autophagosomal compartments, by contrast with LC3B. Since GATE16 acts at a later stage of the autophagosome biogenesis, our results suggest that TRPML3 plays a role in autophagosome maturation through the interaction with GATE16, by providing Ca²⁺ in the fusion process.     

Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors

Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.     

The WD40 domain of ATG16L1 is required for its non‐canonical role in lipidation of LC3 at single membranes

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double‐membrane phagophore, which is driven by the ATG16L1/ATG5‐ATG12 complex. In contrast, non‐canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3‐associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non‐canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat‐containing C‐terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non‐canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non‐canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD. Further, we demonstrate activation of non‐canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non‐canonical use of autophagy machinery.     

PFKP facilitates ATG4B phosphorylation during amino acid deprivation-induced autophagy

ATG4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of ATG4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how ATG4B phosphorylation is involved in amino acid deprivation-induced autophagy is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) and identified the ATG4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between ATG4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP loss reduced the degradation of LC3-II and p62 due to a partial block in autophagic flux. Furthermore, MS analysis of Flag-tagged ATG4B immunoprecipitates identified phosphorylation of ATG4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated ATG4B at S34. This phosphorylation could enhance ATG4B activity and p62 degradation. In addition, PFKP S386 phosphorylation was important to ATG4B S34 phosphorylation and autophagy in HEK293T cells. In brief, our findings describe that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for ATG4B to regulate ATG4B activity and autophagy under amino acid deprivation condition.     

Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers

Autophagy is an evolutionarily conserved degradation pathway characterized by dynamic rearrangement of membranes that sequester cytoplasm, protein aggregates, organelles, and pathogens for delivery to the vacuole and lysosome, respectively. The ability of autophagosomal membranes to act selectively toward specific cargo is dependent on the small ubiquitin-like modifier ATG8/LC3 and the LC3-interacting region (LIR) present in autophagy receptors. Here, we describe a comprehensive protein-protein interaction analysis of TBC (Tre2, Bub2, and Cdc16) domain-containing Rab GTPase-activating proteins (GAPs) as potential autophagy adaptors. We identified 14 TBC domain-containing Rab GAPs that bind directly to ATG8 modifiers and that colocalize with LC3-positive autophagy membranes in cells. Intriguingly, one of our screening hits, TBC1D5, contains two LIR motifs. The N-terminal LIR was critical for interaction with the retromer complex and transport of cargo. Direct binding of the retromer component VPS29 to TBC1D5 could be titrated out by LC3, indicating a molecular switch between endosomes and autophagy. Moreover, TBC1D5 could bridge the endosome and autophagosome via its C-terminal LIR motif. During starvation-induced autophagy, TBC1D5 was relocalized from endosomal localization to the LC3-positive autophagosomes. We propose that LC3-interacting Rab GAPs are implicated in the reprogramming of the endocytic trafficking events under starvation-induced autophagy.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p     

WIPI2B links PtdIns3P to LC3 lipidation through binding ATG16L1

WIPI proteins, phosphatidylinositol 3-phosphate (PtdIns3P) binding proteins with β-propeller folds, are recruited to the omegasome following PtdIns3P production. The functions of the WIPI proteins in autophagosome formation are poorly understood. In a recent study, we reported that WIPI2B directly binds ATG16L1 and functions by recruiting the ATG12–ATG5-ATG16L1 complex to forming autophagosomes during starvation- or pathogen-induced autophagy. Our model of WIPI2 function provides an explanation for the PtdIns3P-dependent recruitment of the ATG12–ATG5-ATG16L1 complex during initiation of autophagy.     

Swapping of interaction partners with ATG5 for autophagosome maturation

Autophagy is a tightly regulated lysosome-mediated catabolic process in eukaryotes that maintains cellular homeostasis. A distinguishable feature of autophagy is the formation of double-membrane structures, autophagosome, which envelopes the intracellular cargoes and finally degrades them by fusion with lysosomes. So far, many structures of Atg proteins working on the autophagosome formation have been reported, however those involved in autophagosome maturation, a fusion with lysosome, are relatively unknown. One of the molecules in autophagosome maturation, TECPR1, has been identified and recently, structural studies on both ATG5-TECPR1 and ATG5-ATG16L1 complexes revealed that TECPR1 and ATG16L1 share the same binding site on ATG5. These results, in combination with supporting biochemical and cellular biological data, provide an insight into a model for swapping ATG5 partners for autophagosome maturation. [BMB Reports 2015; 48(3): 129-130]