| 2003 |
Mouse Atg16L (ATG16L1) is a novel WD-repeat protein that interacts with both Atg5 and additional Atg16L monomers (neither interaction requiring the WD-repeat domain), and together with the Atg12-Atg5 conjugate forms an ~800 kDa complex that associates with the autophagic isolation membrane for the duration of autophagosome formation. Membrane targeting of Atg16L requires Atg5 but not Atg12. |
Co-immunoprecipitation, size-exclusion chromatography, fluorescence microscopy of GFP-tagged proteins |
Journal of cell science |
High |
12665549
|
| 2008 |
The Atg16L complex acts as an E3-like enzyme that determines the site of LC3 lipidation by directing membrane localization. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. The interaction of Atg12 with Atg3 (the E2 enzyme for LC3 lipidation) is also required for specifying the lipidation site. |
Overexpression/mislocalization experiments, immunofluorescence, biochemical LC3 lipidation assays |
Molecular biology of the cell |
High |
18321988
|
| 2008 |
Atg16L1 deficiency disrupts recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in loss of LC3 conjugation to phosphatidylethanolamine and severely impaired autophagosome formation. In LPS-stimulated macrophages, Atg16L1 deficiency causes TRIF-dependent caspase-1 activation leading to increased IL-1β and IL-18 production. |
Atg16L1 knockout mice, immunoblot for LC3 lipidation, electron microscopy, caspase-1 activation assays, cytokine ELISA |
Nature |
High |
18849965
|
| 2008 |
ATG16L1 (and ATG5) are selectively required for Paneth cell granule exocytosis in the ileal epithelium; ATG16L1- and ATG5-deficient Paneth cells exhibit notable abnormalities in the granule exocytosis pathway. ATG16L1-deficient Paneth cells show a gain-of-function transcriptional program including increased PPAR signaling, lipid metabolism genes, and adipocytokines (leptin, adiponectin). |
Hypomorphic ATG16L1 mouse generation, electron microscopy of Paneth cell granules, transcriptional profiling, immunohistochemistry of human Crohn's disease tissue |
Nature |
High |
18849966
|
| 2008 |
Golgi-resident small GTPase Rab33B (and Rab33A) specifically interacts with Atg16L via the coiled-coil domain of Atg16L in a GTP-dependent manner. Expression of GTPase-deficient Rab33B-Q92L induces LC3 lipidation under nutrient-rich conditions, and overexpression of the Rab33B-binding domain of Atg16L suppresses autophagosome formation. |
Co-immunoprecipitation, GTP-agarose pulldown, LC3 lipidation assay, p62 degradation assay, dominant-negative/overexpression experiments |
Molecular biology of the cell |
High |
18448665
|
| 2010 |
ATG16L1 and NOD2 function in an autophagy-dependent antibacterial pathway; MDP stimulation activates autophagy and increases intracellular Salmonella killing in a manner requiring both ATG16L1 and NOD2 expression. The ATG16L1 T300A variant blocks the MDP-mediated increase in Salmonella killing in epithelial cell lines but not in primary macrophages or dendritic cells. |
siRNA knockdown, confocal microscopy, flow cytometry, gentamicin protection assay, primary human macrophages and dendritic cells |
Gastroenterology |
Medium |
20637199
|
| 2011 |
ATG16L2, a paralog of ATG16L1, forms an ~800 kDa Atg12-5-16L2 complex but is not recruited to phagophores and resides mostly in the cytosol. The difference in autophagic function between ATG16L1 and ATG16L2 maps entirely to their middle regions (coiled-coil domain, particularly around residues 229-242), which is required for phagophore targeting. |
Biochemical fractionation, immunofluorescence, chimeric protein analysis, ATG16L2 knockdown |
Autophagy |
Medium |
22082872
|
| 2012 |
A direct interaction between FIP200 (ULK1 complex) and ATG16L1 is mediated by a short FIP200-binding domain (FBD) in ATG16L1 that is distinct from its ATG5-interaction and self-dimerization domains. An FBD-deleted ATG16L1 mutant is defective in amino acid starvation-induced (ULK1-dependent) autophagy but retains function in glucose deprivation-induced (ULK1-independent) autophagy. |
Co-immunoprecipitation, domain deletion mutants, autophagy induction assays under different starvation conditions |
