Discovery of Small-Molecule Autophagy Inhibitors by Disrupting the Protein-Protein Interactions Involving Autophagy-Related 5.

One possible strategy for modulating autophagy is to disrupt the critical protein-protein interactions (PPIs) formed during this process. Our attention is on the autophagy-related 12 (ATG12)-autophagy-related 5 (ATG5)-autophagy-related 16-like 1 (ATG16L1) heterotrimer complex, which is responsible for ATG8 translocation from ATG3 to phosphatidylethanolamine. In this work, we discovered a compound with an (E)-3-(2-furanylmethylene)-2-pyrrolidinone core moiety (T1742) that blocked the ATG5-ATG16L1 and ATG5-TECAIR interactions in the in vitro binding assay (IC50 = 1-2 µM) and also exhibited autophagy inhibition in cellular assays. The possible binding mode of T1742 to ATG5 was predicted through molecular modeling, and a batch of derivatives sharing essentially the same core moiety were synthesized and tested. The outcomes of the in vitro binding assay and the flow cytometry assay of those newly synthesized compounds were generally consistent. This work has validated our central hypothesis that small-molecule inhibitors of the PPIs involving ATG5 can tune down autophagy effectively, and their pharmaceutical potential may be further explored.

Atg5 Regulates Selective Autophagy of the Parental Macronucleus during Tetrahymena Sexual Reproduction.

Nuclear autophagy is an important selective autophagy process. The selective autophagy of sexual development micronuclei (MICs) and the programmed nuclear degradation of parental macronucleus (paMAC) occur during sexual reproduction in Tetrahymena thermophila. The molecular regulatory mechanism of nuclear selective autophagy is unclear. In this study, the autophagy-related protein Atg5 was identified from T. thermophila. Atg5 was localized in the cytoplasm in the early sexual-development stage and was localized in the paMAC in the late sexual-development stage. During this stage, the degradation of meiotic products of MIC was delayed in atg5i mutants. Furthermore, paMAC was abnormally enlarged and delayed or failed to degrade. The expression level and lipidation of Atg8.2 significantly decreased in the mutants. All these results indicated that Atg5 was involved in the regulation of the selective autophagy of paMAC by regulating Atg8.2 in Tetrahymena.

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast.

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12-Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12-Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113-131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64-99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.

Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.

Structural Studies of Mammalian Autophagy Lipidation Complex.

Members of the autophagy-related protein 8 (Atg8) family of ubiquitin-like proteins (ublps), including mammalian LC3 and GABARAP proteins, play crucial roles in autophagosome biogenesis, as well as selective autophagy. Upon induction of autophagy, the autophagic ublps are covalently attached to a phosphatidylethanolamine (PE) molecule of the autophagosomal membrane. This unique lipid conjugation of the autophagic ublps, which is essential for their functions, occurs in a ubiquitination-like reaction cascade consisting of the E1 enzyme ATG7, the E2 ATG3, and the E3 ATG12~ATG5-ATG16L1 complex (~denotes a covalent linkage). These enzymes are structurally unique among those of the canonical ubiquitination cascades, necessitating structural and biochemical studies of these molecules for understanding the molecular mechanisms underlying the lipidation cascade. Here, we will describe methods that were employed in our previous studies (Otomo et al., Nat Struct Mol Biol 20:59-66, 2013; Metlagel et al., Proc Natl Acad Sci U S A 110:18844-18849, 2013; Ohashi and Otomo, Biochem Biophys Res Commun 463:447-452, 2015), including the production of recombinant enzymes, in vitro enzymatic reactions, the crystallization of the E3 complexes, and the NMR-based investigations of E1-E2 and E2-E3 interactions.

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.

CERKL regulates autophagy via the NAD-dependent deacetylase SIRT1.

Macroautophagy/autophagy is an important intracellular mechanism for the maintenance of cellular homeostasis. Here we show that the CERKL (ceramide kinase like) gene, a retinal degeneration (RD) pathogenic gene, plays a critical role in regulating autophagy by stabilizing SIRT1. In vitro and in vivo, suppressing CERKL results in impaired autophagy. SIRT1 is one of the main regulators of acetylation/deacetylation in autophagy. In CERKL-depleted retinas and cells, SIRT1 is downregulated. ATG5 and ATG7, 2 essential components of autophagy, show a higher degree of acetylation in CERKL-depleted cells. Overexpression of SIRT1 rescues autophagy in CERKL-depleted cells, whereas CERKL loses its function of regulating autophagy in SIRT1-depleted cells, and overexpression of CERKL upregulates SIRT1. Finally, we show that CERKL directly interacts with SIRT1, and may regulate its phosphorylation at Ser27 to stabilize SIRT1. These results show that CERKL is an important regulator of autophagy and it plays this role by stabilizing the deacetylase SIRT1.

