{"gene":"ATG16L1","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2003,"finding":"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.","method":"Co-immunoprecipitation, size-exclusion chromatography, fluorescence microscopy of GFP-tagged proteins","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, biochemical fractionation, and live-cell imaging, independently establishing complex composition and membrane targeting; foundational paper replicated by many subsequent studies","pmids":["12665549"],"is_preprint":false},{"year":2008,"finding":"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.","method":"Overexpression/mislocalization experiments, immunofluorescence, biochemical LC3 lipidation assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution-style forced-localization experiment directly establishing E3-like scaffold function; replicated conceptually in multiple subsequent studies","pmids":["18321988"],"is_preprint":false},{"year":2008,"finding":"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.","method":"Atg16L1 knockout mice, immunoblot for LC3 lipidation, electron microscopy, caspase-1 activation assays, cytokine ELISA","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO mouse model with multiple orthogonal readouts (autophagosome formation, LC3 lipidation, cytokine production, caspase activation); widely replicated","pmids":["18849965"],"is_preprint":false},{"year":2008,"finding":"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).","method":"Hypomorphic ATG16L1 mouse generation, electron microscopy of Paneth cell granules, transcriptional profiling, immunohistochemistry of human Crohn's disease tissue","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic hypomorphic mouse model validated in human tissue samples, multiple orthogonal readouts; widely replicated","pmids":["18849966"],"is_preprint":false},{"year":2008,"finding":"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.","method":"Co-immunoprecipitation, GTP-agarose pulldown, LC3 lipidation assay, p62 degradation assay, dominant-negative/overexpression experiments","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — GTP-dependent pulldown, reciprocal co-IP, functional mutant analysis with defined autophagy readouts; replicated structurally in PMID 32960676","pmids":["18448665"],"is_preprint":false},{"year":2010,"finding":"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.","method":"siRNA knockdown, confocal microscopy, flow cytometry, gentamicin protection assay, primary human macrophages and dendritic cells","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell types and methods (siRNA, microscopy, bacterial killing assay), but single lab","pmids":["20637199"],"is_preprint":false},{"year":2011,"finding":"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.","method":"Biochemical fractionation, immunofluorescence, chimeric protein analysis, ATG16L2 knockdown","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-swap chimera analysis with functional readouts, single lab","pmids":["22082872"],"is_preprint":false},{"year":2012,"finding":"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.","method":"Co-immunoprecipitation, domain deletion mutants, autophagy induction assays under different starvation conditions","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct interaction mapped to specific domain with direct functional consequence distinguishing two autophagy pathways; multiple orthogonal methods in single study","pmids":["23262492"],"is_preprint":false},{"year":2013,"finding":"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.","method":"siRNA knockdown, ubiquitination assays, complex assembly analysis, cytokine ELISA, autophagy-incompetent ATG16L1 truncation mutants","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — autophagy-independent function established using truncation mutants incapable of autophagy, Rip2 ubiquitination mechanistically defined, multiple orthogonal methods","pmids":["24238340"],"is_preprint":false},{"year":2015,"finding":"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.","method":"In vitro kinase assay, site-directed mutagenesis (S139A/D), co-immunoprecipitation, phospho-specific antibody, CSNK2 inhibitor treatment, shRNA knockdown","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis identifying specific phosphorylation site and functional consequence; multiple orthogonal methods in single lab","pmids":["26083323"],"is_preprint":false},{"year":2018,"finding":"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.","method":"WD40 domain deletion mutants, LC3 lipidation assays, MHC II antigen presentation assays in dendritic cells from transgenic mice, influenza infection model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-deletion approach distinguishing canonical vs non-canonical autophagy with in vivo mouse genetic validation and multiple functional readouts","pmids":["29317426"],"is_preprint":false},{"year":2018,"finding":"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.","method":"In vitro methylation assay, site-directed mutagenesis (K151), co-immunoprecipitation, SETD7 shRNA knockdown, small molecule SETD7 inhibitor","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro methylation assay with mutagenesis, crosstalk with phosphorylation mechanistically defined, multiple orthogonal approaches in single lab","pmids":["29634390"],"is_preprint":false},{"year":2018,"finding":"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.","method":"ATG16L1 KO cells, lysosome fusion assays, cholesterol staining, blebbing assays, Listeria cell-to-cell spread assay, T300A variant cells","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple KO and variant cell models with defined mechanistic readouts (lysosome fusion, cholesterol, blebbing) establishing autophagy-independent plasma membrane repair function","pmids":["30478389"],"is_preprint":false},{"year":2019,"finding":"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.","method":"CRISPR screen, bacterial transposon screen, co-immunoprecipitation, SopF enzymatic ADP-ribosylation assay, site-directed mutagenesis (Q124), WD40 domain deletion","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical ADP-ribosylation assay, CRISPR screen, domain mapping, and mutagenesis all converging on the same axis; published in Cell with multiple orthogonal methods","pmids":["31327526"],"is_preprint":false},{"year":2019,"finding":"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.","method":"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","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components, mutagenesis, and rescue experiments in knockout cells; multiple orthogonal approaches","pmids":["30936093","31122169"],"is_preprint":false},{"year":2019,"finding":"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.","method":"Co-immunoprecipitation, ubiquitination assay, Gigaxonin KO/knockdown, ATG16L1 turnover assay, LC3 lipidation and p62 degradation readouts","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, ubiquitination assay, genetic KO with multiple downstream autophagy readouts; published in Nature Communications","pmids":["30770803"],"is_preprint":false},{"year":2019,"finding":"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.","method":"In vitro ULK1 kinase assay, phospho-dead ATG16L1 stable cell lines, bacterial infection xenophagy assay, ATG16L1 T300A variant comparison","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay establishing direct phosphorylation, stable phospho-dead cell lines with xenophagy readout, T300A comparison","pmids":["31267703"],"is_preprint":false},{"year":2019,"finding":"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.","method":"Chemical genetic PKA screen, mass spectrometry, peptide spot arrays, biochemical validation, mouse in vivo genetics, pharmacological autophagy inhibition","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — chemical genetic screen, MS-identified phosphorylation sites, in vivo mouse validation, multiple orthogonal methods","pmids":["31580256"],"is_preprint":false},{"year":2019,"finding":"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.","method":"ATG16L1 KO MEFs, BioID proximity labeling, co-immunoprecipitation, proteasome inhibitor treatment, siRNA knockdown of Klhl9/Klhl13/Cul3, insulin signaling assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — BioID and co-IP identifying novel complex, siRNA rescue experiments, proteasome inhibitor confirmation; multiple orthogonal methods","pmids":["31515271"],"is_preprint":false},{"year":2020,"finding":"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.","method":"STING agonist treatment, WD40 domain deletion/mutation, bafilomycin A1 and SopF inhibition, LC3B lipidation assays, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-specific genetic approach, pharmacological inhibition, bacterial effector tool; multiple independent lines of evidence in one study","pmids":["33201170"],"is_preprint":false},{"year":2020,"finding":"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.","method":"Crystal structure determination, pulldown assays, microscale thermophoresis (binding affinity), fluorescence