Nature structural & molecular biology |
High |
23262492
|
| 2013 |
ATG16L1 negatively regulates Nod1- and Nod2-driven inflammatory responses in an autophagy-independent manner. Knockdown of ATG16L1 (but not ATG5 or ATG9a) enhances Nod-driven cytokine production. Mechanistically, ATG16L1 interferes with poly-ubiquitination of the Rip2 adaptor and prevents Rip2 recruitment into large signaling complexes. The CD-associated ATG16L1 T300A allele is impaired in regulating Nod-driven cytokine responses. |
siRNA knockdown, ubiquitination assays, complex assembly analysis, cytokine ELISA, autophagy-incompetent ATG16L1 truncation mutants |
Immunity |
High |
24238340
|
| 2015 |
CSNK2 (casein kinase 2) phosphorylates ATG16L1 at Ser139, and this phosphorylation is required for ATG16L1 association with the ATG12-ATG5 conjugate and for hypoxia/reoxygenation-induced autophagy in cardiomyocytes. PPP1 (protein phosphatase 1) dephosphorylates ATG16L1, antagonizing CSNK2. PPP1 binds an RVxF motif in the C-terminal tail of ATG16L1; mutation of this site disrupts PPP1 association. |
In vitro kinase assay, site-directed mutagenesis (S139A/D), co-immunoprecipitation, phospho-specific antibody, CSNK2 inhibitor treatment, shRNA knockdown |
Autophagy |
High |
26083323
|
| 2018 |
The WD40 C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 lipidation onto single membranes (non-canonical autophagy/LC3-associated phagocytosis) but dispensable for canonical (double-membrane) autophagy. Mice lacking the WD40 CTD show reduced MHC class II antigen presentation in dendritic cells and impaired non-canonical autophagy during influenza A virus infection. |
WD40 domain deletion mutants, LC3 lipidation assays, MHC II antigen presentation assays in dendritic cells from transgenic mice, influenza infection model |
The EMBO journal |
High |
29317426
|
| 2018 |
SETD7 methylates ATG16L1 at lysine 151, and KDM1A/LSD1 removes this methyl mark. Methylation at K151 impairs ATG16L1 binding to the ATG12-ATG5 conjugate, inhibiting autophagy and increasing apoptosis in hypoxia/reoxygenation-treated cardiomyocytes. This methylation also inhibits CSNK2-mediated phosphorylation at S139, establishing a methylation-phosphorylation switch. |
In vitro methylation assay, site-directed mutagenesis (K151), co-immunoprecipitation, SETD7 shRNA knockdown, small molecule SETD7 inhibitor |
Autophagy |
High |
29634390
|
| 2018 |
ATG16L1 (with ATG5 and ATG12) is required for plasma membrane repair through a pathway independent of macroautophagy. ATG16L1 is required for lysosome fusion with the plasma membrane and blebbing responses that promote repair. ATG16L1 deficiency causes cholesterol accumulation in lysosomes contributing to defective membrane repair. The ATG16L1 T300A allele also accumulates cholesterol and is defective in plasma membrane repair. |
ATG16L1 KO cells, lysosome fusion assays, cholesterol staining, blebbing assays, Listeria cell-to-cell spread assay, T300A variant cells |
Nature microbiology |
High |
30478389
|
| 2019 |
The V-ATPase recruits ATG16L1 onto bacteria-containing vacuoles upon vacuolar damage during xenophagy, a process blocked by the bacterial effector SopF. ATG16L1's WD40 domain is required for interacting with the V-ATPase. SopF ADP-ribosylates Gln124 of ATP6V0C in the V-ATPase; mutation of Gln124 blocks xenophagy but not canonical autophagy. This V-ATPase–ATG16L1 axis is essential for autophagic recognition of intracellular pathogens. |
CRISPR screen, bacterial transposon screen, co-immunoprecipitation, SopF enzymatic ADP-ribosylation assay, site-directed mutagenesis (Q124), WD40 domain deletion |
Cell |
High |
31327526
|
| 2019 |
The full-length ATG12-ATG5-ATG16L1 complex reconstituted in vitro reveals membrane-binding regions within ATG16L1 that contribute to membrane targeting and LC3/GABARAP lipidation. ATG16L1 intrinsically binds phosphoinositides including PI3P via conserved residues in its coiled-coil domain; mutating these residues abrogates ATG16L1 localization to the pre-autophagosomal structure (PAS) and inhibits LC3 lipidation. |
Purification of full-length complex, in vitro reconstitution of LC3 lipidation, phosphoinositide-binding assays, structural and mutational analysis of CCD lipid-binding residues, rescue experiments in KO cells |