Exploring the Role of Autophagy-Related Gene 5 (ATG5) Yields Important Insights Into Autophagy in Autoimmune/Autoinflammatory Diseases.

Autophagy is a highly conserved process that degrades certain intracellular contents in both physiological and pathological conditions. Autophagy-related proteins (ATG) are key players in this pathway, among which ATG5 is indispensable in both canonical and non-canonical autophagy. Recent studies demonstrate that ATG5 modulates the immune system and crosstalks with apoptosis. However, our knowledge of the pathogenesis and regulatory mechanisms of autophagy in various immune related diseases is lacking. Thus, a deeper understanding of ATG5's role in the autophagy mechanism may shed light on the link between autophagy and the immune response, and lead to the development of new therapies for autoimmune diseases and autoinflammatory diseases. In this focused review, we discuss the latest insights into the role of ATG5 in autoimmunity. Although these studies are at a relatively early stage, ATG5 may eventually come to be regarded as a "guardian of immune integrity." Notably, accumulating evidence indicates that other ATG genes may have similar functions.

Exosomes from MiR-30d-5p-ADSCs Reverse Acute Ischemic Stroke-Induced, Autophagy-Mediated Brain Injury by Promoting M2 Microglial/Macrophage Polarization.

BACKGROUND/AIMS: Recent studies have indicated that exosomes secreted from adipose-derived stem cells (ADSCs) have important effects in the treatment of ischemic injury. However, the treatment mechanism is unclear. This study aimed to investigate whether ADSC-derived exosomes enriched with microRNA (miR)-30d-5p have a protective effect on acute ischemic stroke (AIS). METHODS: In the current study, inflammatory factors and miR-30d-5p expression were assessed in 70 subjects with AIS and 35 healthy controls. Exosomes were characterized by transmission electron microscopy and further examined using nanoparticle tracking analyses. A rat model of AIS and an in vitro model of oxygen- and glucose-deprived (OGD) primary microglia were established to study the protective mechanism of exosomes from miR-30d-5p-overexpressing ADSCs in ischemia-induced nerve injury. RESULTS: The results showed that following AIS, the expression of inflammatory cytokines increased, while the anti-inflammatory cytokines IL-4, IL-10, and miR-30d-5p decreased both in patients and in animal models. Moreover, in vitro studies demonstrated that suppression of autophagy significantly reduced the OGD-induced inflammatory response. In addition, exosome treatment was more effective in suppressing the inflammatory response by reversing OGD-induced and autophagy-mediated microglial polarization to M1. Furthermore, in vivo studies showed that exosomes derived from ADSCs significantly decreased the cerebral injury area of infarction by suppressing autophagy and promoting M2 microglia/macrophage polarization. CONCLUSIONS: Our results suggest that miR-30d-5p-enhanced ADSC-derived exosomes prevent cerebral injury by inhibiting autophagy-mediated microglial polarization to M1.

MiR-181a mediates Ang II-induced myocardial hypertrophy by mediating autophagy.