lifetime imaging (FLIM/FRET), site-directed mutagenesis, LC3 lipidation assay","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of RAB33B-ATG16L1 CCD complex with mutagenesis and functional validation; multiple orthogonal structural and biochemical methods","pmids":["32960676"],"is_preprint":false},{"year":2021,"finding":"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.","method":"X-ray crystallography (1.85 Å), interface mutagenesis, in vitro LC3B lipidation to synthetic membranes, cell-based autophagy assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus in vitro reconstitution plus cellular validation; multiple orthogonal methods in one study","pmids":["34505572"],"is_preprint":false},{"year":2021,"finding":"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.","method":"WD40 domain proteomic screen (ATG16L1 WDD interactome), co-immunoprecipitation, conditional double-KO mice, intestinal phenotyping","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteomic screen validated by co-IP, in vivo double-KO mouse model with defined pathological phenotype","pmids":["31015422"],"is_preprint":false},{"year":2021,"finding":"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.","method":"Co-immunoprecipitation, domain mapping (WD40 deletion, IFT20 motif mutation), phosphoinositide staining, confocal microscopy of ATG16L1-KO cells, INPP5E trafficking assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP with domain mapping, KO cells, lipid and protein trafficking readouts; multiple orthogonal methods","pmids":["33910006"],"is_preprint":false},{"year":2022,"finding":"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.","method":"Co-immunoprecipitation in HEK293 cells and primary human dendritic cells, domain mapping, NF-κB reporter assay, ubiquitination assay, cytokine ELISA","journal":"International immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP in two cell types, ubiquitination assay, NF-κB reporter; single lab","pmids":["32909611"],"is_preprint":false},{"year":2022,"finding":"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.","method":"ATG16L1 T300A knockin mice and cells, SQSTM1 KO rescue experiments, polyubiquitination assays, NF-κB activation assays, cytokine measurement","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockin mouse model, genetic rescue (SQSTM1 KO), mechanistic link to ubiquitination; multiple orthogonal methods","pmids":["35220902"],"is_preprint":false},{"year":2023,"finding":"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.","method":"X-ray crystallography of WIPI2b-ATG16L1 WBS1 and WBS2 complexes, mutagenesis at each binding site, autophagic flux assays in cells","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Strong — dual crystal structures plus mutagenesis plus cellular flux assays; multiple orthogonal methods in one study","pmids":["36857448"],"is_preprint":false},{"year":2024,"finding":"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.","method":"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","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding established biochemically, structural loop mapped, validated in multiple cell systems including neurons; published in Molecular Cell","pmids":["39089251"],"is_preprint":false},{"year":2024,"finding":"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.","method":"Acyl-biotin exchange (ABE) palmitoylation assay, ZDHHC7 KO/overexpression, C153S site-directed mutagenesis, rescue in ATG16L1-KO cells, co-immunoprecipitation for WIPI2B/RAB33B interactions","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro palmitoylation assay, site-directed mutagenesis, KO rescue, mechanistic complex formation analysis; multiple orthogonal methods","pmids":["39087410"],"is_preprint":false},{"year":2024,"finding":"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.","method":"ATG16L1 WD40 domain deletion, LRRK2 recruitment assays to lysosomes, lysosomal damage models, lysosomal secretion and size readouts, independence from canonical autophagy initiation complex confirmed","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-specific ATG16L1 mutants, LRRK2 recruitment and activation assays, multiple lysosomal functional readouts; published in JCB","pmids":["38227290"],"is_preprint":false},{"year":2024,"finding":"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.","method":"Two complementary in vitro membrane reconstitution approaches, electron microscopy of membrane cups, truncation mutants of ATG16L1, comparison across ATG8 family members","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components on membranes, EM validation, truncation series establishing domain requirements; published in Nature Structural & Molecular Biology","pmids":["38834913"],"is_preprint":false},{"year":2024,"finding":"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.","method":"Molecular dynamics simulations, in vitro LC3 lipidation assays on synthetic membranes, cell-based validation experiments","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Strong — MD simulation integrated with in vitro reconstitution and cellular validation; mechanistic pathway defined with multiple approaches","pmids":["38324698"],"is_preprint":false},{"year":2017,"finding":"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).","method":"X-ray crystallography (1.55 Å resolution)","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure; single structural study without extensive functional mutagenesis in the same paper","pmids":["28685931"],"is_preprint":false},{"year":2018,"finding":"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.","method":"Domain-deletion mouse genetics (linker+WD40 deletion), LC3 lipidation and p62/SQSTM1 assays in tissues, WIPI2-binding mutant (E230 deletion), neonatal survival assay","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo mouse genetics with domain-deletion approach, multiple tissue and functional readouts distinguishing autophagy from LAP","pmids":["30403914"],"is_preprint":false},{"year":2015,"finding":"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.","method":"ChIP/transcriptional reporter assays establishing VDR binding to ATG16L1 promoter, VDR KO model, ATG16L1 expression correlation","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — transcriptional regulation established by reporter assay, single lab, limited mechanistic follow-up in abstract","pmids":["26218741"],"is_preprint":false},{"year":2015,"finding":"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.","method":"Ultrastructural analysis, proteomics of Atg16L+ vesicles, FACS, lipidomics of Anxa2 KO cells, homotypic fusion assay, LC3 flux in Anxa2-KO dendritic cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (proteomics, lipidomics, ultrastructure, fusion assay, KO), published in Nature Communications","pmids":["25597631"],"is_preprint":false},{"year":2016,"finding":"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.","method":"T cell-specific and Treg-specific Atg16l1 conditional KO mice, intestinal inflammation scoring, T cell subset analysis, metabolic assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional KO with defined cellular and metabolic phenotypes; published in eLife","pmids":["26910010"],"is_preprint":false},{"year":2019,"finding":"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.","method":"Co-immunoprecipitation (14-3-3ζ and ATG16L1), 14-3-3ζ KO/overexpression, ATG16L1 localization by confocal microscopy","journal":"American journal of translational research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP without domain mapping or in vitro validation; single lab","pmids":["29422997"],"is_preprint":false},{"year":2020,"finding":"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.","method":"Co-immunoprecipitation, domain mapping (WD40), TMEM59 interaction assay, Rab6 vesicle trafficking assay, LC3-lipidation mutant analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — bacterial effector molecular mimicry validated biochemically, mechanistic dissection of WD40-dependent vs. LC3-lipidation-independent functions; multiple orthogonal methods","pmids":["33055216"],"is_preprint":false}],"current_model":"ATG16L1 is a core scaffold component of the ~800 kDa ATG12-ATG5-ATG16L1 E3-like complex that lipidates LC3/ATG8 family proteins onto autophagosomal and other membranes: it is recruited to the phagophore via direct interactions with WIPI2 (through both WBS1 and WBS2 sites), Rab33B (via its coiled-coil domain), and the V-ATPase (via the WD40 domain/V1H subunit), and its activity is regulated by phosphorylation (CSNK2 at Ser139, ULK1, PKA at Ser268/269), methylation (SETD7 at Lys151), S-palmitoylation (ZDHHC7 at Cys153), and ubiquitin-dependent turnover (Gigaxonin E3 ligase targeting the WD40 domain); beyond canonical double-membrane autophagy, the WD40 domain specifically mediates non-canonical LC3 lipidation on single membranes (V-ATPase-dependent, triggered by vacuolar damage, STING activation, or pathogen invasion), lysosomal homeostasis via LRRK2 recruitment, plasma membrane repair via lysosome fusion, primary cilia lipid/protein trafficking via IFT20 and INPP5E interactions, and autophagy-independent suppression of NOD/RIP2 and TLR inflammatory signaling."