The EMBO journal |
High |
30936093 31122169
|
| 2019 |
Gigaxonin E3 ligase interacts with the WD40 domain of ATG16L1 and drives its ubiquitination and proteasomal degradation. Gigaxonin depletion induces ATG16L1 aggregate formation and impairs LC3 lipidation, lysosomal fusion, and p62 degradation. |
Co-immunoprecipitation, ubiquitination assay, Gigaxonin KO/knockdown, ATG16L1 turnover assay, LC3 lipidation and p62 degradation readouts |
Nature communications |
High |
30770803
|
| 2019 |
ULK1 directly phosphorylates ATG16L1 in response to infection and starvation. Phosphorylated ATG16L1 localizes to sites of internalized bacteria and is required for xenophagy (phospho-dead mutant impairs xenophagy). ULK1-mediated phosphorylation of the CD-associated T300A ATG16L1 variant drives its destabilization under stress conditions. |
In vitro ULK1 kinase assay, phospho-dead ATG16L1 stable cell lines, bacterial infection xenophagy assay, ATG16L1 T300A variant comparison |
EMBO reports |
High |
31267703
|
| 2019 |
PKA phosphorylates ATG16L1α at Ser268 (ATG16L1β at Ser269), driving phosphorylation-dependent degradation of ATG16L1 protein and thereby limiting endothelial autophagy. Reducing PKA activity increases ATG16L1 levels; autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency in vivo. |
Chemical genetic PKA screen, mass spectrometry, peptide spot arrays, biochemical validation, mouse in vivo genetics, pharmacological autophagy inhibition |
eLife |
High |
31580256
|
| 2019 |
ATG16L1 deficiency leads to insulin resistance through accumulation of KLHL9/KLHL13 (novel IRS1 interactors), which form an E3 ubiquitin ligase complex with CUL3 that promotes proteasomal IRS1 degradation. IRS1 protein levels are rescued by proteasome inhibition or Klhl9/Klhl13/Cul3 siRNA knockdown in ATG16L1 KO MEFs. |
ATG16L1 KO MEFs, BioID proximity labeling, co-immunoprecipitation, proteasome inhibitor treatment, siRNA knockdown of Klhl9/Klhl13/Cul3, insulin signaling assays |
The Journal of biological chemistry |
High |
31515271
|
| 2020 |
STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles via ATG16L1's WD40 domain, bypassing canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 (V-ATPase inhibitor) and by SopF (which catalytically modifies V-ATPase), establishing V-ATPase dependence. |
STING agonist treatment, WD40 domain deletion/mutation, bafilomycin A1 and SopF inhibition, LC3B lipidation assays, immunofluorescence |
The Journal of cell biology |
High |
33201170
|
| 2020 |
RAB33B recruits the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 reveal the recognition mechanism. ATG16L1 acts as a noncanonical RAB-binding protein that can induce RAB33B to adopt an active conformation without nucleotide exchange. RAB33B-ATG16L1 interaction is required for LC3 lipidation and autophagosome formation. |
Crystal structure determination, pulldown assays, microscale thermophoresis (binding affinity), fluorescence lifetime imaging (FLIM/FRET), site-directed mutagenesis, LC3 lipidation assay |
Autophagy |
High |
32960676
|
| 2021 |
Crystal structure of WIPI2d in complex with the WIPI2-interacting region (W2IR) of ATG16L1 (residues 207-230) at 1.85 Å resolution. The ATG16L1 W2IR adopts an alpha-helical conformation binding in an electropositive/hydrophobic groove between WIPI2 β-propeller blades 2 and 3. Mutations at the interface reduce recruitment of ATG12-5-16L1 and LC3B conjugation to membranes and decrease starvation-induced autophagy. |
X-ray crystallography (1.85 Å), interface mutagenesis, in vitro LC3B lipidation to synthetic membranes, cell-based autophagy assays |
eLife |
High |
34505572
|
| 2021 |
ATG16L1's WD40 domain interacts with A20 (anti-inflammatory ubiquitin-editing enzyme). Loss of both A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology. A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or WD40-domain-deficient Atg16l1 stabilizes A20, revealing reciprocal regulation. |
WD40 domain proteomic screen (ATG16L1 WDD interactome), co-immunoprecipitation, conditional double-KO mice, intestinal phenotyping |
Nature communications |
High |
31015422
