HS1BP3 provides a novel mechanism of negative autophagy regulation through membrane lipids

The formation and maturation of the autophagosome, a morphological hallmark of macroautophagy/autophagy, is tightly controlled with regard to location, timing and intensity. Various proteins have been characterized to be essential in regulating autophagosome biogenesis, whereas little is known about the roles of specific lipids and their metabolizing enzymes in this process. In a recent paper, Holland et al. identified the phosphoinositide-binding protein HS1BP3 as a novel negative regulator of autophagosome formation. HS1BP3 is proposed to act by inhibiting PLD1 (phospholipase D1) activity and localization to ATG16L1 and TFRC (transferrin receptor)-positive vesicles thereby modulating the phosphatidic acid (PA) levels and lipid composition of autophagosome precursor membranes.     

ATG16L1 meets ATG9 in recycling endosomes

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

Commentary: ATG16L1 meets ATG9 in recycling endosomes: Additional roles for the plasma membrane and endocytosis in autophagosome biogenesis

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

Transiently expressed ATG16L1 inhibits autophagosome biogenesis and aberrantly targets RAB11-positive recycling endosomes

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12–ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1⁺ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.     

RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.     

RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.     

Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p

Multiple genetic studies have implicated the autophagy-related gene, ATG16L1, in the pathogenesis of Crohn disease (CD). While CD-related research on ATG16L1 has focused on the functional significance of ATG16L1 genetic variations, the mechanisms underlying the regulation of ATG16L1 expression are unclear. Our laboratory has described that microRNAs (miRNAs), key regulators of gene expression, are dysregulated in CD. Here, we report miRNA-mediated regulation of ATG16L1 in colonic epithelial cells as well as Jurkat T cells. Dual luciferase reporter assays following the transfection of vectors containing the ATG16L1 3′-untranslated region (3′UTR) or truncated 3′UTR fragments suggest that the first half of ATG16L1 3′UTR in the 5′ end is more functional for miRNA targeting. Of 5 tested miRNAs with putative binding sites within the region, MIR142-3p, upon transient overexpression in the cells, resulted in decreased ATG16L1 mRNA and protein levels. Further observation demonstrated that the luciferase reporter vector with a mutant MIR142-3p binding sequence in the 3′UTR was unresponsive to the inhibitory effect of MIR142-3p, suggesting ATG16L1 is a gene target of MIR142-3p. Moreover, the regulation of ATG16L1 expression by a MIR142-3p mimic blunted starvation- and L18-MDP-induced autophagic activity in HCT116 cells. Additionally, we found that a MIR142-3p inhibitor enhanced starvation-induced autophagy in Jurkat T cells. Our study reveals MIR142-3p as a new autophagy-regulating small molecule by targeting ATG16L1, implying a role of this miRNA in intestinal inflammation and CD.     

Local detection of PtdIns3P at autophagosome biogenesis membrane platforms

Phosphatidylinositol 3-phosphate (PtdIns3P) is a key player of membrane trafficking regulation, mostly synthesized by the PIK3C3 lipid kinase. The presence of PtdIns3P on endosomes has been demonstrated; however, the role and dynamics of the pool of PtdIns3P dedicated to macroautophagy/autophagy remains elusive. Here we addressed this question by studying the mobilization of PtdIns3P in time and space during autophagosome biogenesis. We compared different dyes known to specifically detect PtdIns3P by fluorescence microscopy analysis, based on PtdIns3P-binding FYVE and PX domains, and show that these transfected dyes induce defects in endosomal dynamics as well as artificial and sustained autophagosome formation. In contrast, indirect use of recombinant FYVE enabled us to track and discriminate endosomal and autophagosomal pools of PtdIns3P. We used this method to analyze localization and dynamics of PtdIns3P subdomains on the endoplasmic reticulum, at sites of pre-autophagosome associated protein recruitment such as the PtdIns3P-binding ZFYVE1/DFCP1 and WIPI2 autophagy regulators. This approach thus revealed the presence of a specific pool of PtdIns3P at the site where autophagosome assembly is initiated.     

TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1

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MicroRNA-129-5p inhibits 3T3-L1 preadipocyte proliferation by targeting G3BP1

MicroRNAs have been regarded to play a crucial role in the proliferation of different cell types including preadipocytes. In our study, we observed that miR-129-5p was down-regulated during 3T3-L1 preadipocyte proliferation, while the expression of G3BP1 showed a contrary tendency. 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay and flow cytometry showed that overexpression of miR-129-5p could bring about a reduction in S-phase cells and G2-phase arrest. Additional study indicated that miR-129-5p impaired cell cycle-related genes in 3T3-L1 preadipocytes. Importantly, it showed that miR-129-5p directly targeted the 3′UTR of G3BP1 and the expression of G3BP1 was inhibited by miR-129-5p mimic. Moreover, miR-129-5p mimic activated the p38 signaling pathway through up-regulating p38 and the phosphorylation level of p38. In a word, results in our study revealed that miR-129-5p suppressed preadipocyte proliferation via targeting G3BP1 and activating the p38 signaling pathway.     

Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway

ABSTRACT

Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication.     

The NLR protein,NLRX1, and its partner,TUFM, reduce type I interferon,and enhance autophagy

The NLR (nucleotide-binding domain leucine-rich repeat containing) proteins serve as regulators of inflammatory signaling pathways. NLRX1, a mitochondria-localized NLR protein, has been previously shown to negatively regulate inflammatory cytokine production activated via the MAVS-DDX58 (RIG-I) pathway. The literature also indicates that DDX58 has a negative impact upon autophagy. Consistent with the inhibitory role of NLRX1 on DDX58, our recent study indicates a role of NLRX1 in augmenting virus-induced autophagy. This effect is through its interaction with another mitochondrial protein TUFM (Tu translation elongation factor, mitochondrial, also known as EF-TuMT, COXPD4, and P43). TUFM also reduces DDX58-activated cytokines but augments autophagy. Additionally it interacts with ATG12–ATG5-ATG16L1 to form a molecular complex that modulates autophagy. The work shows that both NLRX1 and TUFM work in concert to reduce cytokine response and augment autophagy.     

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.     

TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3

Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19‐amino‐acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD‐repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1‐binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.     

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.     

WIPI2B links PtdIns3P to LC3 lipidation through binding ATG16L1

WIPI proteins, phosphatidylinositol 3-phosphate (PtdIns3P) binding proteins with β-propeller folds, are recruited to the omegasome following PtdIns3P production. The functions of the WIPI proteins in autophagosome formation are poorly understood. In a recent study, we reported that WIPI2B directly binds ATG16L1 and functions by recruiting the ATG12–ATG5-ATG16L1 complex to forming autophagosomes during starvation- or pathogen-induced autophagy. Our model of WIPI2 function provides an explanation for the PtdIns3P-dependent recruitment of the ATG12–ATG5-ATG16L1 complex during initiation of autophagy.     

Receptor‐mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy

Autophagosome formation requires multiple autophagy‐related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome‐wide CRISPR screens identifying genes required for NBR1 flux in ATG7^KO cells. We find that ATG7‐independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1''s ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin‐independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor‐mediated clustering of upstream autophagy factors drives continued autophagosome formation.