},"narrative":{"mechanistic_narrative":"ATG16L1 is the scaffold subunit of the ~800 kDa ATG12–ATG5–ATG16L1 E3-like ligase that determines where the ATG8/LC3 family is conjugated to membrane phosphatidylethanolamine, thereby governing autophagosome biogenesis and several autophagy-related membrane processes [PMID:12665549, PMID:18321988]. It self-dimerizes and binds ATG5 through domains distinct from its C-terminal WD40 region, and ATG5-dependent membrane targeting positions the complex on the isolation membrane for the duration of autophagosome formation; loss of ATG16L1 abolishes ATG12–ATG5 recruitment, LC3 lipidation, and autophagosome formation [PMID:12665549, PMID:18849965]. The complex functions as a membrane-acting E3 that dictates the lipidation site, working with the ATG3 E2 to position ATG3~LC3 at the membrane, and ATG16L1 together with LC3B can remodel flat membranes into phagophore-like cups via its C-terminal membrane-binding domain [PMID:18321988, PMID:38834913, PMID:38324698]. Recruitment to the phagophore is achieved through a dual-site interaction with the PI3P effector WIPI2 (WBS1 and WBS2), GTP-dependent binding to Rab33B via the coiled-coil domain, and intrinsic phosphoinositide binding by the coiled-coil domain, with these inputs feeding canonical, ULK1/FIP200-dependent starvation autophagy [PMID:23262492, PMID:30936093, PMID:31122169, PMID:32960676, PMID:34505572, PMID:36857448]. The activity and abundance of ATG16L1 are tuned by post-translational modification: CSNK2 phosphorylation at Ser139 and SETD7 methylation at Lys151 form a mutually antagonistic switch controlling association with the ATG12–ATG5 conjugate, ULK1 and PKA phosphorylation regulate its localization and degradative turnover, ZDHHC7-mediated S-palmitoylation at Cys153 promotes WIPI2B and RAB33B complex assembly, and the Gigaxonin E3 ligase drives WD40-dependent ubiquitination and proteasomal turnover [PMID:26083323, PMID:29634390, PMID:31267703, PMID:31580256, PMID:39087410, PMID:30770803]. The WD40 domain defines a separable arm of ATG16L1 function: it mediates V-ATPase-dependent non-canonical LC3 lipidation onto single membranes triggered by vacuolar damage, STING activation, or pathogen invasion, recruits LRRK2 to stressed lysosomes, and supports LC3-associated phagocytosis, all dispensable for canonical double-membrane autophagy [PMID:29317426, PMID:31327526, PMID:33201170, PMID:39089251, PMID:38227290, PMID:30403914]. ATG16L1 also carries autophagy-independent roles, negatively regulating NOD1/NOD2–RIP2 and TLR inflammatory signaling by limiting RIP2 ubiquitination and recruitment, supporting lysosome-fusion-dependent plasma membrane repair, and directing ciliary phosphoinositide trafficking through IFT20 and INPP5E [PMID:24238340, PMID:30478389, PMID:33910006, PMID:32909611]. Genetic loss of ATG16L1 disrupts Paneth cell granule exocytosis and intestinal immune homeostasis, and the Crohn's-disease-associated T300A variant is selectively impaired across multiple of these pathways [PMID:18849966, PMID:20637199, PMID:24238340, PMID:35220902].","teleology":[{"year":2003,"claim":"Established that ATG16L1 is a WD-repeat scaffold that bridges the ATG12–ATG5 conjugate into a large complex on the isolation membrane, defining its core architecture.","evidence":"Co-IP, size-exclusion chromatography, and GFP imaging in mouse cells","pmids":["12665549"],"confidence":"High","gaps":["Did not define how membrane targeting is achieved beyond ATG5 dependence","No structural detail of the WD40 domain or self-dimerization interface"]},{"year":2008,"claim":"Showed the complex acts as an E3-like enzyme that specifies the LC3 lipidation site by directing membrane localization, reframing ATG16L1 as a positional determinant rather than a passive scaffold.","evidence":"Forced-mislocalization to plasma membrane and biochemical LC3 lipidation assays","pmids":["18321988"],"confidence":"High","gaps":["Did not identify the physiological membrane-recruitment receptors","Catalytic contribution of ATG3 only partially defined"]},{"year":2008,"claim":"Demonstrated in vivo that ATG16L1 is required for autophagosome formation and additionally restrains inflammatory IL-1β/IL-18 production and supports Paneth cell biology, linking it to intestinal immunity.","evidence":"Atg16l1 KO and hypomorphic mice, LC3 immunoblot, EM, cytokine assays, human Crohn's tissue","pmids":["18849965","18849966"],"confidence":"High","gaps":["Did not separate autophagy-dependent from autophagy-independent contributions to inflammation","Mechanism of Paneth granule defect unresolved"]},{"year":2008,"claim":"Identified Rab33B as a GTP-dependent coiled-coil-domain partner controlling LC3 lipidation, providing an early recruitment input.","evidence":"GTP-agarose pulldown, co-IP, dominant-negative and LC3 lipidation assays","pmids":["18448665"],"confidence":"High","gaps":["Structural basis of recognition not yet resolved","Relationship to other recruitment factors unclear"]},{"year":2010,"claim":"Connected ATG16L1 to NOD2-dependent antibacterial autophagy and showed the T300A variant impairs MDP-driven bacterial killing in epithelial cells.","evidence":"siRNA knockdown, gentamicin protection assay, primary macrophages/dendritic cells","pmids":["20637199"],"confidence":"Medium","gaps":["Cell-type-specific effect of T300A not mechanistically explained","Single lab"]},{"year":2011,"claim":"Mapped phagophore-targeting specificity to the coiled-coil/middle region by contrasting ATG16L1 with its cytosolic paralog ATG16L2.","evidence":"Chimeric domain-swap analysis, fractionation, knockdown","pmids":["22082872"],"confidence":"Medium","gaps":["Did not identify the receptor recognized by the middle region","Single lab"]},{"year":2012,"claim":"Defined a dedicated FIP200-binding domain in ATG16L1 that selectively routes ULK1-dependent starvation autophagy, distinguishing it from ULK1-independent autophagy.","evidence":"Co-IP, domain-deletion mutants, condition-specific autophagy assays","pmids":["23262492"],"confidence":"High","gaps":["Structural detail of the FBD–FIP200 interface not resolved","Did not address how this integrates with WIPI2/Rab33B inputs"]},{"year":2013,"claim":"Established an autophagy-independent role: ATG16L1 directly suppresses NOD1/NOD2–RIP2 inflammatory signaling by blocking RIP2 ubiquitination, and the T300A allele is impaired in this function.","evidence":"Autophagy-incompetent truncation mutants, ubiquitination assays, cytokine ELISA","pmids":["24238340"],"confidence":"High","gaps":["Domain region responsible not fully mapped here","Structural basis of RIP2 binding unknown"]},{"year":2015,"claim":"Showed phosphoregulation of ATG16L1: CSNK2 phosphorylation at Ser139 promotes ATG12–ATG5 association and stress autophagy, antagonized by PP1 docking at a C-terminal RVxF motif.","evidence":"In vitro kinase assay, S139 mutagenesis, phospho-antibody, co-IP in cardiomyocytes","pmids":["26083323"],"confidence":"High","gaps":["In vivo relevance beyond cardiomyocytes not tested","Single lab"]},{"year":2015,"claim":"Identified upstream regulators of ATG16L1 supply: VDR as a transcriptional activator and Annexin A2 as a driver of ATG16L1-positive vesicle biogenesis and homotypic fusion.","evidence":"ChIP/reporter assays, VDR KO; proteomics/lipidomics and fusion assays in Anxa2 KO cells","pmids":["26218741","25597631"],"confidence":"Medium","gaps":["VDR regulation has limited mechanistic follow-up","How Annexin A2 vesicles relate to WIPI2/Rab33B recruitment unclear"]},{"year":2016,"claim":"Showed cell-type-specific ATG16L1 (distinct from ATG5/ATG9a) sustains intestinal Treg survival and restrains aberrant TH2 inflammation.","evidence":"T cell- and Treg-specific conditional KO mice, subset and metabolic analysis","pmids":["26910010"],"confidence":"High","gaps":["Molecular basis of the ATG16L1-selective requirement not defined","Link to its non-canonical functions untested"]},{"year":2017,"claim":"Provided a high-resolution WD40-domain structure, supplying the scaffold for understanding non-canonical protein-interaction functions.","evidence":"X-ray crystallography at 1.55 Å","pmids":["28685931"],"confidence":"High","gaps":["No co-complex structures with partners in this study","Functional residues not mutationally tested here"]},{"year":2018,"claim":"Genetically separated WD40-dependent single-membrane LC3 lipidation (non-canonical autophagy/LAP) from canonical double-membrane autophagy, with consequences for antigen presentation and viral infection.","evidence":"WD40 and linker+WD domain-deletion mice, LC3 lipidation/p62 assays, influenza and MHC-II readouts","pmids":["29317426","30403914"],"confidence":"High","gaps":["Did not identify the WD40 membrane-recruitment receptor (later V-ATPase)","Full repertoire of WD40-dependent processes incomplete"]},{"year":2018,"claim":"Established a methylation–phosphorylation switch (SETD7 K151 methylation opposing CSNK2 S139 phosphorylation) and an autophagy-independent role in lysosome-mediated plasma membrane repair.","evidence":"In vitro methylation assay, K151 mutagenesis; KO/T300A cells with lysosome fusion, cholesterol, and repair