Individual Atg8 paralogs exhibit unique properties in streptococcus pneumoniae-induced hierarchical autophagy.

Individual Atg8 (autophagy related 8) paralogs, comprising MAP1LC3A/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2/GATE16, play a crucial role in canonical macroautophagy/autophagy. However, their functions remain unclear owing to functional redundancy. In a previous study, we reported that intracellular Streptococcus pneumoniae triggers hierarchical autophagy in response to bacterial infection. This process commences with the induction of conjugation of Atg8 paralogs (Atg8s) to single membranes (CASM), followed by CASM shedding and subsequent induction of xenophagy. In our recent study, we performed functional analysis of Atg8s during pneumococci-induced hierarchical autophagy. Our findings suggest that LC3A and GABARAPL1 are crucial for CASM induction, whereas GABARAPL2 and GABARAP play sequential roles in CASM shedding and subsequent induction of xenophagy, respectively.

An APEX2-based proximity-dependent biotinylation assay with temporal specificity to study protein interactions during autophagy in the yeast Saccharomyces cerevisiae.

Autophagosome biogenesis is a complex process orchestrated by dynamic interactions between Atg (autophagy-related) proteins and characterized by the turnover of specific cargoes, which can differ over time and depending on how autophagy is stimulated. Proteomic analyses are central to uncover protein-protein interaction networks and when combined with proximity-dependent biotinylation or proximity labeling (PL) approaches, they also permit to detect transient and weak interactions. However, current PL procedures for yeast Saccharomyces cerevisiae, one of the leading models for the study of autophagy, do not allow to keep temporal specificity and thus identify interactions and cargoes at a precise time point upon autophagy induction. Here, we present a new ascorbate peroxidase 2 (APEX2)-based PL protocol adapted to yeast that preserves temporal specificity and allows uncovering neighbor proteins by either western blot or proteomics. As a proof of concept, we applied this new method to identify Atg8 and Atg9 interactors and detected known binding partners as well as potential uncharacterized ones in rich and nitrogen starvation conditions. Also, as a proof of concept, we confirmed the spatial proximity interaction between Atg8 and Faa1. We believe that this protocol will be a new important experimental tool for all those researchers studying the mechanism and roles of autophagy in yeast, but also other cellular pathways in this model organism.Abbreviations: APEX2, ascorbate peroxidase 2, Atg, autophagy-related; BP, biotin phenol; Cvt, cytoplasm-to-vacuole targeting; ER, endoplasmic reticulum; LN2, liquid nitrogen; MS, mass spectrometry; PAS, phagophore assembly site; PL, proximity labeling; PE, phosphatidylethanolamine; PPINs, protein-protein interaction networks; PPIs, protein-protein interactions; RT, room temperature; SARs, selective autophagy receptors; WT, wild-type.

Computational and Experimental Approaches Towards Understanding the Role of ATG8 in Autophagy: A Therapeutic Paradigm in Leishmaniasis.

In the realm of parasitology, autophagy has emerged as a critical focal point, particularly in combating Leishmaniasis. Central to this endeavour is the recognition of the protein ATG8 as pivotal for the survival and infectivity of the parasitic organism Leishmania major, thereby making it a potential target for therapeutic intervention. Consequently, there is a pressing need to delve into the structural characteristics of ATG8 to facilitate the design of effective drugs. In this study, our efforts centered on the purification of ATG8 from Leishmania major, which enabled novel insights into its structural features through meticulous spectroscopic analysis. We aimed to comprehensively assess the stability and behaviour of ATG8 in the presence of various denaturants, including urea, guanidinium chloride, and SDS-based chemicals. Methodically, our approach included secondary structural analysis utilizing CD spectroscopy, which not only validated but also augmented computationally predicted structures of ATG8 reported in previous investigations. Remarkably, our findings unveiled that the purified ATG8 protein retained its folded conformation, exhibiting the anticipated secondary structure. Moreover, our exploration extended to the influence of lipids on ATG8 stability, yielding intriguing revelations. We uncovered a nuanced perspective suggesting that targeting both the lipid composition of Leishmania major and ATG8 could offer a promising strategy for future therapeutic approaches in combating leishmaniasis. Collectively, our study underscores the importance of understanding the structural intricacies of ATG8 in driving advancements towards the development of targeted therapies against Leishmaniasis, thereby providing a foundation for future investigations in this field.

Comprehensive analysis of non-selective and selective autophagy in yeast atg mutants and characterization of autophagic activity in the absence of the Atg8 conjugation system.