OBJECTIVE: To investigate the relationship between miR-181a and cardiac hypertrophy and autophagy in rats with myocardial hypertrophy, and whether miR-181a regulates the autophagy through ATG5, thereby participating in the occurrence and development of myocardial hypertrophy. MATERIALS AND METHODS: The rat model of myocardial hypertrophy was established via the abdominal aortic coarctation. The expression of miR-181a in cardiac tissues was detected via reverse transcription-polymerase chain reaction (RT-PCR). The expressions of autophagy-related proteins, ATG5 and LC3II/LC3I, in cardiac tissues, were detected via Western blotting (WB). After the primary culture of myocardial cells in rats, they were stimulated via Angiotensin II (Ang II) to observe the effects of autophagy inhibitor 3-methyladenine (3-MA) and overexpression of ATG5 on the expression of hypertrophic genes in myocardial cells, respectively. The expressions of autophagy-related proteins ATG5 and LC3II/LC3I were detected via WB, the autophagic rate was observed via flow cytometry and the changes in autophagic vacuoles of myocardial cells were observed using the transmission electron microscope. The changes in mRNA and protein expressions of ATG in myocardial cells were observed after the overexpression of miR-181a or the inhibition of miR-181a activity. The changes in miR-181a and the expression of hypertrophic genes in myocardial cells after Ang II stimulation were observed via RT-PCR. RESULTS: In rats with myocardial hypertrophy, the cardiac autophagy was increased and the expression of miR-181a in hypertrophic myocardium was downregulated. 3-MA inhibited the ATG5-induced autophagy and improved the Ang II-induced myocardial hypertrophy, while the overexpression of ATG5 enhanced the myocardial autophagy and the expression of hypertrophic genes. MiR-181a regulated the ATG5-induced myocardial autophagy, and its downregulation mediated the Ang II-induced myocardial hypertrophy. CONCLUSIONS: The enhancement of ATG5-induced myocardial autophagy mediates the Ang II-induced myocardial hypertrophy. ATG5 is the target gene of miR-181a, it can regulate the myocardial autophagy via ATG5, thus mediating the Ang II-induced myocardial hypertrophy.

Identification and characterization of the autophagy-related genes Atg12 and Atg5 in hydra.

Autophagy is an evolutionarily conserved process in eukaryotic cells that is involved in the degradation of cytoplasmic contents including organelles via the lysosome. Hydra is an early metazoan which exhibits simple tissue grade organization, a primitive nervous system, and is one of the classical non-bilaterian models extensively used in evo-devo research. Here, we describe the characterization of two core autophagy genes, Atg12 and Atg5, from hydra. In silico analyses including sequence similarity, domain analysis, and phylogenetic analysis demonstrate the conservation of these genes across eukaryotes. The predicted 3D structure of hydra Atg12 showed very little variance when compared to human Atg12 and yeast Atg12, whereas the hydra Atg5 predicted 3D structure was found to be variable, when compared with its human and yeast homologs. Strikingly, whole mount in situ hybridization showed high expression of Atg12 transcripts specifically in nematoblasts, whereas Atg5 transcripts were found to be expressed strongly in budding region and growing buds. This study may provide a framework to understand the evolution of autophagy networks in higher eukaryotes.

Crystallographic Characterization of ATG Proteins and Their Interacting Partners.

Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid phosphatidylethanolamine. Atg8 lipidation is mediated by distinctive UBL cascades involving autophagy-specific E1, E2, and E3 enzymes that differ substantially in sequence from components of other UBL conjugation cascades. Structural studies are important for elucidating the roles of Atg proteins that regulate multiple steps involved in autophagy. This chapter describes methods to prepare and crystallize selected proteins and complexes involved in autophagy UBL conjugation pathways, as a guide for strategies for structural and biochemical characterization of Atg proteins.

Mechanism of cargo-directed Atg8 conjugation during selective autophagy.

Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.

MOLECULAR CHARACTERIZATION OF AUTOPHAGY-RELATED GENE 5 FROM Spodoptera exigua AND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS.

Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry-SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl-HP cells and partially co-localized with BmATG6-GFP, but it almost has no significant co-localization with GFP-HaATG8. Then, the Western blot analysis demonstrated that GFP-SeATG5 conjugated with ATG12. Moreover, real-time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.

Promotion of Proapoptotic Signals by Lysosomal Photodamage: Mechanistic Aspects and Influence of Autophagy.

Prior studies demonstrated that a low level (LD10-15 ) of lysosomal photodamage can sensitize cells to the apoptotic death that results from subsequent mitochondrial photodamage. We have proposed that this process occurs via a calpain-catalyzed cleavage of the autophagy-associated protein ATG5 to form a proapoptotic fragment. In this report, we provide evidence for the postulated ATG5 cleavage and show that the sequential photodynamic therapy (PDT) protocol can also partly overcome the adverse effect of hypoxia on the initiation of apoptosis. While autophagy can offer cytoprotection after mitochondrial photodamage, this does not appear to apply when lysosomes are the target. This may account for the ability of very low PDT doses directed at lysosomes to evoke ATG5 cleavage. The resulting proapoptotic effect overcomes intrinsic cytoprotection from mitochondrial photodamage along with a further stimulation of phototoxicity.