assays","pmids":["29634390","30478389"],"confidence":"High","gaps":["In vivo significance of K151 switch not established","How cholesterol accumulation arises mechanistically unresolved"]},{"year":2019,"claim":"Defined the V-ATPase as the WD40-domain receptor that recruits ATG16L1 to damaged vacuoles for xenophagy, a step targeted by the bacterial effector SopF.","evidence":"CRISPR and transposon screens, co-IP, ADP-ribosylation assay, domain deletion","pmids":["31327526"],"confidence":"High","gaps":["Precise V-ATPase subunit contact not yet defined (later V1H)","Coupling to membrane damage sensing incomplete"]},{"year":2019,"claim":"Reconstituted the full ATG12–ATG5–ATG16L1 complex in vitro and showed ATG16L1 intrinsically binds phosphoinositides (including PI3P) via coiled-coil residues required for PAS localization and LC3 lipidation.","evidence":"Purified full-length complex, in vitro lipidation, lipid-binding and mutagenesis, KO rescue","pmids":["30936093","31122169"],"confidence":"High","gaps":["Relative contribution of intrinsic lipid binding vs WIPI2/Rab33B unclear","Membrane curvature preferences not defined"]},{"year":2019,"claim":"Expanded the regulatory network: ULK1, PKA phosphorylation, and Gigaxonin-mediated ubiquitination control ATG16L1 localization and turnover, and ATG16L1 loss drives IRS1 degradation via a KLHL9/KLHL13–CUL3 ligase.","evidence":"In vitro kinase assays, phospho-dead lines, chemical-genetic PKA screen with in vivo validation, ubiquitination/turnover assays, BioID in KO MEFs","pmids":["31267703","31580256","30770803","31515271"],"confidence":"High","gaps":["How multiple modifications are integrated temporally is unresolved","Physiological hierarchy of degradation pathways unclear"]},{"year":2020,"claim":"Showed the V-ATPase–ATG16L1–WD40 axis mediates STING-induced single-membrane LC3B lipidation, generalizing non-canonical lipidation beyond pathogen invasion.","evidence":"STING agonist, WD40 mutants, bafilomycin and SopF inhibition, lipidation/imaging","pmids":["33201170"],"confidence":"High","gaps":["Functional output of STING-driven lipidation not fully defined","Trigger sensing upstream of V-ATPase unclear"]},{"year":2020,"claim":"Solved the Rab33B–ATG16L1 coiled-coil structure, revealing ATG16L1 as a noncanonical Rab effector that can drive an active Rab33B conformation without nucleotide exchange.","evidence":"Crystallography, MST, FLIM/FRET, mutagenesis, LC3 lipidation","pmids":["32960676"],"confidence":"High","gaps":["Physiological timing of Rab33B vs WIPI2 engagement unresolved","In vivo requirement not tested"]},{"year":2020,"claim":"Revealed an LC3-lipidation-independent WD40 function in trafficking: the Chlamydia effector CT622/TaiP mimics the ATG16L1-binding motif to block ATG16L1–TMEM59 interaction and reroute Rab6 compartments.","evidence":"Co-IP, domain mapping, TMEM59/Rab6 trafficking and lipidation-mutant analyses","pmids":["33055216"],"confidence":"High","gaps":["Endogenous role of the ATG16L1–TMEM59 interaction beyond infection unclear","Single pathogen system"]},{"year":2021,"claim":"Defined the WIPI2–ATG16L1 recruitment interface at atomic resolution and additional WD40 functions: A20 reciprocal regulation in intestinal homeostasis and IFT20/INPP5E-dependent ciliary phosphoinositide trafficking.","evidence":"WIPI2d–W2IR crystal structure with interface mutagenesis; WD40 interactome, double-KO mice, ciliary lipid/trafficking assays","pmids":["34505572","31015422","33910006"],"confidence":"High","gaps":["How a single WD40 domain coordinates many partners spatially is unresolved","In vivo ciliary relevance not fully established"]},{"year":2022,"claim":"Extended inflammatory regulation to TLR2–RIP2 signaling and showed the T300A variant amplifies TRAF6/RIPK2 ubiquitination and NF-κB responses through SQSTM1/p62 accumulation.","evidence":"Co-IP, NF-κB reporter and ubiquitination assays; T300A knockin mice with SQSTM1-KO rescue","pmids":["32909611","35220902"],"confidence":"High","gaps":["Direct vs autophagy-mediated contributions to p62 accumulation not fully separated in all systems","TLR2 finding from single lab"]},{"year":2023,"claim":"Showed ATG16L1 engages WIPI2 through two distinct sites (WBS1 and WBS2), both needed for efficient autophagic flux, refining the recruitment mechanism.","evidence":"Dual WIPI2b–ATG16L1 crystal structures, site-specific mutagenesis, flux assays","pmids":["36857448"],"confidence":"High","gaps":["How the two sites are used sequentially or cooperatively unresolved","Regulation of bivalent binding unknown"]},{"year":2024,"claim":"Resolved the molecular mechanism of LC3 lipidation and membrane shaping: a three-step WIPI2/ATG16L1/ATG3 docking model, ATG16L1+LC3B membrane-coat-driven cup formation, V1H as the assembled-V-ATPase contact, palmitoylation control, and WD40-dependent lysosomal LRRK2 recruitment.","evidence":"MD simulations with in vitro lipidation; reconstitution/EM of membrane cups; V1H direct binding in neurons; ABE palmitoylation/C153S rescue; WD40-deletion LRRK2 recruitment assays","pmids":["38324698","38834913","39089251","39087410","38227290"],"confidence":"High","gaps":["Catalytic histidines in ATG3 proposed but not definitively validated","How distinct WD40 outputs (LRRK2, non-canonical lipidation, trafficking) are selected remains unresolved"]},{"year":null,"claim":"How the diverse inputs converging on ATG16L1 — multiple post-translational modifications, WIPI2/Rab33B/V-ATPase receptors, and competing WD40 partners — are integrated to select between canonical autophagy, single-membrane lipidation, trafficking, and inflammatory regulation in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model coordinating modification state with partner selection","Mechanism distinguishing competing WD40-domain functions undefined","In vivo hierarchy of canonical vs non-canonical roles unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,31]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,7,20,21,26]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[14,30]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[8,24]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,30]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,6]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,35]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[12,29]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,12]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[23]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0,2,7,14]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,8,13,24,25]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,5,25]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[12,35]}],"complexes":["ATG12-ATG5-ATG16L1 E3-like complex","V-ATPase (interacting recruitment platform via V1H)"],"partners":["ATG5","WIPI2","RAB33B","FIP200","ATG3","RIP2","IFT20","ATP6V1H"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q676U5","full_name":"Autophagy-related protein 16-1","aliases":["APG16-like 1"],"length_aa":607,"mass_kda":68.3,"function":"Plays an essential role in both canonical and non-canonical autophagy: interacts with ATG12-ATG5 to mediate the lipidation to ATG8 family proteins (MAP1LC3A, MAP1LC3B, MAP1LC3C, GABARAPL1, GABARAPL2 and GABARAP) (PubMed:23376921, PubMed:23392225, PubMed:24553140, PubMed:24954904, PubMed:27273576, PubMed:29317426, PubMed:30778222, PubMed:33909989). Acts as a molecular hub, coordinating autophagy pathways via distinct domains that support either canonical or non-canonical signaling (PubMed:29317426, PubMed:30778222). During canonical autophagy, interacts with ATG12-ATG5 to mediate the conjugation of phosphatidylethanolamine (PE) to ATG8 proteins, to produce a membrane-bound activated form of ATG8 (PubMed:23376921, PubMed:23392225, PubMed:24553140, PubMed:24954904, PubMed:27273576). Thereby, controls the elongation of the nascent autophagosomal membrane (PubMed:23376921, PubMed:23392225, PubMed:24553140, PubMed:24954904, PubMed:27273576). As part of the ATG8 conjugation system with ATG5 and ATG12, required for recruitment of LRRK2 to stressed lysosomes and induction of LRRK2 kinase activity in response to lysosomal stress (By similarity). Also involved in non-canonical autophagy, a parallel pathway involving conjugation of ATG8 proteins to single membranes at endolysosomal compartments, probably by catalyzing conjugation of phosphatidylserine (PS) to ATG8 (PubMed:33909989). Non-canonical autophagy plays a key role in epithelial cells to limit lethal infection by influenza A (IAV) virus (By similarity). Regulates mitochondrial antiviral signaling (MAVS)-dependent type I interferon (IFN-I) production (PubMed:22749352, PubMed:25645662). Negatively regulates NOD1- and NOD2-driven inflammatory cytokine response (PubMed:24238340). Instead, promotes an autophagy-dependent antibacterial pathway together with NOD1 or NOD2 (PubMed:20637199). Plays a role in regulating morphology and function of Paneth cell (PubMed:18849966)","subcellular_location":"Cytoplasm; Preautophagosomal structure membrane; Endosome membrane; Lysosome