Most autophagy-related genes, or ATG genes, have been identified in studies using budding yeast. Although the functions of the ATG genes are well understood, the contributions of individual genes to non-selective and various types of selective autophagy remain to be fully elucidated. In this study, we quantified the activity of non-selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy, endoplasmic reticulum (ER)-phagy, and pexophagy in all Saccharomyces cerevisiae atg mutants. Among the mutants of the core autophagy genes considered essential for autophagy, the atg13 mutant and mutants of the genes involved in the two ubiquitin-like conjugation systems retained residual autophagic functionality. In particular, mutants of the Atg8 ubiquitin-like conjugation system (the Atg8 system) exhibited substantial levels of non-selective autophagy, the Cvt pathway, and pexophagy, although mitophagy and ER-phagy were undetectable. Atg8-system mutants also displayed intravacuolar vesicles resembling autophagic bodies, albeit at significantly reduced size and frequency. Thus, our data suggest that membranous sequestration and vacuolar delivery of autophagic cargo can occur in the absence of the Atg8 system. Alongside these findings, the comprehensive analysis conducted here provides valuable datasets for future autophagy research.

DDHD2 promotes lipid droplet catabolism by acting as a TAG lipase and a cargo receptor for lipophagy.

Mutations in the DDHD2 (DDHD domain containing 2) gene cause autosomal recessive spastic paraplegia type 54 (SPG54), a rare neurodegenerative disorder characterized by the early childhood onset of progressive spastic paraplegia. DDHD2 is reported as the principal brain triacylglycerol (TAG) lipase whose dysfunction causes massive lipid droplet (LD) accumulation in the brains of SPG54 patients. However, the precise functions of DDHD2 in regulating LD catabolism are not yet fully understood. In a recent study, we demonstrate that DDHD2 interacts with multiple members of the Atg8-family proteins (MAP1LC3/LC3s, GABARAPs), which play crucial roles in lipophagy. DDHD2 possesses two LC3-interacting region (LIR) motifs that contribute to its LD-eliminating activity. Moreover, DDHD2 enhances the colocalization between LC3B and LDs to promote lipophagy. LD·ATTEC, a compound that tethers LC3 to LDs to enhance their macroautophagic/autophagic clearance, effectively counteracts DDHD2 deficiency-induced LD accumulation. These findings provide insights into the dual functions of DDHD2 as a TAG lipase and cargo receptor for lipophagy in neuronal LD catabolism, and also suggest a potential therapeutic approach for treating SPG54 patients.

Last but not least: emerging roles of the autophagy-related protein ATG4D.

The evolutionarily conserved ATG4 cysteine proteases regulate macroautophagy/autophagy through the priming and deconjugation of the Atg8-family proteins. In mammals there are four ATG4 family members (ATG4A, ATG4B, ATG4C, ATG4D) but ATG4D has been relatively understudied. Heightened interest in ATG4D has been stimulated by recent links to human disease. Notably, genetic variations in human ATG4D were implicated in a heritable neurodevelopmental disorder. Genetic analyses in dogs, along with loss-of-function zebrafish and mouse models, further support a neuroprotective role for ATG4D. Here we discuss the evidence connecting ATG4D to neurological diseases and other pathologies and summarize its roles in both autophagy-dependent and autophagy-independent cellular processes.Abbrevation: ATG: autophagy related; BafA1: bafilomycin A1; BCL2: BCL2 apoptosis regulator; BH3: BCL2 homology region 3; CASP3: caspase 3; EV: extracellular vesicle; GABA: gamma aminobutyric acid; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; LIR: LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MYC: MYC proto-oncogene, bHLH transcription factor; PE: phosphatidylethanolamine; PS: phosphatidylserine; QKO: quadruple knockout; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel; SQSTM1: sequestosome 1.

The Autophagy-Related Protein ATG8 Orchestrates Asexual Development and AFB1 Biosynthesis in Aspergillus flavus.

Autophagy, a conserved cellular recycling process, plays a crucial role in maintaining homeostasis under stress conditions. It also regulates the development and virulence of numerous filamentous fungi. In this study, we investigated the specific function of ATG8, a reliable autophagic marker, in the opportunistic pathogen Aspergillus flavus. To investigate the role of atg8 in A. flavus, the deletion and complemented mutants of atg8 were generated according to the homologous recombination principle. Deletion of atg8 showed a significant decrease in conidiation, spore germination, and sclerotia formation compared to the WT and atg8C strains. Additionally, aflatoxin production was found severely impaired in the ∆atg8 mutant. The stress assays demonstrated that ATG8 was important for A. flavus response to oxidative stress. The fluorescence microscopy showed increased levels of reactive oxygen species in the ∆atg8 mutant cells, and the transcriptional result also indicated that genes related to the antioxidant system were significantly reduced in the ∆atg8 mutant. We further found that ATG8 participated in regulating the pathogenicity of A. flavus on crop seeds. These results revealed the biological role of ATG8 in A. flavus, which might provide a potential target for the control of A. flavus and AFB1 biosynthesis.

