WD40 Domain Of ATG16L1 Research Articles

Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus–host interactions and potential antiviral strategies.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

V-ATPase is a universal regulator of LC3-associated phagocytosis and non-canonical autophagy.

Non-canonical autophagy is a key cellular pathway in immunity, cancer, and neurodegeneration, characterized by conjugation of ATG8 to endolysosomal single membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1), and infection (influenza), dependent on K490 in the ATG16L1 WD40-domain. However, factors associated with non-canonical ATG16L1 recruitment and CASM induction remain unknown. Here, using pharmacological inhibitors, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM and indicate that SopF evolved in part to evade non-canonical autophagy.

The autophagy protein ATG16L1 cooperates with IFT20 and INPP5E to regulate the turnover of phosphoinositides at the primary cilium.

The primary cilium (PC) regulates signalization linked to external stress sensing. Previous works established a functional interplay between the PC and the autophagic machinery. When ciliogenesis is promoted by serum deprivation, the autophagy protein ATG16L1 and the ciliary protein IFT20 are co-transported to the PC. Here, we demonstrate that IFT20 and ATG16L1 are part of the same complex requiring the WD40 domain of ATG16L1 and a Y-E-F-I motif in IFT20. We show that ATG16L1-deficient cells exhibit aberrant ciliary structures, which accumulate PI4,5P2, whereas PI4P, a lipid normally concentrated in the PC, is absent. Finally, we demonstrate that INPP5E, a phosphoinositide-associated phosphatase responsible for PI4P generation, interacts with ATG16L1 and that a perturbation of the ATG16L1/IFT20 complex alters its trafficking to the PC. Altogether, our results reveal a function of ATG16L1 in ciliary lipid and protein trafficking, thus directly contributing to proper PC dynamics and functions.

Regulation of cytokine signaling through direct interaction between cytokine receptors and the ATG16L1 WD40 domain

ATG16L1, an autophagy mediator that specifies the site of LC3 lipidation, includes a C-terminal domain formed by 7 WD40-type repeats (WD40 domain, WDD), the function of which is unclear. Here we show that the WDD interacts with the intracellular domain of cytokine receptors to regulate their signaling output in response to ligand stimulation. Using a refined version of a previously described WDD-binding amino acid motif, here we show that this element is present in the intracellular domain of cytokine receptors. Two of these receptors, IL-10RB and IL-2Rγ, recognize the WDD through the motif and exhibit WDD-dependent LC3 lipidation activity. IL-10 promotes IL-10RB/ATG16L1 interaction through the WDD, and IL-10 signaling is suboptimal in cells lacking the WDD owing to delayed endocytosis and inefficient early trafficking of IL10/IL-10R complexes. Our data reveal WDD-dependent roles of ATG16L1 in the regulation of cytokine receptor trafficking and signaling, and provide a WDD-binding motif that might be used to identify additional WDD activators.

STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain.

Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon β expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase-dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.

The Chlamydia effector CT622/TaiP targets a nonautophagy related function of ATG16L1

The obligate intracellular bacteria Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted diseases, multiply in a vacuolar compartment, the inclusion. From this niche, they secrete "effector" proteins, that modify cellular activities to enable bacterial survival and proliferation. Here, we show that the host autophagy-related protein 16-1 (ATG16L1) restricts inclusion growth and that this effect is counteracted by the secretion of the bacterial effector CT622/TaiP (translocated ATG16L1 interacting protein). ATG16L1 is mostly known for its role in the lipidation of the human homologs of ATG8 (i.e., LC3 and homologs) on double membranes during autophagy as well as on single membranes during LC3-associated phagocytosis and other LC3-lipidation events. Unexpectedly, the LC3-lipidation-related functions of ATG16L1 are not required for restricting inclusion development. We show that the carboxyl-terminal domain of TaiP exposes a mimic of an eukaryotic ATG16L1-binding motif that binds to ATG16L1's WD40 domain. By doing so, TaiP prevents ATG16L1 interaction with the integral membrane protein TMEM59 and allows the rerouting of Rab6-positive compartments toward the inclusion. The discovery that one bacterial effector evolved to target ATG16L1's engagement in intracellular traffic rather than in LC3 lipidation brings this "secondary" activity of ATG16L1 in full light and emphasizes its importance for maintaining host cell homeostasis.

Macrophage scavenger receptor 1 controls Chikungunya virus infection through autophagy in mice

Macrophage scavenger receptor 1 (MSR1) mediates the endocytosis of modified low-density lipoproteins and plays an important antiviral role. However, the molecular mechanism underlying MSR1 antiviral actions remains elusive. We report that MSR1 activates autophagy to restrict infection of Chikungunya virus (CHIKV), an arthritogenic alphavirus that causes acute and chronic crippling arthralgia. Msr1 expression was rapidly upregulated after CHIKV infection in mice. Msr1 knockout mice had elevated viral loads and increased susceptibility to CHIKV arthritis along with a normal type I IFN response. Induction of LC3 lipidation by CHIKV, a marker of autophagy, was reduced in Msr1−/− cells. Mechanistically, MSR1 interacted with ATG12 through its cytoplasmic tail and this interaction was enhanced by CHIKV nsP1 protein. MSR1 repressed CHIKV replication through ATG5-ATG12-ATG16L1 and this was dependent on the FIP200-and-WIPI2-binding domain, but not the WD40 domain of ATG16L1. Our results elucidate an antiviral role for MSR1 involving the autophagic function of ATG5-ATG12-ATG16L1.

Physical and functional interaction between A20 and ATG16L1-WD40 domain in the control of intestinal homeostasis

Prevention of inflammatory bowel disease (IBD) relies on tight control of inflammatory, cell death and autophagic mechanisms, but how these pathways are integrated at the molecular level is still unclear. Here we show that the anti-inflammatory protein A20 and the critical autophagic mediator Atg16l1 physically interact and synergize to regulate the stability of the intestinal epithelial barrier. A proteomic screen using the WD40 domain of ATG16L1 (WDD) identified A20 as a WDD-interacting protein. Loss of A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology, as characterized by severe inflammation and increased intestinal epithelial cell death in both small and large intestine. Mechanistically, absence of A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or expression of WDD-deficient Atg16l1 stabilizes A20. Collectively our data show that A20 and Atg16l1 cooperatively control intestinal homeostasis by acting at the intersection of inflammatory, autophagy and cell death pathways.

Gigaxonin E3 ligase governs ATG16L1 turnover to control autophagosome production

Autophagy is an essential self-digestion machinery for cell survival and homoeostasis. Membrane elongation is fundamental, as it drives the formation of the double-membrane vesicles that engulf cytosolic material. LC3-lipidation, the signature of autophagosome formation, results from a complex ubiquitin-conjugating cascade orchestrated by the ATG16L1 protein, whose regulation is unknown. Here, we identify the Gigaxonin-E3 ligase as the first regulator of ATG16L1 turn-over and autophagosome production. Gigaxonin interacts with the WD40 domain of ATG16L1 to drive its ubiquitination and subsequent degradation. Gigaxonin depletion induces the formation of ATG16L1 aggregates and impairs LC3 lipidation, hence altering lysosomal fusion and degradation of the main autophagy receptor p62. Altogether, we demonstrate that the Gigaxonin-E3 ligase controls the production of autophagosomes by a reversible, ubiquitin-dependent process selective for ATG16L1. Our findings unveil the fundamental mechanisms of the control of autophagosome formation, and provide a molecular switch to fine-tune the activation of autophagy.

Sorting out “non‐canonical” autophagy

Canonically, LC3 lipidation has been associated with autophagy pathways but it becomes increasingly clear that this modification can also occur during autophagy‐unrelated processes. In this issue, Florey and colleagues find that the WD40 domain of ATG16L1 is dispensable for LC3 lipidation during starvation‐induced autophagy but required for its lipidation during several other membrane‐based processes that are different from autophagy. This finding opens the door for the analysis of the functions of LC3 lipidation in these pathways.

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.