|
| 2021 |
ATG16L1 interacts with IFT20 via the WD40 domain of ATG16L1 and a Y-E-F-I motif in IFT20; this complex regulates ciliary phosphoinositide composition. ATG16L1-deficient cells accumulate PI4,5P2 and lack PI4P at the primary cilium. ATG16L1 also interacts with the phosphoinositide phosphatase INPP5E, and disruption of the ATG16L1/IFT20 complex impairs INPP5E trafficking to the primary cilium. |
Co-immunoprecipitation, domain mapping (WD40 deletion, IFT20 motif mutation), phosphoinositide staining, confocal microscopy of ATG16L1-KO cells, INPP5E trafficking assay |
Cell reports |
High |
33910006
|
| 2022 |
ATG16L1 directly binds to the RICK/RIP2 kinase domain and negatively regulates TLR2-mediated NF-κB activation by inhibiting the TLR2-RICK/RIP2 interaction and suppressing RICK/RIP2 polyubiquitination. |
Co-immunoprecipitation in HEK293 cells and primary human dendritic cells, domain mapping, NF-κB reporter assay, ubiquitination assay, cytokine ELISA |
International immunology |
Medium |
32909611
|
| 2022 |
The ATG16L1 T300A polymorphism leads to enhanced polyubiquitination of TRAF6 and RIPK2 due to accumulation of SQSTM1/p62, resulting in increased NF-κB activation and enhanced TLR/NLR cytokine responses. Knockout of Sqstm1 in autophagy-deficient cells almost completely normalizes TRAF6/RIPK2 polyubiquitination and NF-κB activation. |
ATG16L1 T300A knockin mice and cells, SQSTM1 KO rescue experiments, polyubiquitination assays, NF-κB activation assays, cytokine measurement |
Autophagy |
High |
35220902
|
| 2023 |
ATG16L1 adopts a dual-binding-site mode to interact with WIPI2b: a previously known WBS1 (residues ~207-230) and a newly identified WBS2. Crystal structures of WIPI2b with each ATG16L1-binding site were determined. Both WBS1 and WBS2 are required for effective autophagic flux in cells. |
X-ray crystallography of WIPI2b-ATG16L1 WBS1 and WBS2 complexes, mutagenesis at each binding site, autophagic flux assays in cells |
Science advances |
High |
36857448
|
| 2024 |
The V1H subunit of the V-ATPase directly binds ATG16L1; this interaction occurs only within fully assembled V-ATPases, coupling ATG16L1 recruitment to increased V-ATPase assembly following organelle neutralization. A loop within V1H mediates ATG16L1 binding; a neuronal V1H isoform lacking this loop shows attenuated ATG8 targeting in response to ionophores. |
Direct binding assay (V1H-ATG16L1 interaction), structural mapping of binding loop, V1H-KO cells, influenza and STING activation models, primary murine and iPSC-derived neurons |
Molecular cell |
High |
39089251
|
| 2024 |
ZDHHC7 catalyzes S-palmitoylation of ATG16L1 at cysteine 153. The palmitoylation-deficient ATG16L1 C153S mutant fails to rescue LC3 lipidation and autophagosome formation in ATG16L1-KO cells. Mechanistically, palmitoylation at Cys153 enhances formation of ATG16L1-WIPI2B and ATG16L1-RAB33B complexes on the phagophore, promoting LC3 lipidation. |
Acyl-biotin exchange (ABE) palmitoylation assay, ZDHHC7 KO/overexpression, C153S site-directed mutagenesis, rescue in ATG16L1-KO cells, co-immunoprecipitation for WIPI2B/RAB33B interactions |
Autophagy |
High |
39087410
|
| 2024 |
The V-ATPase-ATG16L1 axis recruits LRRK2 onto single membranes of stressed lysosomes/phagosomes (but not double-membrane autophagosomes), requiring the WD40 domain of ATG16L1. This mechanism is responsible for lysosomal stress-induced LRRK2 activation and downstream regulation of lysosomal secretion and enlargement, independently of canonical autophagy. |
ATG16L1 WD40 domain deletion, LRRK2 recruitment assays to lysosomes, lysosomal damage models, lysosomal secretion and size readouts, independence from canonical autophagy initiation complex confirmed |
The Journal of cell biology |
High |
38227290
|
| 2024 |
ATG16L1 together with LC3B forms a membrane coat that remodels flat membranes into cup-shaped structures resembling phagophores in vitro. Cup formation requires collaboration between LC3B and ATG16L1, is specific to LC3B (not other ATG8 family members), and depends on ATG16L1's C-terminal membrane-binding domain; ATG16L1 truncants lacking this domain catalyze LC3B lipidation but fail to form coats or support non-selective autophagosome biogenesis. |
Two complementary in vitro membrane reconstitution approaches, electron microscopy of membrane cups, truncation mutants of ATG16L1, comparison across ATG8 family members |
Nature structural & molecular biology |
High |
38834913
|
| 2024 |
Three-step docking mechanism for LC3 lipidation: (1) WIPI2 recruits ATG12-ATG5-ATG16L1 to the membrane via its PI3P-binding blades, (2) helix α2 of ATG16L1 inserts into the membrane, (3) a membrane-interacting surface of ATG3 positions ATG3∼LC3 near the PE substrate. Two conserved histidines in ATG3 were identified as candidate catalytic residues for LC3 transfer to PE. |
Molecular dynamics simulations, in vitro LC3 lipidation assays on synthetic membranes, cell-based validation experiments |
Science advances |
High |
38324698
|
| 2017 |
Crystal structure of the WD40 domain of human ATG16L1 determined at 1.55 Å resolution, revealing the domain boundaries and structural scaffold for protein-protein interactions in non-canonical ATG16L1 functions (inflammatory control, xenophagy). |
X-ray crystallography (1.55 Å resolution) |
Protein science |
High |
28685931
|
| 2018 |
The ATG5-binding and coiled-coil domains of ATG16L1 (including the WIPI2-binding residue E230) are sufficient for canonical macroautophagy; the WD40 domain and linker are required specifically for LC3-associated phagocytosis (LAP) but not canonical autophagy. Mice lacking the linker and WD domains are LAP-deficient but maintain canonical autophagy and survive postnatal starvation. |
Domain-deletion mouse genetics (linker+WD40 deletion), LC3 lipidation and p62/SQSTM1 assays in tissues, WIPI2-binding mutant (E230 deletion), neonatal survival assay |
Autophagy |
High |
30403914
|
| 2015 |
The vitamin D receptor (VDR) transcriptionally regulates ATG16L1 as a direct VDR target gene. Low VDR levels in the intestine correlate with reduced ATG16L1 expression and impaired Paneth cell autophagy. |
ChIP/transcriptional reporter assays establishing VDR binding to ATG16L1 promoter, VDR KO model, ATG16L1 expression correlation |
Autophagy |
Medium |
26218741
|
| 2015 |
Annexin A2 promotes biogenesis of Atg16L-positive vesicles from the plasma membrane and their homotypic fusion to form phagophores. Annexin A2-deficient cells show reduced Atg16L-positive vesicle formation, impaired homotypic vesicle fusion, reduced LC3 flux, and dampened macroautophagy in dendritic cells. |
Ultrastructural analysis, proteomics of Atg16L+ vesicles, FACS, lipidomics of Anxa2 KO cells, homotypic fusion assay, LC3 flux in Anxa2-KO dendritic cells |
Nature communications |
High |
25597631
|
| 2016 |
ATG16L1 deletion in T cells (but not ATG5 or ATG9a) leads to spontaneous intestinal inflammation characterized by aberrant TH2 responses and loss of Foxp3+ Treg cells. Selective deletion of ATG16L1 in Foxp3+ Treg cells demonstrates that autophagy directly promotes Treg survival and metabolic adaptation in the intestine. |
T cell-specific and Treg-specific Atg16l1 conditional KO mice, intestinal inflammation scoring, T cell subset analysis, metabolic assays |
eLife |
High |
26910010
|
| 2019 |
14-3-3ζ protein (delivered via MSC exosomes) interacts with ATG16L1, promoting localization of ATG16L1 at autophagosome precursors and activating autophagy. ATG16L1 expression is increased by hucMSC exosomes, and 14-3-3ζ knockdown reduces autophagic activity. |
Co-immunoprecipitation (14-3-3ζ and ATG16L1), 14-3-3ζ KO/overexpression, ATG16L1 localization by confocal microscopy |
American journal of translational research |
Low |
29422997
|
| 2020 |
The Chlamydia effector CT622/TaiP contains a eukaryotic ATG16L1-binding motif mimic that binds to ATG16L1's WD40 domain, preventing ATG16L1 interaction with the integral membrane protein TMEM59, thereby allowing rerouting of Rab6-positive compartments toward the bacterial inclusion. The LC3-lipidation functions of ATG16L1 are not required for the restriction of inclusion development. |
Co-immunoprecipitation, domain mapping (WD40), TMEM59 interaction assay, Rab6 vesicle trafficking assay, LC3-lipidation mutant analysis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
33055216
|