membrane","url":"https://www.uniprot.org/uniprotkb/Q676U5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ATG16L1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ATG12","stoichiometry":0.2},{"gene":"ATG3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ATG16L1","total_profiled":1310},"omim":[{"mim_id":"621000","title":"SORTING NEXIN 18; SNX18","url":"https://www.omim.org/entry/621000"},{"mim_id":"619923","title":"PROTEIN PHOSPHATASE 1, REGULATORY SUBUNIT 36; PPP1R36","url":"https://www.omim.org/entry/619923"},{"mim_id":"618990","title":"EVA1 HOMOLOG A, REGULATOR OF PROGRAMMED CELL DEATH; EVA1A","url":"https://www.omim.org/entry/618990"},{"mim_id":"618716","title":"AUTOPHAGY 16-LIKE 2; ATG16L2","url":"https://www.omim.org/entry/618716"},{"mim_id":"618453","title":"INTELLECTUAL DEVELOPMENTAL DISORDER WITH SHORT STATURE AND VARIABLE SKELETAL ANOMALIES; IDDSSA","url":"https://www.omim.org/entry/618453"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Equatorial segment","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ATG16L1"},"hgnc":{"alias_symbol":["WDR30","FLJ10035","ATG16A"],"prev_symbol":["APG16L","ATG16L"]},"alphafold":{"accession":"Q676U5","domains":[{"cath_id":"2.130.10.10","chopping":"314-606","consensus_level":"high","plddt":96.7951,"start":314,"end":606},{"cath_id":"1.20.5","chopping":"104-184","consensus_level":"medium","plddt":96.4791,"start":104,"end":184}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q676U5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q676U5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q676U5-F1-predicted_aligned_error_v6.png","plddt_mean":83.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATG16L1","jax_strain_url":"https://www.jax.org/strain/search?query=ATG16L1"},"sequence":{"accession":"Q676U5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q676U5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q676U5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q676U5"}},"corpus_meta":[{"pmid":"18849965","id":"PMC_18849965","title":"Loss 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shellfish immunology","url":"https://pubmed.ncbi.nlm.nih.gov/31491526","citation_count":14,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":54313,"output_tokens":10822,"usd":0.162634,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":21282,"output_tokens":7530,"usd":0.14733,"stage2_stop_reason":"end_turn"},"total_usd":0.309964,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, size-exclusion chromatography, fluorescence microscopy of GFP-tagged proteins\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, biochemical fractionation, and live-cell imaging, independently establishing complex composition and membrane targeting; foundational paper replicated by many subsequent studies\",\n      \"pmids\": [\"12665549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"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.\",\n      \"method\": \"Overexpression/mislocalization experiments, immunofluorescence, biochemical LC3 lipidation assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution-style forced-localization experiment directly establishing E3-like scaffold function; replicated conceptually in multiple subsequent studies\",\n      \"pmids\": [\"18321988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"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.\",\n      \"method\": \"Atg16L1 knockout mice, immunoblot for LC3 lipidation, electron microscopy, caspase-1 activation assays, cytokine ELISA\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO mouse model with multiple orthogonal readouts (autophagosome formation, LC3 lipidation, cytokine production, caspase activation); widely replicated\",\n      \"pmids\": [\"18849965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"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).\",\n      \"method\": \"Hypomorphic ATG16L1 mouse generation, electron microscopy of Paneth cell granules, transcriptional profiling, immunohistochemistry of human Crohn's disease tissue\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic hypomorphic mouse model validated in human tissue samples, multiple orthogonal readouts; widely replicated\",\n      \"pmids\": [\"18849966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, GTP-agarose pulldown, LC3 lipidation assay, p62 degradation assay, dominant-negative/overexpression experiments\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — GTP-dependent pulldown, reciprocal co-IP, functional mutant analysis with defined autophagy readouts; replicated structurally in PMID 32960676\",\n      \"pmids\": [\"18448665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"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.\",\n      \"method\": \"siRNA knockdown, confocal microscopy, flow cytometry, gentamicin protection assay, primary human macrophages and dendritic cells\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell types and methods (siRNA, microscopy, bacterial killing assay), but single lab\",\n      \"pmids\": [\"20637199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"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.\",\n      \"method\": \"Biochemical fractionation, immunofluorescence, chimeric protein analysis, ATG16L2 knockdown\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-swap chimera analysis with functional readouts, single lab\",\n      \"pmids\": [\"22082872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion mutants, autophagy induction assays under different starvation conditions\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct interaction mapped to specific domain with direct functional consequence distinguishing two autophagy pathways; multiple orthogonal methods in single study\",\n      \"pmids\": [\"23262492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"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.\",\n      \"method\": \"siRNA knockdown, ubiquitination assays, complex assembly analysis, cytokine ELISA, autophagy-incompetent ATG16L1 truncation mutants\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — autophagy-independent function established using truncation mutants incapable of autophagy, Rip2 ubiquitination mechanistically defined, multiple orthogonal methods\",\n      \"pmids\": [\"24238340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (S139A/D), co-immunoprecipitation, phospho-specific antibody, CSNK2 inhibitor treatment, shRNA knockdown\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis identifying specific phosphorylation site and functional consequence; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"26083323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"WD40 domain deletion mutants, LC3 lipidation assays, MHC II antigen presentation assays in dendritic cells from transgenic mice, influenza infection model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-deletion approach distinguishing canonical vs non-canonical autophagy with in vivo mouse genetic validation and multiple functional readouts\",\n      \"pmids\": [\"29317426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro methylation assay, site-directed mutagenesis (K151), co-immunoprecipitation, SETD7 shRNA knockdown, small molecule SETD7 inhibitor\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro methylation assay with mutagenesis, crosstalk with phosphorylation mechanistically defined, multiple orthogonal approaches in single lab\",\n      \"pmids\": [\"29634390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"ATG16L1 KO cells, lysosome fusion assays, cholesterol staining, blebbing assays, Listeria cell-to-cell spread assay, T300A variant cells\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple KO and variant cell models with defined mechanistic readouts (lysosome fusion, cholesterol, blebbing) establishing autophagy-independent plasma membrane repair function\",\n      \"pmids\": [\"30478389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"CRISPR screen, bacterial transposon screen, co-immunoprecipitation, SopF enzymatic ADP-ribosylation assay, site-directed mutagenesis (Q124), WD40 domain deletion\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical ADP-ribosylation assay, CRISPR screen, domain mapping, and mutagenesis all converging on the same axis; published in Cell with multiple orthogonal methods\",\n      \"pmids\": [\"31327526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components, mutagenesis, and rescue experiments in knockout cells; multiple orthogonal approaches\",\n      \"pmids\": [\"30936093\", \"31122169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Gigaxonin KO/knockdown, ATG16L1 turnover assay, LC3 lipidation and p62 degradation readouts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, ubiquitination assay, genetic KO with multiple downstream autophagy readouts; published in Nature Communications\",\n      \"pmids\": [\"30770803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro ULK1 kinase assay, phospho-dead ATG16L1 stable cell lines, bacterial infection xenophagy assay, ATG16L1 T300A variant comparison\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay establishing direct phosphorylation, stable phospho-dead cell lines with xenophagy readout, T300A comparison\",\n      \"pmids\": [\"31267703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"Chemical genetic PKA screen, mass spectrometry, peptide spot arrays, biochemical validation, mouse in vivo genetics, pharmacological autophagy inhibition\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — chemical genetic screen, MS-identified phosphorylation sites, in vivo mouse validation, multiple orthogonal methods\",\n      \"pmids\": [\"31580256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"ATG16L1 KO MEFs, BioID proximity labeling, co-immunoprecipitation, proteasome inhibitor treatment, siRNA knockdown of Klhl9/Klhl13/Cul3, insulin signaling assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BioID and co-IP identifying novel complex, siRNA rescue experiments, proteasome inhibitor confirmation; multiple orthogonal methods\",\n      \"pmids\": [\"31515271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"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.