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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. A living cell must remove unhealthy or excess material from its interior in order to remain healthy and operational. Cells pack this waste into membrane-bound compartments named autophagosomes in a process called autophagy. So-called autophagy proteins make sure that only the unwanted material is eliminated. However, it was not completely clear how these proteins achieve this. What was known was that proteins called cargo receptors recognize and bind to specific waste materials. At the same time, so-called autophagy enzymes tag the membranes of the autophagosome with a protein known as Atg8, so that cargo receptor molecules can bind this membrane. Now, Fracchiolla, Sawa-Makarska et al. report that, in yeast, an autophagy enzyme links these two events by binding to the cargo receptor and promoting the tagging of the autophagosome’s membrane at the same place. The enzyme in question is a complex made from three autophagy proteins (called Atg12, Atg5 and Atg16), and its activity ensures that the membrane is tagged only next to those materials that need to be disposed of. Although it is now clearer how a cell’s waste ends up in the autophagosome, it is still puzzling how this process is regulated and how the other autophagy-related components contribute to this highly coordinated process. In particular, an important next step will be to find out what is the source of membrane that gives rise to the autophagosome.

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.

Macroautophagy deploys a wealth of autophagy-related proteins to synthesize the double-membrane autophagosome, in order to engulf cytosolic components for lysosome-dependent degradation. The recruitment of the ATG12~ATG5-ATG16L1 complex by WIPI family proteins is a crucial step in autophagosome formation. Nevertheless, the molecular mechanism by which WIPI3 facilitates the recruitment of the ATG12~ATG5-ATG16L1 complex remains largely unknown. Here, we uncover that WIPI3 can directly interact with the coiled-coil domain of ATG16L1. By determining the crystal structure of WIPI3 in complex with ATG16L1 coiled-coil, we elucidate the molecular basis underpinning the specific recruitment of the ATG12~ATG5-ATG16L1 complex by WIPI3. Moreover, we demonstrate that WIPI2 and WIPI3 are competitive for interacting with ATG16L1 coiled-coil, and ATG16L1 and ATG2 are mutually exclusive in binding to WIPI3. In all, our findings provide mechanistic insights into the WIPI3/ATG16L1 interaction, and are valuable for further understanding the activation mechanism of the ATG12~ATG5-ATG16L1 complex as well as the working mode of WIPI3 in autophagy.

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.

ATG5 and ATG7 are considered to be essential molecules for the induction of autophagy. However, we found that cells lacking ATG5 or ATG7 can still form autophagosomes/autolysosomes and perform autophagic protein degradation when subjected to certain types of stress. Although the lipidation of LC3 is accepted as a good indicator of autophagy, this did not occur during ATG5/ATG7-independent alternative autophagy. Unlike conventional autophagy, autophagosomes appeared to be generated in a Rab9-dependent manner by the fusion of the phagophores with vesicles derived from the trans-Golgi and late endosomes. Therefore, mammalian autophagy can occur via at least two different pathways; the ATG5/ATG7-dependent conventional pathway and an ATG5/ATG7-independent alternative pathway.

Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)—a master transcriptional regulator of lysosomal biogenesis and autophagy—is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.Nakamura et al. find that the master transcriptional regulator of lysosomal biogenesis and autophagy TFEB is activated following LC3 lipidation during lysosomal damage and show the importance of this mechanism during kidney injury.

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane‐bound vesicles termed autophagosomes. The conserved Atg5–Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5–Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5–Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre‐autophagosomal structure but is essential for autophagy and cytoplasm‐to‐vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5–Atg12/Atg16 complex during autophagosome formation. The conserved Atg5–Atg12/Atg16 complex is essential for autophagosome formation. In vitro reconstitution experiments reveal how the individual subunits of this complex act to tether membranes and to promote subsequent Atg8 conjugation, a key step in autophagy.

Autophagy is a catabolic mechanism to degrade cellular components to maintain cellular energy levels during starvation, a condition where PPARα may be activated. Here we report a reduced autophagic capacity in the liver following chronic activation of PPARα with fenofibrate (FB) in mice. Chronic administration of the PPARα agonist FB substantially reduced the levels of multiple autophagy proteins in the liver (Atg3, Agt4B, Atg5, Atg7 and beclin 1) which were associated with a decrease in the light chain LC3II/LC3I ratio and the accumulation of p62. This was concomitant with an increase in the expression of lipogenic proteins mSREBP1c, ACC, FAS and SCD1. These effects of FB were completely abolished in PPARα-/- mice but remained intact in mice with global deletion of FGF21, a key downstream mediator for PPARα-induced effects. Further studies showed that decreased the content of autophagy proteins by FB was associated with a significant reduction in the level of FoxO1, a transcriptional regulator of autophagic proteins, which occurred independently of both mTOR and Akt. These findings suggest that chronic stimulation of PPARα may suppress the autophagy capacity in the liver as a result of reduced content of a number of autophagy-associated proteins independent of FGF21.

Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34 + AML cells with a large variability in basal autophagy between AMLs observed. The autophagy flux was higher in AMLs classified as poor risk, which are frequently associated with TP53 mutations (TP53 mut ), compared with favorable- and intermediate-risk AMLs. In addition, the higher flux was associated with a higher expression level of several autophagy genes, but was not affected by alterations in p53 expression by knocking down p53 or overexpression of wild-type p53 or p53 R273H . AML CD34 + cells were more sensitive to the autophagy inhibitor hydroxychloroquine (HCQ) than normal bone marrow CD34 + cells. Similar, inhibition of autophagy by knockdown of ATG5 or ATG7 triggered apoptosis, which coincided with increased expression of p53. In contrast to wild-type p53 AML (TP53 wt ), HCQ treatment did not trigger a BAX and PUMA-dependent apoptotic response in AMLs harboring TP53 mut . To further characterize autophagy in the leukemic stem cell-enriched cell fraction AML CD34 + cells were separated into ROS low and ROS high subfractions. The immature AML CD34 + -enriched ROS low cells maintained higher basal autophagy and showed reduced survival upon HCQ treatment compared with ROS high cells. Finally, knockdown of ATG5 inhibits in vivo maintenance of AML CD34 + cells in NSG mice. These results indicate that targeting autophagy might provide new therapeutic options for treatment of AML since it affects the immature AML subfraction.

Despite significant advances in the treatment of human immunodeficiency virus type-1 (HIV) infection, antiretroviral therapy only suppresses viral replication but is unable to eliminate infection. Thus, discontinuation of antiretrovirals results in viral reactivation and disease progression. A major reservoir of HIV latent infection resides in resting central memory CD4 + T cells (T CM ) that escape clearance by current therapeutic regimens and will require novel strategies for elimination. Here, we evaluated the therapeutic potential of autophagy-inducing peptides, Tat-Beclin 1 and Tat-vFLIP-α2, which can induce a novel Na + /K + -ATPase dependent form of cell death (autosis), to kill latently HIV-infected T CM while preventing virologic rebound. In this study, we encapsulated autophagy inducing peptides into biodegradable lipid-coated hybrid PLGA (poly lactic-co-glycolic acid) nanoparticles for controlled intracellular delivery. A single dose of nanopeptides was found to eliminate latent HIV infection in an in vitro primary model of HIV latency and ex vivo using resting CD4 + T cells obtained from peripheral blood mononuclear cells of HIV-infected patients on antiretroviral with fully suppressed virus for greater than 12 months. Notably, increased LC3B lipidation, SQSTM1/p62 degradation and Na + /K + -ATPase activity characteristic of autosis, were detected in nanopeptide treated latently HIV-infected cells compared to untreated uninfected or infected cells. Nanopeptide-induced cell death could be reversed by knockdown of autophagy proteins, ATG5 and ATG7, and inhibition or knockdown of Na + /K + -ATPase. Importantly, viral rebound was not detected following the induction of the Na + /K + -ATPase dependent form of cell death induced by the Tat-Beclin 1 and Tat-vFLIP-α2 nanopeptides. These findings provide a novel strategy to eradicate HIV latently infected resting memory CD4 + T cells, the major reservoir of HIV latency, through the induction of Na + /K + -ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.