HSBP1 Is a Novel Interactor of FIP200 and ATG13 That Promotes Autophagy Initiation and Picornavirus Replication

ATG13 and FIP200 are two subunits of the ULK kinase complex, a key regulatory component of the autophagy machinery. We have previously found that the FIP200-ATG13 subcomplex controls picornavirus replication outside its role in the ULK kinase complex and autophagy. Here, we characterized HSBP1, a very small cytoplasmic coiled-coil protein, as a novel interactor of FIP200 and ATG13 that binds these two proteins via FIP200. HSBP1 is a novel pro-picornaviral host factor since its knockdown or knockout, inhibits the replication of various picornaviruses. The anti-picornaviral function of the FIP200-ATG13 subcomplex was abolished when HSBP1 was depleted, inferring that this subcomplex negatively regulates HSBP1’s pro-picornaviral function during infections. HSBP1depletion also reduces the stability of ULK kinase complex subunits, resulting in an impairment in autophagy induction. Altogether, our data show that HSBP1 interaction with FIP200-ATG13-containing complexes is involved in the regulation of different cellular pathways

­­Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy

During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9ʹs role as a lipid scramblase.Abbreviations: BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.</p

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.Peer reviewe

Sorting the trash:Micronucleophagy gets selective

During micronucleophagy, the nucleolus is targeted by autophagic degradation, but although nucleolar proteins are recycled, ribosomal DNA is spared. Mostofa et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706164) reveal that the separation of these two nucleolar components is mediated by the CLIP and cohibin complexes and is vital for cell survival during starvation

Probing aggrephagy using chemically-induced protein aggregates

Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce and monitor such pathways are limited. Here we introduce the PIM (particles induced by multimerization) assay as a tool for the study of aggrephagy, the autophagic clearance of aggregates. The assay uses an inducible multimerization module to assemble protein clusters, which upon induction recruit ubiquitin, p62, and LC3 before being delivered to lysosomes. Moreover, use of a dual fluorescent tag allows for the direct observation of cluster delivery to the lysosome. Using flow cytometry and fluorescence microscopy, we show that delivery to the lysosome is partially dependent on p62 and ATG7. This assay will help in elucidating the spatiotemporal dynamics and control mechanisms underlying aggregate clearance by the autophagy-lysosomal system

Rôle des récepteurs autophagiques dans la maturation des autophagosomes

Xenophagy relies on the ability of the autophagy process to selectively entrap intracellular pathogens within autophagosomes to degrade them into autolysosomes. The selectivity of the process relies on proteins named autophagy receptors that share the ability to recognise cytosolic cargos on one hand and autophagosome-bound members of the ATG8 family on the other. Among autophagy receptors NDP52 has been described to target Salmonella Typhimurium to the growing autophagosome. We describe a new unexpected role for NDP52, as this receptor also regulates the maturation of Salmonella-containing autophagosomes and during ongoing autophagy. Interestingly, the role of NDP52 in maturation is independent from its role in targeting as they rely on different binding domains and protein partners. We also show that other autophagy receptors also mediate autophagosome maturation such as Optineurin. Therefore, our work shows that NDP52 plays a dual function during xenophagy first by targeting bacteria to growing autophagosomes and then by assuring autophagosome maturation. Moreover, we also provide insights as to how these dual roles are regulated by post-translational modifications of autophagy receptors.This work demonstrates that autophagy receptors have other roles beyond pathogen targeting that are also crucial for an efficient xenophagy. Moreover, autophagy receptors are also necessary for autophagy completion in uninfected cells. These results strengthen our understanding of both ongoing autophagy and xenophagy molecular mechanismsLa xénophagie est une forme d'autophagie sélective permettant de capture des pathogènes dans les autophagosomes et de les dégrader dans les autolysosomes. Cette sélectivité est assurée par une famille de protéines ; les récepteurs autophagiques qui reconnaissent des substrats cytosoliques d'un côté et les membres de la famille LC3 ancrés dans la membrane de l'autophagosome de l'autre. Parmi ces récepteurs, NDP52 cible la bactérie Salmonella Typhimurium vers l'autophagie.Nous décrivons un rôle nouveau et inattendu pour NDP52 ; assurer la maturation d'autophagosomes durant l'infection par Salmonella mais aussi durant l'autophagie basale. De manière intéressante, ce rôle de NDP52 dans la maturation est indépendant de son rôle dans le ciblage de la bactérie puisque ces fonctions nécessitent des domaines et des partenaires moléculaires de NDP52 distincts. Nous montrons aussi que d'autres récepteurs peuvent participer à la maturation comme Optineurine. Ce travail montre donc que NDP52 assure deux rôles durant la xénophagie en ciblant les bactéries vers les autophagosomes en formation puis en promouvant la maturation de l'autophagosome. De plus, nous proposons aussi un possible mécanisme de régulation de ces deux fonctions par des modifications post-traductionnelles des récepteurs autophagiques.Ce travail démontre que les récepteurs autophagiques jouent des rôles au-delà du ciblage des pathogènes qui sont aussi cruciaux pour une xénophagie efficace. De plus, les récepteurs autophagiques sont aussi nécessaires pour le déroulement de l'autophagie basale. Ces travaux offrent une nouvelle compréhension de la régulation moléculaire de l'autophagie et de la xénophagi

Handcuffs for bacteria - NDP52 orchestrates xenophagy of intracellular Salmonella

International audienceEukaryotic cells can selectively target and degrade intracellular pathogens using autophagy, a process referred to as xenophagy. This selectivity is controlled by proteins called autophagy receptors, which can recognise pathogens and address them to the autophagy machinery. Among them, NDP52 can recognise Salmonella Typhimurium on the one hand and the ATG8 family member LC3C on the other hand, thus allowing the docking of the bacteria to a growing autophagosome. Additionally, we recently reported that NDP52 is involved in the maturation of the bacteria-containing autophagosome and hence necessary for the ultimate degradation of the bacteria. These two functions of NDP52 are independent as they rely on distinct binding domains and protein partners. Therefore, NDP52 plays a dual role during xenophagy, first by targeting the bacteria to the autophagy machinery and then by regulating its degradation

Dual function of CALCOCO2/NDP52 during xenophagy

International audienceDuring xenophagy, pathogens are selectively targeted by autophagy receptors to the autophagy machinery for their subsequent degradation. In infected cells, the autophagy receptor CALCOCO2/NDP52 targets Salmonella Typhimurium to the phagophore membrane by concomitantly interacting with LC3C and binding to ubiquitinated cytosolic bacteria or to LGALS8/GALECTIN 8 adsorbed on damaged vacuoles that contain bacteria. We recently reported that in addition, CALCOCO2 is also necessary for the maturation step of Salmonella Typhimurium-containing autophagosomes. Interestingly, the role of CALCOCO2 in maturation is independent of its role in targeting, as these functions rely on distinct binding domains and protein partners. Indeed, to mediate autophagosome maturation CALCOCO2 binds on the one hand to LC3A, LC3B, or GABARAPL2, and on the other hand to MYO6/MYOSIN VI, whereas the interaction with LC3C is dispensable. Therefore, the autophagy receptor CALCOCO2 plays a dual function during xenophagy first by targeting bacteria to nascent autophagosomes and then by promoting autophagosome maturation in order to destroy bacteria

The Interaction between Nidovirales and Autophagy Components

Autophagy is a conserved intracellular catabolic pathway that allows cells to maintain homeostasis through the degradation of deleterious components via specialized double-membrane vesicles called autophagosomes. During the past decades, it has been revealed that numerous pathogens, including viruses, usurp autophagy in order to promote their propagation. Nidovirales are an order of enveloped viruses with large single-stranded positive RNA genomes. Four virus families (Arterividae, Coronaviridae, Mesoniviridae, and Roniviridae) are part of this order, which comprises several human and animal pathogens of medical and veterinary importance. In host cells, Nidovirales induce membrane rearrangements including autophagosome formation. The relevance and putative mechanism of autophagy usurpation, however, remain largely elusive. Here, we review the current knowledge about the possible interplay between Nidovirales and autophagy

NDP52, autophagy and pathogens: "The war then ceased for lack of combatants"

International audienc