\",\n      \"method\": \"STING agonist treatment, WD40 domain deletion/mutation, bafilomycin A1 and SopF inhibition, LC3B lipidation assays, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-specific genetic approach, pharmacological inhibition, bacterial effector tool; multiple independent lines of evidence in one study\",\n      \"pmids\": [\"33201170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"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.\",\n      \"method\": \"Crystal structure determination, pulldown assays, microscale thermophoresis (binding affinity), fluorescence lifetime imaging (FLIM/FRET), site-directed mutagenesis, LC3 lipidation assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of RAB33B-ATG16L1 CCD complex with mutagenesis and functional validation; multiple orthogonal structural and biochemical methods\",\n      \"pmids\": [\"32960676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"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.\",\n      \"method\": \"X-ray crystallography (1.85 Å), interface mutagenesis, in vitro LC3B lipidation to synthetic membranes, cell-based autophagy assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus in vitro reconstitution plus cellular validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"34505572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"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.\",\n      \"method\": \"WD40 domain proteomic screen (ATG16L1 WDD interactome), co-immunoprecipitation, conditional double-KO mice, intestinal phenotyping\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteomic screen validated by co-IP, in vivo double-KO mouse model with defined pathological phenotype\",\n      \"pmids\": [\"31015422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping (WD40 deletion, IFT20 motif mutation), phosphoinositide staining, confocal microscopy of ATG16L1-KO cells, INPP5E trafficking assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP with domain mapping, KO cells, lipid and protein trafficking readouts; multiple orthogonal methods\",\n      \"pmids\": [\"33910006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation in HEK293 cells and primary human dendritic cells, domain mapping, NF-κB reporter assay, ubiquitination assay, cytokine ELISA\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP in two cell types, ubiquitination assay, NF-κB reporter; single lab\",\n      \"pmids\": [\"32909611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"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.\",\n      \"method\": \"ATG16L1 T300A knockin mice and cells, SQSTM1 KO rescue experiments, polyubiquitination assays, NF-κB activation assays, cytokine measurement\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockin mouse model, genetic rescue (SQSTM1 KO), mechanistic link to ubiquitination; multiple orthogonal methods\",\n      \"pmids\": [\"35220902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"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.\",\n      \"method\": \"X-ray crystallography of WIPI2b-ATG16L1 WBS1 and WBS2 complexes, mutagenesis at each binding site, autophagic flux assays in cells\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — dual crystal structures plus mutagenesis plus cellular flux assays; multiple orthogonal methods in one study\",\n      \"pmids\": [\"36857448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding established biochemically, structural loop mapped, validated in multiple cell systems including neurons; published in Molecular Cell\",\n      \"pmids\": [\"39089251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"Acyl-biotin exchange (ABE) palmitoylation assay, ZDHHC7 KO/overexpression, C153S site-directed mutagenesis, rescue in ATG16L1-KO cells, co-immunoprecipitation for WIPI2B/RAB33B interactions\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro palmitoylation assay, site-directed mutagenesis, KO rescue, mechanistic complex formation analysis; multiple orthogonal methods\",\n      \"pmids\": [\"39087410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"ATG16L1 WD40 domain deletion, LRRK2 recruitment assays to lysosomes, lysosomal damage models, lysosomal secretion and size readouts, independence from canonical autophagy initiation complex confirmed\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-specific ATG16L1 mutants, LRRK2 recruitment and activation assays, multiple lysosomal functional readouts; published in JCB\",\n      \"pmids\": [\"38227290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"Two complementary in vitro membrane reconstitution approaches, electron microscopy of membrane cups, truncation mutants of ATG16L1, comparison across ATG8 family members\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components on membranes, EM validation, truncation series establishing domain requirements; published in Nature Structural & Molecular Biology\",\n      \"pmids\": [\"38834913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"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.\",\n      \"method\": \"Molecular dynamics simulations, in vitro LC3 lipidation assays on synthetic membranes, cell-based validation experiments\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — MD simulation integrated with in vitro reconstitution and cellular validation; mechanistic pathway defined with multiple approaches\",\n      \"pmids\": [\"38324698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"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).\",\n      \"method\": \"X-ray crystallography (1.55 Å resolution)\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure; single structural study without extensive functional mutagenesis in the same paper\",\n      \"pmids\": [\"28685931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"Domain-deletion mouse genetics (linker+WD40 deletion), LC3 lipidation and p62/SQSTM1 assays in tissues, WIPI2-binding mutant (E230 deletion), neonatal survival assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo mouse genetics with domain-deletion approach, multiple tissue and functional readouts distinguishing autophagy from LAP\",\n      \"pmids\": [\"30403914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"ChIP/transcriptional reporter assays establishing VDR binding to ATG16L1 promoter, VDR KO model, ATG16L1 expression correlation\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — transcriptional regulation established by reporter assay, single lab, limited mechanistic follow-up in abstract\",\n      \"pmids\": [\"26218741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"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.\",\n      \"method\": \"Ultrastructural analysis, proteomics of Atg16L+ vesicles, FACS, lipidomics of Anxa2 KO cells, homotypic fusion assay, LC3 flux in Anxa2-KO dendritic cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (proteomics, lipidomics, ultrastructure, fusion assay, KO), published in Nature Communications\",\n      \"pmids\": [\"25597631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"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.\",\n      \"method\": \"T cell-specific and Treg-specific Atg16l1 conditional KO mice, intestinal inflammation scoring, T cell subset analysis, metabolic assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional KO with defined cellular and metabolic phenotypes; published in eLife\",\n      \"pmids\": [\"26910010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation (14-3-3ζ and ATG16L1), 14-3-3ζ KO/overexpression, ATG16L1 localization by confocal microscopy\",\n      \"journal\": \"American journal of translational research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP without domain mapping or in vitro validation; single lab\",\n      \"pmids\": [\"29422997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"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.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping (WD40), TMEM59 interaction assay, Rab6 vesicle trafficking assay, LC3-lipidation mutant analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — bacterial effector molecular mimicry validated biochemically, mechanistic dissection of WD40-dependent vs. LC3-lipidation-independent functions; multiple orthogonal methods\",\n      \"pmids\": [\"33055216\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATG16L1 is a core scaffold component of the ~800 kDa ATG12-ATG5-ATG16L1 E3-like complex that lipidates LC3/ATG8 family proteins onto autophagosomal and other membranes: it is recruited to the phagophore via direct interactions with WIPI2 (through both WBS1 and WBS2 sites), Rab33B (via its coiled-coil domain), and the V-ATPase (via the WD40 domain/V1H subunit), and its activity is regulated by phosphorylation (CSNK2 at Ser139, ULK1, PKA at Ser268/269), methylation (SETD7 at Lys151), S-palmitoylation (ZDHHC7 at Cys153), and ubiquitin-dependent turnover (Gigaxonin E3 ligase targeting the WD40 domain); beyond canonical double-membrane autophagy, the WD40 domain specifically mediates non-canonical LC3 lipidation on single membranes (V-ATPase-dependent, triggered by vacuolar damage, STING activation, or pathogen invasion), lysosomal homeostasis via LRRK2 recruitment, plasma membrane repair via lysosome fusion, primary cilia lipid/protein trafficking via IFT20 and INPP5E interactions, and autophagy-independent suppression of NOD/RIP2 and TLR inflammatory signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATG16L1 is the scaffold subunit of the ~800 kDa ATG12–ATG5–ATG16L1 E3-like ligase that determines where the ATG8/LC3 family is conjugated to membrane phosphatidylethanolamine, thereby governing autophagosome biogenesis and several autophagy-related membrane processes [#0, #1]. It self-dimerizes and binds ATG5 through domains distinct from its C-terminal WD40 region, and ATG5-dependent membrane targeting positions the complex on the isolation membrane for the duration of autophagosome formation; loss of ATG16L1 abolishes ATG12–ATG5 recruitment, LC3 lipidation, and autophagosome formation [#0, #2]. The complex functions as a membrane-acting E3 that dictates the lipidation site, working with the ATG3 E2 to position ATG3~LC3 at the membrane, and ATG16L1 together with LC3B can remodel flat membranes into phagophore-like cups via its C-terminal membrane-binding domain [#1, #30, #31]. Recruitment to the phagophore is achieved through a dual-site interaction with the PI3P effector WIPI2 (WBS1 and WBS2), GTP-dependent binding to Rab33B via the coiled-coil domain, and intrinsic phosphoinositide binding by the coiled-coil domain, with these inputs feeding canonical, ULK1/FIP200-dependent starvation autophagy [#7, #14, #20, #21, #26]. The activity and abundance of ATG16L1 are tuned by post-translational modification: CSNK2 phosphorylation at Ser139 and SETD7 methylation at Lys151 form a mutually antagonistic switch controlling association with the ATG12–ATG5 conjugate, ULK1 and PKA phosphorylation regulate its localization and degradative turnover, ZDHHC7-mediated S-palmitoylation at Cys153 promotes WIPI2B and RAB33B complex assembly, and the Gigaxonin E3 ligase drives WD40-dependent ubiquitination and proteasomal turnover [#9, #11, #16, #17, #28, #15]. The WD40 domain defines a separable arm of ATG16L1 function: it mediates V-ATPase-dependent non-canonical LC3 lipidation onto single membranes triggered by vacuolar damage, STING activation, or pathogen invasion, recruits LRRK2 to stressed lysosomes, and supports LC3-associated phagocytosis, all dispensable for canonical double-membrane autophagy [#10, #13, #19, #27, #29, #33]. ATG16L1 also carries autophagy-independent roles, negatively regulating NOD1/NOD2–RIP2 and TLR inflammatory signaling by limiting RIP2 ubiquitination and recruitment, supporting lysosome-fusion-dependent plasma membrane repair, and directing ciliary phosphoinositide trafficking through IFT20 and INPP5E [#8, #12, #23, #24]. Genetic loss of ATG16L1 disrupts Paneth cell granule exocytosis and intestinal immune homeostasis, and the Crohn's-disease-associated T300A variant is selectively impaired across multiple of these pathways [#3, #5, #8, #25].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that ATG16L1 is a WD-repeat scaffold that bridges the ATG12–ATG5 conjugate into a large complex on the isolation membrane, defining its core architecture.\",\n      \"evidence\": \"Co-IP, size-exclusion chromatography, and GFP imaging in mouse cells\",\n      \"pmids\": [\"12665549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how membrane targeting is achieved beyond ATG5 dependence\", \"No structural detail of the WD40 domain or self-dimerization interface\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed the complex acts as an E3-like enzyme that specifies the LC3 lipidation site by directing membrane localization, reframing ATG16L1 as a positional determinant rather than a passive scaffold.\",\n      \"evidence\": \"Forced-mislocalization to plasma membrane and biochemical LC3 lipidation assays\",\n      \"pmids\": [\"18321988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological membrane-recruitment receptors\", \"Catalytic contribution of ATG3 only partially defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated in vivo that ATG16L1 is required for autophagosome formation and additionally restrains inflammatory IL-1β/IL-18 production and supports Paneth cell biology, linking it to intestinal immunity.\",\n      \"evidence\": \"Atg16l1 KO and hypomorphic mice, LC3 immunoblot, EM, cytokine assays, human Crohn's tissue\",\n      \"pmids\": [\"18849965\", \"18849966\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate autophagy-dependent from autophagy-independent contributions to inflammation\", \"Mechanism of Paneth granule defect unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified Rab33B as a GTP-dependent coiled-coil-domain partner controlling LC3 lipidation, providing an early recruitment input.\",\n      \"evidence\": \"GTP-agarose pulldown, co-IP, dominant-negative and LC3 lipidation assays\",\n      \"pmids\": [\"18448665\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of recognition not yet resolved\", \"Relationship to other recruitment factors unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connected ATG16L1 to NOD2-dependent antibacterial autophagy and showed the T300A variant impairs MDP-driven bacterial killing in epithelial cells.\",\n      \"evidence\": \"siRNA knockdown, gentamicin protection assay, primary macrophages/dendritic cells\",\n      \"pmids\": [\"20637199\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-type-specific effect of T300A not mechanistically explained\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapped phagophore-targeting specificity to the coiled-coil/middle region by contrasting ATG16L1 with its cytosolic paralog ATG16L2.\",\n      \"evidence\": \"Chimeric domain-swap analysis, fractionation, knockdown\",\n      \"pmids\": [\"22082872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the receptor recognized by the middle region\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined a dedicated FIP200-binding domain in ATG16L1 that selectively routes ULK1-dependent starvation autophagy, distinguishing it from ULK1-independent autophagy.\",\n      \"evidence\": \"Co-IP, domain-deletion mutants, condition-specific autophagy assays\",\n      \"pmids\": [\"23262492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of the FBD–FIP200 interface not resolved\", \"Did not address how this integrates with WIPI2/Rab33B inputs\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established an autophagy-independent role: ATG16L1 directly suppresses NOD1/NOD2–RIP2 inflammatory signaling by blocking RIP2 ubiquitination, and the T300A allele is impaired in this function.\",\n      \"evidence\": \"Autophagy-incompetent truncation mutants, ubiquitination assays, cytokine ELISA\",\n      \"pmids\": [\"24238340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain region responsible not fully mapped here\", \"Structural basis of RIP2 binding unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed phosphoregulation of ATG16L1: CSNK2 phosphorylation at Ser139 promotes ATG12–ATG5 association and stress autophagy, antagonized by PP1 docking at a C-terminal RVxF motif.\",\n      \"evidence\": \"In vitro kinase assay, S139 mutagenesis, phospho-antibody, co-IP in cardiomyocytes\",\n      \"pmids\": [\"26083323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance beyond cardiomyocytes not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified upstream regulators of ATG16L1 supply: VDR as a transcriptional activator and Annexin A2 as a driver of ATG16L1-positive vesicle biogenesis and homotypic fusion.\",\n      \"evidence\": \"ChIP/reporter assays, VDR KO; proteomics/lipidomics and fusion assays in Anxa2 KO cells\",\n      \"pmids\": [\"26218741\", \"25597631\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"VDR regulation has limited mechanistic follow-up\", \"How Annexin A2 vesicles relate to WIPI2/Rab33B recruitment unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed cell-type-specific ATG16L1 (distinct from ATG5/ATG9a) sustains intestinal Treg survival and restrains aberrant TH2 inflammation.\",\n      \"evidence\": \"T cell- and Treg-specific conditional KO mice, subset and metabolic analysis\",\n      \"pmids\": [\"26910010\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the ATG16L1-selective requirement not defined\", \"Link to its non-canonical functions untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided a high-resolution WD40-domain structure, supplying the scaffold for understanding non-canonical protein-interaction functions.\",\n      \"evidence\": \"X-ray crystallography at 1.55 Å\",\n      \"pmids\": [\"28685931\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-complex structures with partners in this study\", \"Functional residues not mutationally tested here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Genetically separated WD40-dependent single-membrane LC3 lipidation (non-canonical autophagy/LAP) from canonical double-membrane autophagy, with consequences for antigen presentation and viral infection.\",\n      \"evidence\": \"WD40 and linker+WD domain-deletion mice, LC3 lipidation/p62 assays, influenza and MHC-II readouts\",\n      \"pmids\": [\"29317426\", \"30403914\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the WD40 membrane-recruitment receptor (later V-ATPase)\", \"Full repertoire of WD40-dependent processes incomplete\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established a methylation–phosphorylation switch (SETD7 K151 methylation opposing CSNK2 S139 phosphorylation) and an autophagy-independent role in lysosome-mediated plasma membrane repair.\",\n      \"evidence\": \"In vitro methylation assay, K151 mutagenesis; KO/T300A cells with lysosome fusion, cholesterol, and repair assays\",\n      \"pmids\": [\"29634390\", \"30478389\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of K151 switch not established\", \"How cholesterol accumulation arises mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the V-ATPase as the WD40-domain receptor that recruits ATG16L1 to damaged vacuoles for xenophagy, a step targeted by the bacterial effector SopF.\",\n      \"evidence\": \"CRISPR and transposon screens, co-IP, ADP-ribosylation assay, domain deletion\",\n      \"pmids\": [\"31327526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise V-ATPase subunit contact not yet defined (later V1H)\", \"Coupling to membrane damage sensing incomplete\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Reconstituted the full ATG12–ATG5–ATG16L1 complex in vitro and showed ATG16L1 intrinsically binds phosphoinositides (including PI3P) via coiled-coil residues required for PAS localization and LC3 lipidation.\",\n      \"evidence\": \"Purified full-length complex, in vitro lipidation, lipid-binding and mutagenesis, KO rescue\",\n      \"pmids\": [\"30936093\", \"31122169\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of intrinsic lipid binding vs WIPI2/Rab33B unclear\", \"Membrane curvature preferences not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expanded the regulatory network: ULK1, PKA phosphorylation, and Gigaxonin-mediated ubiquitination control ATG16L1 localization and turnover, and ATG16L1 loss drives IRS1 degradation via a KLHL9/KLHL13–CUL3 ligase.\",\n      \"evidence\": \"In vitro kinase assays, phospho-dead lines, chemical-genetic PKA screen with in vivo validation, ubiquitination/turnover assays, BioID in KO MEFs\",\n      \"pmids\": [\"31267703\", \"31580256\", \"30770803\", \"31515271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How multiple modifications are integrated temporally is unresolved\", \"Physiological hierarchy of degradation pathways unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed the V-ATPase–ATG16L1–WD40 axis mediates STING-induced single-membrane LC3B lipidation, generalizing non-canonical lipidation beyond pathogen invasion.\",\n      \"evidence\": \"STING agonist, WD40 mutants, bafilomycin and SopF inhibition, lipidation/imaging\",\n      \"pmids\": [\"33201170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional output of STING-driven lipidation not fully defined\", \"Trigger sensing upstream of V-ATPase unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Solved the Rab33B–ATG16L1 coiled-coil structure, revealing ATG16L1 as a noncanonical Rab effector that can drive an active Rab33B conformation without nucleotide exchange.\",\n      \"evidence\": \"Crystallography, MST, FLIM/FRET, mutagenesis, LC3 lipidation\",\n      \"pmids\": [\"32960676\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological timing of Rab33B vs WIPI2 engagement unresolved\", \"In vivo requirement not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed an LC3-lipidation-independent WD40 function in trafficking: the Chlamydia effector CT622/TaiP mimics the ATG16L1-binding motif to block ATG16L1–TMEM59 interaction and reroute Rab6 compartments.\",\n      \"evidence\": \"Co-IP, domain mapping, TMEM59/Rab6 trafficking and lipidation-mutant analyses\",\n      \"pmids\": [\"33055216\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous role of the ATG16L1–TMEM59 interaction beyond infection unclear\", \"Single pathogen system\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the WIPI2–ATG16L1 recruitment interface at atomic resolution and additional WD40 functions: A20 reciprocal regulation in intestinal homeostasis and IFT20/INPP5E-dependent ciliary phosphoinositide trafficking.\",\n      \"evidence\": \"WIPI2d–W2IR crystal structure with interface mutagenesis; WD40 interactome, double-KO mice, ciliary lipid/trafficking assays\",\n      \"pmids\": [\"34505572\", \"31015422\", \"33910006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single WD40 domain coordinates many partners spatially is unresolved\", \"In vivo ciliary relevance not fully established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended inflammatory regulation to TLR2–RIP2 signaling and showed the T300A variant amplifies TRAF6/RIPK2 ubiquitination and NF-κB responses through SQSTM1/p62 accumulation.\",\n      \"evidence\": \"Co-IP, NF-κB reporter and ubiquitination assays; T300A knockin mice with SQSTM1-KO rescue\",\n      \"pmids\": [\"32909611\", \"35220902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs autophagy-mediated contributions to p62 accumulation not fully separated in all systems\", \"TLR2 finding from single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed ATG16L1 engages WIPI2 through two distinct sites (WBS1 and WBS2), both needed for efficient autophagic flux, refining the recruitment mechanism.\",\n      \"evidence\": \"Dual WIPI2b–ATG16L1 crystal structures, site-specific mutagenesis, flux assays\",\n      \"pmids\": [\"36857448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the two sites are used sequentially or cooperatively unresolved\", \"Regulation of bivalent binding unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the molecular mechanism of LC3 lipidation and membrane shaping: a three-step WIPI2/ATG16L1/ATG3 docking model, ATG16L1+LC3B membrane-coat-driven cup formation, V1H as the assembled-V-ATPase contact, palmitoylation control, and WD40-dependent lysosomal LRRK2 recruitment.\",\n      \"evidence\": \"MD simulations with in vitro lipidation; reconstitution/EM of membrane cups; V1H direct binding in neurons; ABE palmitoylation/C153S rescue; WD40-deletion LRRK2 recruitment assays\",\n      \"pmids\": [\"38324698\", \"38834913\", \"39089251\", \"39087410\", \"38227290\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic histidines in ATG3 proposed but not definitively validated\", \"How distinct WD40 outputs (LRRK2, non-canonical lipidation, trafficking) are selected remains unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse inputs converging on ATG16L1 — multiple post-translational modifications, WIPI2/Rab33B/V-ATPase receptors, and competing WD40 partners — are integrated to select between canonical autophagy, single-membrane lipidation, trafficking, and inflammatory regulation in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model coordinating modification state with partner selection\", \"Mechanism distinguishing competing WD40-domain functions undefined\", \"In vivo hierarchy of canonical vs non-canonical roles unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 31]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 7, 20, 21, 26]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [14, 30]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [8, 24]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 30]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 35]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [12, 29]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 12]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0, 2, 7, 14]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 8, 13, 24, 25]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 5, 25]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [12, 35]}\n    ],\n    \"complexes\": [\n      \"ATG12-ATG5-ATG16L1 E3-like complex\",\n      \"V-ATPase (interacting recruitment platform via V1H)\"\n    ],\n    \"partners\": [\n      \"ATG5\",\n      \"WIPI2\",\n      \"RAB33B\",\n      \"FIP200\",\n      \"ATG3\",\n      \"RIP2\",\n      \"IFT20\",\n      \"ATP6V1H\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}