Vesicle-vesicle fusion promoted by the Atg8-family autophagy protein LC3C: relevance of the N-terminal region.

Among the MAP1LC3/LC3 subfamily of Atg8 proteins, LC3B and LC3C constitute the most and least studied members, respectively, LC3B being generally considered as an autophagosomal marker and a canonical representative of the LC3 subfamily. In several recent studies, LC3C has emerged as an important modulator in various processes of cell homeostasis. Our own research data demonstrate that LC3C induces higher levels of tethering and of intervesicular lipid mixing than LC3B. LC3C contains a peculiar N-terminal region, different from the other Atg8-family protein members. Using a series of mutants, we have shown that the N-terminal region of LC3C is responsible for the enhanced vesicle tethering, membrane perturbation and vesicle-vesicle fusion activities of LC3C as compared to LC3B.Abbreviations: ATG: autophagy related; GABARAP: gamma-aminobutyric acid receptor associated protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PC: phosphatidyl choline; PE: phosphatidylethanolamine; PEmal: maleimide-derivatized PE; PtdIns: phosphatidylinositol.

Upregulation of neuronal ER-phagy improves organismal fitness and alleviates APP toxicity.

ER-phagy, a selective autophagy targeting the endoplasmic reticulum (ER) for lysosomal degradation through cargo receptors, plays a critical role in ER quality control and is linked to various diseases. However, its physiological and pathological roles remain largely unclear due to a lack of animal model studies. This study establishes Drosophila as an in vivo ER-phagy model. Starvation triggers ER-phagy across multiple fly tissues. Disturbing ER-phagy by either globally upregulating or downregulating ER-phagy receptors, Atl or Rtnl1, harms the fly. Notably, moderate upregulation of ER-phagy in fly brains by overexpressing Atl or Rtnl1 significantly attenuates age-associated neurodegenerations. Furthermore, in a Drosophila model of Alzheimer's disease expressing human amyloid precursor protein (APP), impaired ER-phagy is observed. Enhancing ER-phagy in the APP-expressing fly brain facilitates APP degradation, significantly alleviating disease symptoms. Therefore, our findings suggest that modulating ER-phagy may offer a therapeutic strategy to treat aging and diseases associated with ER protein aggregation.

The function of the ATG8 in the cilia and cortical microtubule maintenance of Euplotes amieti.

Autophagy regulates the formation of primary cilia, which in turn affects autophagy. The relationship between autophagy and cilia is known to be bidirectional although the specific mechanisms involved have yet to be elucidated. In this study, we found for the first time that ATG8 protein localizes in the basal body of the dorsal kineties and the base of the ventral cirri in Euplotes amieti. ATG8 protein maintains the structural integrity of cilia and plays a role in the construction of the cortical ciliature and microtubule cytoskeleton associated with cilia. ATG8 gene interference leads to the degradation of IFT88, the transport protein in cilia, thus inhibiting the generation of cilia, and affecting the swing of cilia. This influences the swimming speed and cilia pattern, leading to death in Euplotes amieti.

When an underdog becomes a major player: the role of protein structural disorder in the Atg8 conjugation system.

The noncanonical ubiquitin-like conjugation cascade involving the E1 (Atg7), E2 (Atg3, Atg10), and E3 (Atg12-Atg5-Atg16 complex) enzymes is essential for incorporation of Atg8 into the growing phagophore via covalent linkage to PE. This process is an indispensable step in autophagy. Atg8 and E1-E3 enzymes are the first subset from the core autophagy protein machinery structures that were investigated in earlier studies by crystallographic analyses of globular domains. However, research over the past decade shows that many important functions in the conjugation machinery are mediated by intrinsically disordered protein regions (IDPRs) - parts of the protein that do not adopt a stable secondary or tertiary structure, which are inherently dynamic and well suited for protein-membrane interactions but are invisible in protein crystals. Here, we summarize earlier and recent findings on the autophagy conjugation machinery by focusing on the IDPRs. This summary reveals that IDPRs, originally considered dispensable, are in fact major players and a driving force in the function of the autophagy conjugation system. Abbreviation: AD, activation domain of Atg7; AH, amphipathic helix; AIM, Atg8-family interacting motif; CL, catalytic loop (of Atg7); CTD, C-terminal domain; FR, flexible region (of Atg3 or Atg10); GUV, giant unilammelar vesicles; HR, handle region (of Atg3); IDPR, intrinsically disordered protein region; IDPs: intrinsically disordered proteins; LIR, LC3-interacting region; NHD: N-terminal helical domain; NMR, nuclear magnetic resonance; PE, phosphatidylethanolamine; UBL, ubiquitin like.

Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

Receptor-mediated cargo hitchhiking on bulk autophagy.

While the molecular mechanism of autophagy is well studied, the cargoes delivered by autophagy remain incompletely characterized. To examine the selectivity of autophagy cargo, we conducted proteomics on isolated yeast autophagic bodies, which are intermediate structures in the autophagy process. We identify a protein, Hab1, that is highly preferentially delivered to vacuoles. The N-terminal 42 amino acid region of Hab1 contains an amphipathic helix and an Atg8-family interacting motif, both of which are necessary and sufficient for the preferential delivery of Hab1 by autophagy. We find that fusion of this region with a cytosolic protein results in preferential delivery of this protein to the vacuole. Furthermore, attachment of this region to an organelle allows for autophagic delivery in a manner independent of canonical autophagy receptor or scaffold proteins. We propose a novel mode of selective autophagy in which a receptor, in this case Hab1, binds directly to forming isolation membranes during bulk autophagy.

The two autophagy-related proteins 8a and 8b play distinct physiological roles in Drosophila.

Atg8 family proteins play crucial roles in autophagy to maintain cellular homeostasis. However, the physiological roles of Atg8 family proteins have not been systematically determined. In this study, we generated Atg8a and Atg8b (homologs of Atg8 in Drosophila melanogaster) knockout flies. We found that the loss of Atg8a affected autophagy and resulted in partial lethality, abnormal wings, decreased lifespan, and decreased climbing ability in flies. Furthermore, the loss of Atg8a resulted in reduced muscle integrity and the progressive degeneration of the neuron system. We also found that the phosphorylation at Ser88 of Atg8a is important for autophagy and neuronal integrity. The loss of Atg8b did not affect autophagy but induced male sterility in flies. Here, we take full advantage of the fly system to elucidate the physiological function of Atg8a and Atg8b in Drosophila.

A RabGAP negatively regulates plant autophagy and immune trafficking.

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as a key membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL reduces autophagic flux in three model plant species, suggesting that its negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4+ T lymphocytes.

HIV-1 entry into CD4+ T lymphocytes relies on the viral and cellular membranes' fusion, leading to viral capsid delivery in the target cell cytoplasm. Atg8/LC3B conjugation to lipids, process named Atg8ylation mainly studied in the context of macroautophagy/autophagy, occurs transiently in the early stages of HIV-1 replication in CD4+ T lymphocytes. Despite numerous studies investigating the HIV-1-autophagy interplays, the Atg8ylation impact in these early stages of infection remains unknown. Here we found that HIV-1 exposure leads to the rapid LC3B enrichment toward the target cell plasma membrane, in close proximity with the incoming viral particles. Furthermore, we demonstrated that Atg8ylation is a key event facilitating HIV-1 entry in target CD4+ T cells. Interestingly, this effect is independent of canonical autophagy as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical step of its replication cycle.Abbreviations: BafA1: bafilomycin A1; BlaM: beta-lactamase; CD4+ TL: CD4+ T lymphocytes; PtdIns3K-BECN1 complex: BECN1-containing class III phosphatidylinositol 3-kinase complex; Env: HIV-1 envelope glycoproteins; HIV-1: type 1 human immunodeficiency virus; PM: plasma membrane; PtdIns3P: phosphatidylinositol-3-phosphate; VLP: virus-like particle.

Individual Atg8 paralogs and a bacterial metabolite sequentially promote hierarchical CASM-xenophagy induction and transition.

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H2O2.

MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrial outer membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.

CASM mediates LRRK2 recruitment and activation under lysosomal stress.

Conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments has attracted attention as the non-autophagic function of the Atg8-family protein conjugation system, and the V-ATPase-ATG16L1 axis has emerged as a core mechanism. Our recent research has revealed that this mechanism contributes to the lysosomal recruitment and activation of LRRK2, a Parkinson disease-associated kinase that phosphorylates a subset of RAB GTPases. The activated LRRK2 under CASM-causing lysosomal stress acts to regulate lysosomal morphology and stimulate extracellular secretion of lysosomal contents, thereby promoting the lysosomal stress response.

A novel workflow for unbiased quantification of autophagosomes in 3D in Arabidopsis thaliana roots.

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most likely does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume correlated with the intensity of autophagy induction. Compared to the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers.