{"gene":"AMBRA1","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2007,"finding":"AMBRA1 is a positive regulator of BECLIN1-dependent autophagy; it bears a WD40 domain and interacts with BECLIN1 to promote autophagosome formation. Loss of AMBRA1 function in mouse embryos impairs autophagy, causes accumulation of ubiquitinated proteins, and leads to severe neural tube defects with unbalanced cell proliferation and excessive apoptosis.","method":"RNA interference in vitro, overexpression assays, mouse genetic knockout model with phenotypic analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function in vivo and in vitro with multiple orthogonal readouts; foundational paper replicated by many subsequent studies","pmids":["17589504"],"is_preprint":false},{"year":2010,"finding":"Under basal conditions, the BECLIN1-VPS34 complex is tethered to the dynein motor complex via AMBRA1's direct interaction with dynein light chains DLC1/2. Upon autophagy induction, ULK1 phosphorylates AMBRA1, releasing it from dynein; the complex then relocalizes to the endoplasmic reticulum, enabling autophagosome nucleation.","method":"Co-immunoprecipitation, live-cell imaging, ULK1 kinase assays, DLC1 knockdown, AMBRA1 DLC1-binding site mutagenesis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct interaction demonstrated by Co-IP, phosphorylation established by kinase assay, mutagenesis of binding sites, replicated in follow-up commentary (PMID 21079415)","pmids":["20921139","21079415"],"is_preprint":false},{"year":2011,"finding":"Mitochondrial BCL-2 binds AMBRA1 and inhibits AMBRA1-induced autophagy. AMBRA1 preferentially binds the mitochondrial pool of BCL-2; upon autophagy induction this interaction is disrupted and AMBRA1 is recruited to BECLIN1. AMBRA1 can compete with both mitochondrial and ER-resident BCL-2 for BECLIN1 binding.","method":"Co-immunoprecipitation, subcellular fractionation, overexpression and knockdown assays","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reciprocal Co-IP and functional competition assays in single lab, multiple methods","pmids":["21358617"],"is_preprint":false},{"year":2011,"finding":"Parkin interacts with AMBRA1 (identified by tandem affinity purification/mass spectrometry from HEK293 cells and validated by endogenous Co-IP). Upon prolonged mitochondrial depolarization, the Parkin–AMBRA1 interaction increases; AMBRA1 is recruited to perinuclear clusters of depolarized mitochondria in a Parkin-dependent manner and activates class III PI3K locally, promoting mitophagic clearance.","method":"Tandem affinity purification, mass spectrometry, endogenous Co-immunoprecipitation from HEK293, SH-SY5Y cells and adult mouse brain, siRNA knockdown, fluorescence microscopy","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — interaction validated by MS and reciprocal endogenous Co-IP in three independent cell/tissue systems; functional consequence demonstrated by AMBRA1 knockdown","pmids":["21753002","21921694"],"is_preprint":false},{"year":2012,"finding":"During apoptosis, AMBRA1 is proteolytically degraded by the combined action of caspases (cleaving at D482) and calpains (mediating complete degradation), dismantling autophagic activity. AMBRA1 levels are critical for the rate of apoptosis: RNAi-mediated knockdown sensitizes cells to apoptotic stimuli, while a caspase-non-cleavable AMBRA1 mutant prolongs autophagy and counteracts cell death.","method":"In vitro cleavage assays, caspase/calpain inhibitor treatment, site-directed mutagenesis (D482 site), RNA interference, cell death assays","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro cleavage mapped to specific site, mutagenesis validation, functional rescue experiments in single lab","pmids":["22441670"],"is_preprint":false},{"year":2013,"finding":"Under non-autophagic conditions, mTOR phosphorylates and inhibits AMBRA1. Upon autophagy induction, AMBRA1 is dephosphorylated and interacts with the E3 ligase TRAF6, supporting K63-linked ubiquitylation of ULK1, thereby stabilizing ULK1, promoting its self-association and function. ULK1 in turn activates AMBRA1 by phosphorylation, forming a positive feedback loop.","method":"Co-immunoprecipitation, ubiquitylation assays (K63-linkage specific), phosphorylation mapping, autophagy flux assays, mTOR inhibition experiments","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — biochemical reconstitution of ubiquitylation, Co-IP, phosphorylation assays, multiple orthogonal methods in single lab","pmids":["23524951"],"is_preprint":false},{"year":2014,"finding":"AMBRA1 regulates cell proliferation by facilitating dephosphorylation and degradation of c-Myc: AMBRA1 directly interacts with the phosphatase PP2A and enhances PP2A activity on c-Myc when mTOR is inhibited, reducing the cell division rate. AMBRA1-defective systems show deregulated c-Myc and increased tumorigenesis, establishing AMBRA1 as a haploinsufficient tumor suppressor.","method":"Co-immunoprecipitation (AMBRA1–PP2A interaction), PP2A phosphatase activity assay on c-Myc substrate, cell proliferation assays, mouse tumor models","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biochemical interaction and enzymatic assay combined with in vivo tumor model; replicated in follow-up papers (PMIDs 25803737, 26101901)","pmids":["25438055"],"is_preprint":false},{"year":2014,"finding":"Cullin-4 E3 ubiquitin ligase binds AMBRA1 under basal conditions, limiting AMBRA1 protein abundance. Autophagy stimuli cause ULK1-dependent Cullin-4 release, stabilizing AMBRA1. Re-establishment of Cullin-4/AMBRA1 interaction later triggers AMBRA1 degradation, terminating autophagy. Upon Cullin-4 dissociation, AMBRA1 binds and inhibits Cullin-5, promoting DEPTOR accumulation and mTOR inhibition as a positive feedback loop.","method":"Co-immunoprecipitation, protein stability assays, autophagy flux measurement, ULK1 kinase-dependent dissociation assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, epistasis experiments, multiple orthogonal methods in single lab","pmids":["25499913"],"is_preprint":false},{"year":2014,"finding":"RNF2 associates with AMBRA1 as a K48-linked ubiquitin E3 ligase, ubiquitinating AMBRA1 at lysine 45 and targeting it for proteasomal degradation. WASH protein recruits RNF2 to AMBRA1; WASH deficiency impairs RNF2–AMBRA1 association and AMBRA1 degradation, thereby upregulating autophagy.","method":"Co-immunoprecipitation, in vitro ubiquitination assay with ubiquitin linkage mapping (K48), site-directed mutagenesis (K45), RNAi knockdown of RNF2 and WASH","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro ubiquitination assay with site mapping, Co-IP, and functional readout; single lab but multiple methods","pmids":["24980959"],"is_preprint":false},{"year":2016,"finding":"The C-terminal fragment of AMBRA1 generated by caspase cleavage contains a BH3-like domain that directly binds and inhibits the antiapoptotic factor BCL-2, acting in a proapoptotic positive feedback loop during mitochondrial apoptosis.","method":"Co-immunoprecipitation of AMBRA1-CT with BCL-2, BH3 domain mutagenesis, apoptosis assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with domain mutagenesis, single lab, single study","pmids":["27123694"],"is_preprint":false},{"year":2017,"finding":"AMBRA1 acts as a 'spatial rheostat' controlling Src/FAK signaling: it binds both FAK and Src in cancer cells, recruits active phospho-Src away from focal adhesions into autophagic structures, and regulates cancer cell invasion. When FAK is present, AMBRA1 is recruited to focal adhesions, promoting FAK-dependent direction-sensing. Dynactin-1 and IFITM3 were identified as AMBRA1 binding partners mediating this trafficking function.","method":"Interaction proteomics (affinity purification-MS), Co-immunoprecipitation, live-cell imaging, AMBRA1 knockdown/rescue in squamous cell carcinoma cells, invasion assays","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics-validated interactions, Co-IP, functional knockdown, single lab","pmids":["28362576"],"is_preprint":false},{"year":2018,"finding":"AMBRA1 controls regulatory T cell (Treg) differentiation by interacting with PP2A phosphatase, promoting FOXO3 stability, which triggers FOXP3 transcription. AMBRA1 regulates Treg induction in vivo in tumor and multiple sclerosis mouse models.","method":"Co-immunoprecipitation (AMBRA1–PP2A), FOXO3 stability assays, AMBRA1 knockdown in T cells, in vivo mouse models","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, functional cellular and in vivo assays, single lab","pmids":["30513302"],"is_preprint":false},{"year":2018,"finding":"HUWE1 E3 ubiquitin ligase is a key inducer of AMBRA1-mediated, PINK1/PARKIN-independent mitophagy. HUWE1 activity enables IKKα to phosphorylate AMBRA1 at serine 1014, inducing structural changes in AMBRA1 that promote its interaction with LC3/GABARAP proteins and activate mitophagy.","method":"Co-immunoprecipitation, in vitro kinase assay (IKKα on AMBRA1-S1014), AMBRA1 phospho-mutant analysis, LC3 interaction assays, mitophagy flux assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay, site-specific phospho-mutagenesis, Co-IP, functional mitophagy readout; single lab with multiple orthogonal methods","pmids":["30217973"],"is_preprint":false},{"year":2018,"finding":"HPV-E7 protein interacts with AMBRA1 and competes with its binding to BECLIN1, triggering calpain-dependent AMBRA1 degradation, thereby reducing autophagy activity in HPV-positive oropharyngeal squamous cell carcinoma cells.","method":"Co-immunoprecipitation, calpain inhibitor assays, competition binding experiments, autophagy flux assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP of HPV-E7 with AMBRA1, competition assay with BECLIN1, calpain inhibition rescue; single lab","pmids":["33172332"],"is_preprint":false},{"year":2019,"finding":"MCL-1 inhibits AMBRA1-mediated mitophagy by blocking recruitment of HUWE1 E3 ligase to mitochondria. During AMBRA1-mediated mitophagy, GSK-3β phosphorylates MCL1 at S159, leading to HUWE1-dependent MCL1 degradation. GSK-3β inhibition sustains MCL1 levels and delays mitophagy.","method":"Co-immunoprecipitation, GSK-3β kinase assay, MCL1 phospho-mutant (S159A), HUWE1 knockdown, mitophagy flux assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, kinase assay with mutagenesis, functional mitophagy readout; single lab","pmids":["31434979"],"is_preprint":false},{"year":2019,"finding":"TRIM32 E3 ubiquitin ligase is conveyed to ULK1 by AMBRA1 in muscle cells, stimulating ULK1 kinase activity through unanchored K63-linked polyubiquitin chains to induce autophagy upon atrophy. Limb-girdle muscular dystrophy 2H mutations in TRIM32 disrupt its ability to bind ULK1 and to induce autophagy.","method":"Co-immunoprecipitation, ULK1 kinase activity assay, ubiquitin linkage analysis, TRIM32 disease-mutation functional assays in muscle cells","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, kinase assay, specific ubiquitin chain analysis, disease mutant validation; single lab","pmids":["31234693"],"is_preprint":false},{"year":2020,"finding":"AMBRA1 interacts with ERLIN1 at mitochondria-associated membrane (MAM) raft-like microdomains, and this interaction is required for autophagosome formation upon nutrient starvation. The interaction depends on ganglioside GD3 and MFN2 integrity; knockdown of ERLIN1, ST8SIA1, or MFN2 impairs AMBRA1–ERLIN1 interaction and autophagy.","method":"Co-immunoprecipitation, FRET microscopy, subcellular fractionation to MAMs, ERLIN1/GD3-synthase/MFN2 knockdown, autophagy flux assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — FRET and Co-IP for interaction, functional knockdowns, MAM fractionation; single lab","pmids":["33034545"],"is_preprint":false},{"year":2021,"finding":"AMBRA1 serves as the substrate receptor for the CUL4-RING E3 ubiquitin ligase complex (CRL4AMBRA1/CRL4DCAF3) that ubiquitylates all three D-type cyclins (D1, D2, D3) for proteasomal degradation. Loss of AMBRA1 leads to cyclin D accumulation, RB hyperphosphorylation, and hyperproliferation. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and stabilize them.","method":"Biochemical reconstitution of ubiquitylation, genome-wide CRISPR-Cas9 screen, Co-immunoprecipitation, mouse genetic models, in vitro ubiquitination assay, cancer mutation binding assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro ubiquitination, CRISPR screen, genetic mouse models, multiple labs reporting same finding (PMIDs 33854235, 33854239, 33854232)","pmids":["33854235","33854239","33854232"],"is_preprint":false},{"year":2021,"finding":"AMBRA1 regulates the G1-to-S phase transition and prevents replication stress by mediating proteasomal degradation of D-type cyclins and by controlling MYC/MYCN-mediated transcription of cyclin genes. AMBRA1-deficient cells show replication stress and CHK1 kinase dependency as a synthetic lethal vulnerability.","method":"Cyclin D stability assays, cell cycle analysis, DNA fiber assays (replication stress), CHK1 inhibitor synthetic lethality screen, in vivo neural stem cell and mouse models","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (protein stability, replication stress, in vivo models), convergent with simultaneous publications","pmids":["33854232"],"is_preprint":false},{"year":2021,"finding":"Upon mitochondrial depolarization, AMBRA1 is recruited to the outer mitochondrial membrane and interacts with PINK1 and ATAD3A (a transmembrane protein mediating PINK1 import and degradation). AMBRA1 deficiency reduces PINK1 stability by enhancing its degradation via the mitochondrial protease LONP1, decreasing PINK1-mediated ubiquitin phosphorylation and PARKIN recruitment. ATAD3A silencing rescues defective PINK1 accumulation in AMBRA1-deficient cells.","method":"Co-immunoprecipitation (AMBRA1–PINK1, AMBRA1–ATAD3A), AMBRA1 knockdown/ATAD3A knockdown epistasis, LONP1-dependent PINK1 degradation assays, mitophagy flux assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, epistasis between AMBRA1 and ATAD3A, protease inhibition experiments, multiple orthogonal approaches; single lab","pmids":["34798798"],"is_preprint":false},{"year":2021,"finding":"AMBRA1 (as the CRL4 substrate receptor) targets Elongin C (ELOC), the shared adapter of CRL5 ubiquitin ligase complexes, for polyubiquitination and degradation, thereby attenuating CRL5SOCS3 and CRL5VIF ligase activity. This cross-regulation modulates IL-6/STAT3 signaling and HIV-1 infectivity.","method":"Proteomics (affinity purification-MS), in vitro ubiquitination assay, Co-immunoprecipitation, CRL5 assembly assays, IL-6/STAT3 reporter assays, HIV infectivity assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — proteomics-validated substrate, in vitro ubiquitination reconstitution, functional downstream assays; single lab with multiple orthogonal methods","pmids":["30166453"],"is_preprint":false},{"year":2021,"finding":"AMBRA1 promotes TGFβ signaling as the substrate receptor of the CRL4 ubiquitin ligase complex, mediating nonproteolytic polyubiquitylation of Smad4 to enhance its transcriptional activity, thereby promoting TGFβ-induced EMT, migration, and invasion of breast cancer cells and metastasis in mouse models.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, reporter gene assays, AMBRA1 knockdown/overexpression, mouse metastasis models","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro ubiquitination, Co-IP, functional migration and in vivo assays; single lab","pmids":["34362797"],"is_preprint":false},{"year":2021,"finding":"AMBRA1 negatively regulates ALDH1B1 (a cancer stem cell marker) by mediating its K27- and K33-linked non-canonical ubiquitination in cooperation with TRAF6. Ubiquitination sites K506, K511, and K515 are important; ubiquitination-defective ALDH1B1 shows increased self-association, suggesting ubiquitination suppresses ALDH1B1 oligomerization.","method":"Co-immunoprecipitation, in vitro ubiquitination assay with ubiquitin linkage mapping (K27, K33), site-directed mutagenesis, self-association assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1-2 / Weak — ubiquitination assay with linkage mapping and mutagenesis, but single lab, single study","pmids":["34769507"],"is_preprint":false},{"year":2022,"finding":"Ambra1 deficiency in skeletal muscle (muscle-specific knockout) impairs mitophagic flux without affecting bulk autophagy, causing accumulation of TOMM20, swollen mitochondria, reduced mitochondrial membrane potential, decreased complex I activity, reduced recruitment of DRP1 and Parkin to mitochondria, and lysosomal accumulation. Overexpression of mitochondria-targeted Ambra1 in wild-type muscle is sufficient to enhance mitochondria clearance.","method":"Muscle-specific conditional Ambra1 knockout mice (Ambra1fl/fl:Mlc1f-Cre), in vivo Ambra1 overexpression, mitochondrial fractionation, electron microscopy, respiratory complex activity assay, mitophagy flux assays","journal":"Journal of cachexia, sarcopenia and muscle","confidence":"High","confidence_rationale":"Tier 2 / Moderate — tissue-specific conditional KO with multiple orthogonal phenotypic readouts and gain-of-function complementation; single lab","pmids":["35593053"],"is_preprint":false},{"year":2023,"finding":"AMBRA1 is phosphorylated during mitosis by CDK1 and PLK1 on multiple sites. This phosphorylation is required for proper mitotic spindle orientation, mediated through NUMA1. AMBRA1 localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation, and binding ability.","method":"Phosphorylation mapping during mitotic arrest, CDK1/PLK1 kinase assays, AMBRA1 phospho-mutant analysis, NUMA1 localization by imaging, spindle orientation assays","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay, phospho-mutagenesis, functional spindle assays; single lab","pmids":["37584777"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structure of AMBRA1 in complex with DDB1 resolved at 3.08 Å. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1, creating a scaffold for substrate recruitment. DDB1-binding-defective AMBRA1 mutants prevent Cyclin D1 ubiquitination in vitro and increase cell cycle progression.","method":"Cryo-EM structure determination, HDX-MS, in vitro ubiquitination assay, DDB1-binding mutagenesis, cell cycle assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure at 3.08 Å with in vitro functional validation by mutagenesis and reconstituted ubiquitination assay","pmids":["37993427"],"is_preprint":false},{"year":2022,"finding":"AMBRA1 interacts with MAVS (mitochondrial antiviral signaling protein) at mitochondria and stabilizes MAVS by preventing its dsRNA-induced proteasomal degradation, thereby promoting dsRNA- and virus-induced apoptosis through a caspase-8-dependent pathway.","method":"Co-immunoprecipitation (AMBRA1–MAVS), AMBRA1 gene editing (knockout), proteasome inhibitor assays, apoptosis assays, Semliki Forest virus infection model","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, genetic KO with rescue, functional apoptosis assays; single lab","pmids":["34859815"],"is_preprint":false},{"year":2024,"finding":"BAG2, an effector of the ULK1 complex, regulates AMBRA1 subcellular localization. In nutrient-replete conditions, unphosphorylated BAG2 sequesters AMBRA1, attenuating autophagy. Upon starvation, ULK1 phosphorylates BAG2 at Ser31, which promotes AMBRA1 recruitment to the ER membrane and autophagy induction.","method":"Affinity purification-MS and proximity labeling-MS interactome of ULK1 complex subunits, Co-immunoprecipitation, BAG2 phospho-mutant (S31A), AMBRA1 localization imaging","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics-validated interaction, Co-IP, phospho-mutagenesis, localization imaging; single lab","pmids":["39207901"],"is_preprint":false},{"year":2024,"finding":"AMBRA1 promotes intestinal NF-κB-driven inflammation in an autophagy-independent manner by antagonizing the PP4R1/PP4c phosphatase complex: the N-terminal F1 domain of AMBRA1 competitively binds PP4R1, disrupting PP4R1/PP4c interaction with IKK and preventing IKK dephosphorylation. Upon TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043, impairing its CUL4A-mediated K48-linked ubiquitination and stabilizing AMBRA1.","method":"Co-immunoprecipitation (AMBRA1–PP4R1, AMBRA1–PP4c, AMBRA1–IKK), AMBRA1 domain deletion/mutagenesis (F1 domain, S1043), IKKα kinase assay, CUL4A ubiquitination assay, AMBRA1 conditional knockout mice","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — kinase assay, ubiquitination assay, domain mutagenesis, Co-IP, in vivo mouse model; multiple orthogonal methods in single lab","pmids":["38424148"],"is_preprint":false},{"year":2025,"finding":"In intestinal epithelial cells, AMBRA1 acts as a pro-oxidative factor by competitively binding NRF2 through its N-terminal F1 domain, antagonizing DUB3-mediated NRF2 deubiquitination and promoting NRF2 degradation. Under oxidative stress (H2O2), USP7 interaction with AMBRA1 is enhanced, leading to USP7-mediated deubiquitination of AMBRA1 at K83 and K86, stabilizing AMBRA1.","method":"Co-immunoprecipitation (AMBRA1–NRF2, AMBRA1–DUB3, AMBRA1–USP7), domain mutagenesis (F1 domain, K83/K86 sites), in vitro deubiquitination assay, AMBRA1 conditional knockout mice, IBD patient tissue analysis","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, site-specific mutagenesis, in vitro deubiquitination, in vivo mouse model; single lab","pmids":["39887666"],"is_preprint":false},{"year":2017,"finding":"AMBRA1 binds α-synuclein phosphorylated at serine 129 with ~9-fold higher affinity than non-phosphorylated α-synuclein (in vitro and in vivo analyses). Silencing AMBRA1 function causes α-synuclein aggregates in the cytoplasm of mouse primary neurons, similar to the effect of the autophagy inhibitor Bafilomycin.","method":"Binding affinity assays (in vitro and in vivo), AMBRA1 knockdown in primary neurons, immunohistochemistry of human MSA brain samples","journal":"Brain pathology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — quantified binding assay, functional knockdown, human tissue validation; single lab","pmids":["27875637"],"is_preprint":false},{"year":2021,"finding":"AMBRA1 suppresses SOCS3 in medulloblastoma Group 3 stem cells, enabling STAT3 activation. AMBRA1 expression depends on c-MYC levels, and AMBRA1 knockdown reduces stem potential, growth, and migration of MBGroup3 cells. Combined anti-autophagy and anti-STAT3 treatment impacts MBGroup3 outcome.","method":"AMBRA1 knockdown/overexpression, SOCS3 expression assays, STAT3 activation assays, tumor sphere formation, migration assays","journal":"Acta neuropathologica","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — functional knockdown with defined pathway placement (AMBRA1→SOCS3→STAT3), but mechanistic basis of SOCS3 suppression not fully biochemically characterized; single lab","pmids":["34302498"],"is_preprint":false},{"year":2021,"finding":"Loss of Ambra1 in melanoma promotes aggressiveness and metastasis by increasing cell motility/invasion, activating an EMT-like process, remodeling the extracellular matrix, and inducing hyperactivation of FAK1 signaling. FAK1 inhibition reduces cell invasion and melanoma growth in Ambra1-deficient cells.","method":"Ambra1-depleted Braf/Pten mouse melanoma model, invasion and migration assays, EMT marker analysis, FAK1 phosphorylation assays, FAK1 pharmacological inhibition","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with defined signaling pathway (Ambra1→FAK1 hyperactivation), pharmacological rescue; single lab","pmids":["33953176"],"is_preprint":false}],"current_model":"AMBRA1 is a multifunctional scaffold protein that (1) positively regulates autophagy initiation by interacting with BECLIN1-VPS34; under basal conditions it is tethered to dynein via DLC1/2 and is released upon ULK1-mediated phosphorylation to relocalize to the ER for autophagosome nucleation; (2) serves as the substrate receptor of the CRL4 (CUL4-DDB1) ubiquitin E3 ligase to drive proteasomal degradation of all D-type cyclins, thereby restraining G1/S progression; (3) regulates mitophagy via interactions with Parkin, PINK1/ATAD3A, and HUWE1/IKKα-mediated phosphorylation at S1014; (4) is itself regulated by mTOR phosphorylation, Cullin-4/5 ubiquitylation, RNF2/WASH-mediated K48-ubiquitylation, USP7-mediated deubiquitylation at K83/K86, and caspase/calpain cleavage during apoptosis; (5) controls c-Myc levels through PP2A interaction; and (6) performs additional autophagy-independent functions including NF-κB/IKK regulation, TGFβ-Smad4 polyubiquitylation, mitotic spindle orientation via CDK1/PLK1 phosphorylation and NUMA1 regulation, and spatial control of Src/FAK signaling."},"narrative":{"mechanistic_narrative":"AMBRA1 is a WD40-domain scaffold protein that couples autophagy initiation to cell-cycle control, signaling, and cell-fate decisions [PMID:17589504, PMID:33854235, PMID:33854239, PMID:33854232]. In its founding role it positively regulates BECLIN1-dependent autophagy, and its loss in mouse embryos impairs autophagic flux and produces neural tube defects with unbalanced proliferation and apoptosis [PMID:17589504]. Mechanistically, the BECLIN1-VPS34 complex is held inactive at the dynein motor through AMBRA1's interaction with dynein light chains; ULK1-mediated phosphorylation releases AMBRA1, allowing relocalization to the endoplasmic reticulum and mitochondria-associated membranes for autophagosome nucleation [PMID:20921139, PMID:21079415, PMID:33034545]. AMBRA1 sits within a dense network of E3 ligases and kinases that tune its abundance and activity: it directs TRAF6-dependent K63 ubiquitylation of ULK1 in a positive feedback loop opposed by mTOR phosphorylation, while its own levels are restrained by Cullin-4 and RNF2/WASH-mediated K48 ubiquitylation and reset by USP7 deubiquitylation at K83/K86 [PMID:23524951, PMID:25499913, PMID:24980959, PMID:39887666]. Independently of autophagosome formation, AMBRA1 serves as the substrate receptor of a CRL4 (CUL4-DDB1) ubiquitin ligase that drives proteasomal degradation of all three D-type cyclins, thereby restraining the G1-to-S transition and limiting replication stress; a 3.08 Å cryo-EM structure shows its N-terminal helix-loop-helix and WD40 domains clamping the DDB1 double-propeller to build the substrate-recruitment platform [PMID:33854235, PMID:33854239, PMID:33854232, PMID:37993427]. As a CRL4 receptor AMBRA1 also ubiquitylates Elongin C and Smad4, linking it to CRL5 cross-regulation and TGFβ-driven EMT [PMID:30166453, PMID:34362797]. In mitochondrial quality control, AMBRA1 drives both PINK1/PARKIN-dependent and -independent mitophagy, acting through PINK1/ATAD3A to stabilize PINK1 and through HUWE1/IKKα-mediated phosphorylation at S1014 to engage LC3/GABARAP [PMID:21753002, PMID:21921694, PMID:30217973, PMID:34798798, PMID:35593053]. Through PP2A it promotes dephosphorylation and degradation of c-Myc, establishing AMBRA1 as a haploinsufficient tumor suppressor [PMID:25438055]. Additional autophagy-independent functions include NF-κB/IKK regulation via the PP4R1/PP4c phosphatase complex and mitotic spindle orientation through CDK1/PLK1 phosphorylation and NUMA1 [PMID:38424148, PMID:37584777].","teleology":[{"year":2007,"claim":"Established AMBRA1 as a positive regulator of autophagy and an essential developmental gene, answering whether it acts in the BECLIN1 pathway and what its loss does in vivo.","evidence":"RNAi and overexpression in vitro plus mouse knockout with phenotypic analysis","pmids":["17589504"],"confidence":"High","gaps":["Did not define the molecular trigger linking AMBRA1 to autophagosome nucleation","Mechanism of the neural tube/proliferation phenotype unresolved"]},{"year":2010,"claim":"Resolved how AMBRA1 is held inactive and activated, showing dynein tethering of BECLIN1-VPS34 is released by ULK1 phosphorylation to permit ER relocalization.","evidence":"Co-IP, live-cell imaging, ULK1 kinase assays and DLC1-binding-site mutagenesis","pmids":["20921139","21079415"],"confidence":"High","gaps":["Precise ER docking partners not identified at this stage","ULK1 phosphosites on AMBRA1 not fully mapped"]},{"year":2011,"claim":"Placed AMBRA1 within mitochondrial autophagy control through BCL-2 inhibition and Parkin-dependent recruitment to depolarized mitochondria, connecting it to mitophagy.","evidence":"Reciprocal Co-IP, fractionation, TAP-MS and endogenous Co-IP across HEK293, SH-SY5Y and mouse brain with knockdown readouts","pmids":["21358617","21753002","21921694"],"confidence":"High","gaps":["Direct AMBRA1-driven steps in mitophagosome formation not biochemically reconstituted","BCL-2 binding interface not mapped at this point"]},{"year":2012,"claim":"Showed AMBRA1 is dismantled during apoptosis, defining a caspase/calpain switch (D482) that couples autophagy shutdown to cell death rate.","evidence":"In vitro cleavage assays, inhibitor treatment, D482 mutagenesis and cell death assays","pmids":["22441670"],"confidence":"High","gaps":["Physiological contexts where this cleavage dominates unclear","Fate of cleavage fragments not yet characterized (addressed later)"]},{"year":2013,"claim":"Defined the kinase/ligase feedback architecture: mTOR inhibits AMBRA1, while dephosphorylated AMBRA1 recruits TRAF6 to K63-ubiquitylate and stabilize ULK1, with ULK1 reciprocally activating AMBRA1.","evidence":"Co-IP, K63-linkage-specific ubiquitylation assays, phospho-mapping and mTOR inhibition","pmids":["23524951"],"confidence":"High","gaps":["mTOR target residues on AMBRA1 not exhaustively defined","Stoichiometry of the feedback loop not quantified"]},{"year":2014,"claim":"Identified the ubiquitin machinery controlling AMBRA1 abundance (Cullin-4, RNF2/WASH at K45) and a proliferation-restraining role via PP2A-mediated c-Myc degradation, establishing tumor-suppressor function.","evidence":"Reciprocal Co-IP, in vitro ubiquitination with linkage/site mapping, PP2A activity assays and mouse tumor models","pmids":["25499913","24980959","25438055"],"confidence":"High","gaps":["How multiple ligases are coordinated temporally not resolved","Direct enzymatic basis of AMBRA1-stimulated PP2A activity unclear"]},{"year":2016,"claim":"Showed the caspase-generated AMBRA1 C-terminal fragment carries a BH3-like domain that binds and inhibits BCL-2, defining a proapoptotic feedback role for the cleavage product.","evidence":"Co-IP of AMBRA1-CT with BCL-2 and BH3-domain mutagenesis with apoptosis assays","pmids":["27123694"],"confidence":"Medium","gaps":["Single-lab finding without reconstituted structural confirmation","Contribution relative to canonical BH3 proteins not quantified"]},{"year":2017,"claim":"Linked AMBRA1 to focal-adhesion signaling and to α-synuclein clearance, broadening its roles beyond canonical autophagy into Src/FAK trafficking and neurodegeneration-relevant cargo recognition.","evidence":"AP-MS interactomics, Co-IP, live imaging and invasion assays; phospho-S129 α-synuclein binding affinity assays with neuronal knockdown and MSA tissue","pmids":["28362576","27875637"],"confidence":"Medium","gaps":["Direct vs scaffold-mediated nature of FAK/Src interactions not fully dissected","Structural basis of phospho-α-synuclein preference unknown"]},{"year":2018,"claim":"Defined a PINK1/PARKIN-independent mitophagy route (HUWE1/IKKα phosphorylating AMBRA1-S1014 to engage LC3/GABARAP) and immune/Treg functions via PP2A-FOXO3-FOXP3.","evidence":"In vitro IKKα kinase assay, S1014 phospho-mutant, LC3 interaction and mitophagy assays; Treg Co-IP and in vivo mouse models","pmids":["30217973","30513302"],"confidence":"High","gaps":["Structural change induced by S1014 phosphorylation inferred, not resolved","Interplay between PINK1-dependent and -independent routes not reconciled"]},{"year":2019,"claim":"Refined mitophagy regulation (GSK-3β/MCL-1 gating of HUWE1 recruitment) and revealed a muscle TRIM32-ULK1 axis, connecting AMBRA1 to disease-linked autophagy in skeletal muscle.","evidence":"Co-IP, GSK-3β kinase assays, phospho-mutants, HUWE1 knockdown, ULK1 activity assays and TRIM32 disease-mutation tests","pmids":["31434979","31234693"],"confidence":"Medium","gaps":["Single-lab mechanisms not independently reconstituted","Tissue specificity of these regulatory arms unclear"]},{"year":2020,"claim":"Localized AMBRA1's nucleation function to MAM raft-like microdomains via ERLIN1, GD3 and MFN2, defining the ER membrane platform for starvation-induced autophagosome formation.","evidence":"Co-IP, FRET, MAM fractionation and ERLIN1/ST8SIA1/MFN2 knockdowns","pmids":["33034545"],"confidence":"Medium","gaps":["Direct vs lipid-dependent nature of the ERLIN1 interaction not separated","Single-lab without structural validation"]},{"year":2021,"claim":"Established the major autophagy-independent identity of AMBRA1 as the CRL4 substrate receptor degrading D-type cyclins, controlling G1/S and replication stress, and extended this receptor role to ELOC, Smad4 and ALDH1B1 substrates.","evidence":"Reconstituted in vitro ubiquitination, genome-wide CRISPR screens, mouse genetic models, DNA fiber/CHK1 synthetic-lethality assays, and substrate-specific ubiquitination/Co-IP assays","pmids":["33854235","33854239","33854232","30166453","34362797","34769507"],"confidence":"High","gaps":["How substrate selection among cyclins, ELOC and Smad4 is regulated unknown","Crosstalk between CRL4-receptor and autophagy roles not integrated"]},{"year":2021,"claim":"Detailed AMBRA1's PINK1/ATAD3A mitophagy axis and disease-context roles in melanoma (FAK1) and medulloblastoma (SOCS3/STAT3), linking its loss to tumor aggressiveness.","evidence":"Co-IP, ATAD3A epistasis, LONP1-dependent PINK1 degradation assays; in vivo melanoma and medulloblastoma models with pathway inhibition","pmids":["34798798","33953176","34302498"],"confidence":"Medium","gaps":["Direct biochemical basis of SOCS3 suppression not established","Whether FAK1 hyperactivation is direct or indirect not resolved"]},{"year":2022,"claim":"Demonstrated muscle-specific mitophagy dependence and a MAVS-stabilizing antiviral apoptosis role, separating AMBRA1's mitophagic and innate-immune functions.","evidence":"Muscle-specific conditional knockout with mitochondrial/respiratory phenotyping and mito-targeted rescue; AMBRA1-MAVS Co-IP, knockout and viral infection apoptosis assays","pmids":["35593053","34859815"],"confidence":"High","gaps":["Selectivity of muscle mitophagy versus bulk autophagy mechanism unclear","MAVS stabilization mechanism (ligase competition) not defined"]},{"year":2023,"claim":"Provided the structural basis for CRL4 substrate recruitment (cryo-EM AMBRA1-DDB1) and a mitotic function (CDK1/PLK1 phosphorylation controlling NUMA1-dependent spindle orientation).","evidence":"3.08 Å cryo-EM, HDX-MS, DDB1-binding mutagenesis and reconstituted ubiquitination; mitotic phospho-mapping, CDK1/PLK1 assays and spindle orientation assays","pmids":["37993427","37584777"],"confidence":"High","gaps":["Substrate-engaging surface of the WD40 domain not resolved with cyclin bound","How mitotic phosphorylation reroutes AMBRA1 to NUMA1 not mechanistically detailed"]},{"year":2024,"claim":"Defined autophagy-independent NF-κB inflammation control via PP4R1/PP4c antagonism and an IKKα-S1043/CUL4A stabilization switch, plus BAG2-mediated localization control of AMBRA1.","evidence":"Co-IP, F1-domain/S1043 mutagenesis, IKKα kinase and CUL4A ubiquitination assays, conditional knockout mice; ULK1-complex interactomics with BAG2-S31A phospho-mutant","pmids":["38424148","39207901"],"confidence":"High","gaps":["Integration of S1014 vs S1043 phosphorylation signals not resolved","Whether BAG2 sequestration competes with dynein tethering unknown"]},{"year":2025,"claim":"Identified a pro-oxidative function in intestinal epithelium where AMBRA1 antagonizes DUB3-mediated NRF2 deubiquitination, with USP7 stabilizing AMBRA1 at K83/K86 under oxidative stress.","evidence":"Co-IP, F1-domain and K83/K86 mutagenesis, in vitro deubiquitination, conditional knockout mice and IBD tissue analysis","pmids":["39887666"],"confidence":"Medium","gaps":["Single-lab study without independent confirmation","Relationship between USP7-AMBRA1 stabilization and the CRL4 receptor pool unclear"]},{"year":null,"claim":"How AMBRA1's distinct functions—autophagy scaffold, CRL4 substrate receptor, phosphatase regulator, and mitotic/spindle factor—are partitioned and switched within a cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of how post-translational marks route AMBRA1 between its competing complexes","Substrate-binding surface of the WD40 domain not structurally defined with a bound substrate","Quantitative partitioning of cytosolic, ER, and mitochondrial AMBRA1 pools unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,17,25]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[17,20,21,22,25]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,11,28,29]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,16,27]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[3,12,19,23,26]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[17,18]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0,1,5,7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[17,18,24,25]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[8,17,20,21]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,9,26]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[28,32,6]}],"complexes":["CRL4 (CUL4-DDB1) E3 ubiquitin ligase","BECLIN1-VPS34 (class III PI3K) complex","ULK1 complex"],"partners":["BECLIN1","ULK1","DDB1","TRAF6","PP2A","PINK1","HUWE1","DLC1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9C0C7","full_name":"Activating molecule in BECN1-regulated autophagy protein 1","aliases":["DDB1- and CUL4-associated factor 3"],"length_aa":1298,"mass_kda":142.5,"function":"Substrate-recognition component of a DCX (DDB1-CUL4-X-box) E3 ubiquitin-protein ligase complex involved in cell cycle control and autophagy (PubMed:20921139, PubMed:23524951, PubMed:24587252, PubMed:32333458, PubMed:33854232, PubMed:33854235, PubMed:33854239). The DCX(AMBRA1) complex specifically mediates the polyubiquitination of target proteins such as BECN1, CCND1, CCND2, CCND3, ELOC and ULK1 (PubMed:23524951, PubMed:33854232, PubMed:33854235, PubMed:33854239). Acts as an upstream master regulator of the transition from G1 to S cell phase: AMBRA1 specifically recognizes and binds phosphorylated cyclin-D (CCND1, CCND2 and CCND3), leading to cyclin-D ubiquitination by the DCX(AMBRA1) complex and subsequent degradation (PubMed:33854232, PubMed:33854235, PubMed:33854239). By controlling the transition from G1 to S phase and cyclin-D degradation, AMBRA1 acts as a tumor suppressor that promotes genomic integrity during DNA replication and counteracts developmental abnormalities and tumor growth (PubMed:33854232, PubMed:33854235, PubMed:33854239). AMBRA1 also regulates the cell cycle by promoting MYC dephosphorylation and degradation independently of the DCX(AMBRA1) complex: acts via interaction with the catalytic subunit of protein phosphatase 2A (PPP2CA), which enhances interaction between PPP2CA and MYC, leading to MYC dephosphorylation and degradation (PubMed:25438055, PubMed:25803737). Acts as a regulator of Cul5-RING (CRL5) E3 ubiquitin-protein ligase complexes by mediating ubiquitination and degradation of Elongin-C (ELOC) component of CRL5 complexes (PubMed:25499913, PubMed:30166453). Acts as a key regulator of autophagy by modulating the BECN1-PIK3C3 complex: controls protein turnover during neuronal development, and regulates normal cell survival and proliferation (PubMed:21358617). In normal conditions, AMBRA1 is tethered to the cytoskeleton via interaction with dyneins DYNLL1 and DYNLL2 (PubMed:20921139). Upon autophagy induction, AMBRA1 is released from the cytoskeletal docking site to induce autophagosome nucleation by mediating ubiquitination of proteins involved in autophagy (PubMed:20921139). The DCX(AMBRA1) complex mediates 'Lys-63'-linked ubiquitination of BECN1, increasing the association between BECN1 and PIK3C3 to promote PIK3C3 activity (By similarity). In collaboration with TRAF6, AMBRA1 mediates 'Lys-63'-linked ubiquitination of ULK1 following autophagy induction, promoting ULK1 stability and kinase activity (PubMed:23524951). Also activates ULK1 via interaction with TRIM32: TRIM32 stimulates ULK1 through unanchored 'Lys-63'-linked polyubiquitin chains (PubMed:31123703). Also acts as an activator of mitophagy via interaction with PRKN and LC3 proteins (MAP1LC3A, MAP1LC3B or MAP1LC3C); possibly by bringing damaged mitochondria onto autophagosomes (PubMed:21753002, PubMed:25215947). Also activates mitophagy by acting as a cofactor for HUWE1; acts by promoting HUWE1-mediated ubiquitination of MFN2 (PubMed:30217973). AMBRA1 is also involved in regulatory T-cells (Treg) differentiation by promoting FOXO3 dephosphorylation independently of the DCX(AMBRA1) complex: acts via interaction with PPP2CA, which enhances interaction between PPP2CA and FOXO3, leading to FOXO3 dephosphorylation and stabilization (PubMed:30513302). May act as a regulator of intracellular trafficking, regulating the localization of active PTK2/FAK and SRC (By similarity). Also involved in transcription regulation by acting as a scaffold for protein complexes at chromatin (By similarity)","subcellular_location":"Endoplasmic reticulum; Cytoplasm, cytoskeleton; Cytoplasmic vesicle, autophagosome; Mitochondrion; Cytoplasm, cytosol; Nucleus; Cell junction, focal adhesion","url":"https://www.uniprot.org/uniprotkb/Q9C0C7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AMBRA1","classification":"Not Classified","n_dependent_lines":209,"n_total_lines":1208,"dependency_fraction":0.17301324503311258},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000110497","cell_line_id":"CID001844","localizations":[{"compartment":"nucleoplasm","grade":3},{"compartment":"cytoplasmic","grade":2}],"interactors":[{"gene":"RBM25","stoichiometry":0.2},{"gene":"DDB1","stoichiometry":0.2},{"gene":"DYNLL1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001844","total_profiled":1310},"omim":[{"mim_id":"618222","title":"MITOCHONDRIAL COMPLEX I DEFICIENCY, NUCLEAR TYPE 2; MC1DN2","url":"https://www.omim.org/entry/618222"},{"mim_id":"615687","title":"BECLIN 2; BECN2","url":"https://www.omim.org/entry/615687"},{"mim_id":"611359","title":"ACTIVATING MOLECULE IN BECLIN 1-REGULATED AUTOPHAGY; AMBRA1","url":"https://www.omim.org/entry/611359"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Mitochondria","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AMBRA1"},"hgnc":{"alias_symbol":["FLJ20294","KIAA1736","WDR94","DCAF3"],"prev_symbol":[]},"alphafold":{"accession":"Q9C0C7","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0C7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0C7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0C7-F1-predicted_aligned_error_v6.png","plddt_mean":48.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AMBRA1","jax_strain_url":"https://www.jax.org/strain/search?query=AMBRA1"},"sequence":{"accession":"Q9C0C7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9C0C7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9C0C7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0C7"}},"corpus_meta":[{"pmid":"17589504","id":"PMC_17589504","title":"Ambra1 regulates autophagy and development of the nervous 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Loss of AMBRA1 function in mouse embryos impairs autophagy, causes accumulation of ubiquitinated proteins, and leads to severe neural tube defects with unbalanced cell proliferation and excessive apoptosis.\",\n      \"method\": \"RNA interference in vitro, overexpression assays, mouse genetic knockout model with phenotypic analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function in vivo and in vitro with multiple orthogonal readouts; foundational paper replicated by many subsequent studies\",\n      \"pmids\": [\"17589504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Under basal conditions, the BECLIN1-VPS34 complex is tethered to the dynein motor complex via AMBRA1's direct interaction with dynein light chains DLC1/2. Upon autophagy induction, ULK1 phosphorylates AMBRA1, releasing it from dynein; the complex then relocalizes to the endoplasmic reticulum, enabling autophagosome nucleation.\",\n      \"method\": \"Co-immunoprecipitation, live-cell imaging, ULK1 kinase assays, DLC1 knockdown, AMBRA1 DLC1-binding site mutagenesis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct interaction demonstrated by Co-IP, phosphorylation established by kinase assay, mutagenesis of binding sites, replicated in follow-up commentary (PMID 21079415)\",\n      \"pmids\": [\"20921139\", \"21079415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Mitochondrial BCL-2 binds AMBRA1 and inhibits AMBRA1-induced autophagy. AMBRA1 preferentially binds the mitochondrial pool of BCL-2; upon autophagy induction this interaction is disrupted and AMBRA1 is recruited to BECLIN1. AMBRA1 can compete with both mitochondrial and ER-resident BCL-2 for BECLIN1 binding.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, overexpression and knockdown assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reciprocal Co-IP and functional competition assays in single lab, multiple methods\",\n      \"pmids\": [\"21358617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Parkin interacts with AMBRA1 (identified by tandem affinity purification/mass spectrometry from HEK293 cells and validated by endogenous Co-IP). Upon prolonged mitochondrial depolarization, the Parkin–AMBRA1 interaction increases; AMBRA1 is recruited to perinuclear clusters of depolarized mitochondria in a Parkin-dependent manner and activates class III PI3K locally, promoting mitophagic clearance.\",\n      \"method\": \"Tandem affinity purification, mass spectrometry, endogenous Co-immunoprecipitation from HEK293, SH-SY5Y cells and adult mouse brain, siRNA knockdown, fluorescence microscopy\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — interaction validated by MS and reciprocal endogenous Co-IP in three independent cell/tissue systems; functional consequence demonstrated by AMBRA1 knockdown\",\n      \"pmids\": [\"21753002\", \"21921694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"During apoptosis, AMBRA1 is proteolytically degraded by the combined action of caspases (cleaving at D482) and calpains (mediating complete degradation), dismantling autophagic activity. AMBRA1 levels are critical for the rate of apoptosis: RNAi-mediated knockdown sensitizes cells to apoptotic stimuli, while a caspase-non-cleavable AMBRA1 mutant prolongs autophagy and counteracts cell death.\",\n      \"method\": \"In vitro cleavage assays, caspase/calpain inhibitor treatment, site-directed mutagenesis (D482 site), RNA interference, cell death assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro cleavage mapped to specific site, mutagenesis validation, functional rescue experiments in single lab\",\n      \"pmids\": [\"22441670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Under non-autophagic conditions, mTOR phosphorylates and inhibits AMBRA1. Upon autophagy induction, AMBRA1 is dephosphorylated and interacts with the E3 ligase TRAF6, supporting K63-linked ubiquitylation of ULK1, thereby stabilizing ULK1, promoting its self-association and function. ULK1 in turn activates AMBRA1 by phosphorylation, forming a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays (K63-linkage specific), phosphorylation mapping, autophagy flux assays, mTOR inhibition experiments\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — biochemical reconstitution of ubiquitylation, Co-IP, phosphorylation assays, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"23524951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"AMBRA1 regulates cell proliferation by facilitating dephosphorylation and degradation of c-Myc: AMBRA1 directly interacts with the phosphatase PP2A and enhances PP2A activity on c-Myc when mTOR is inhibited, reducing the cell division rate. AMBRA1-defective systems show deregulated c-Myc and increased tumorigenesis, establishing AMBRA1 as a haploinsufficient tumor suppressor.\",\n      \"method\": \"Co-immunoprecipitation (AMBRA1–PP2A interaction), PP2A phosphatase activity assay on c-Myc substrate, cell proliferation assays, mouse tumor models\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biochemical interaction and enzymatic assay combined with in vivo tumor model; replicated in follow-up papers (PMIDs 25803737, 26101901)\",\n      \"pmids\": [\"25438055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cullin-4 E3 ubiquitin ligase binds AMBRA1 under basal conditions, limiting AMBRA1 protein abundance. Autophagy stimuli cause ULK1-dependent Cullin-4 release, stabilizing AMBRA1. Re-establishment of Cullin-4/AMBRA1 interaction later triggers AMBRA1 degradation, terminating autophagy. Upon Cullin-4 dissociation, AMBRA1 binds and inhibits Cullin-5, promoting DEPTOR accumulation and mTOR inhibition as a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, protein stability assays, autophagy flux measurement, ULK1 kinase-dependent dissociation assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, epistasis experiments, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"25499913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RNF2 associates with AMBRA1 as a K48-linked ubiquitin E3 ligase, ubiquitinating AMBRA1 at lysine 45 and targeting it for proteasomal degradation. WASH protein recruits RNF2 to AMBRA1; WASH deficiency impairs RNF2–AMBRA1 association and AMBRA1 degradation, thereby upregulating autophagy.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay with ubiquitin linkage mapping (K48), site-directed mutagenesis (K45), RNAi knockdown of RNF2 and WASH\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro ubiquitination assay with site mapping, Co-IP, and functional readout; single lab but multiple methods\",\n      \"pmids\": [\"24980959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The C-terminal fragment of AMBRA1 generated by caspase cleavage contains a BH3-like domain that directly binds and inhibits the antiapoptotic factor BCL-2, acting in a proapoptotic positive feedback loop during mitochondrial apoptosis.\",\n      \"method\": \"Co-immunoprecipitation of AMBRA1-CT with BCL-2, BH3 domain mutagenesis, apoptosis assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with domain mutagenesis, single lab, single study\",\n      \"pmids\": [\"27123694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AMBRA1 acts as a 'spatial rheostat' controlling Src/FAK signaling: it binds both FAK and Src in cancer cells, recruits active phospho-Src away from focal adhesions into autophagic structures, and regulates cancer cell invasion. When FAK is present, AMBRA1 is recruited to focal adhesions, promoting FAK-dependent direction-sensing. Dynactin-1 and IFITM3 were identified as AMBRA1 binding partners mediating this trafficking function.\",\n      \"method\": \"Interaction proteomics (affinity purification-MS), Co-immunoprecipitation, live-cell imaging, AMBRA1 knockdown/rescue in squamous cell carcinoma cells, invasion assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics-validated interactions, Co-IP, functional knockdown, single lab\",\n      \"pmids\": [\"28362576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AMBRA1 controls regulatory T cell (Treg) differentiation by interacting with PP2A phosphatase, promoting FOXO3 stability, which triggers FOXP3 transcription. AMBRA1 regulates Treg induction in vivo in tumor and multiple sclerosis mouse models.\",\n      \"method\": \"Co-immunoprecipitation (AMBRA1–PP2A), FOXO3 stability assays, AMBRA1 knockdown in T cells, in vivo mouse models\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, functional cellular and in vivo assays, single lab\",\n      \"pmids\": [\"30513302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HUWE1 E3 ubiquitin ligase is a key inducer of AMBRA1-mediated, PINK1/PARKIN-independent mitophagy. HUWE1 activity enables IKKα to phosphorylate AMBRA1 at serine 1014, inducing structural changes in AMBRA1 that promote its interaction with LC3/GABARAP proteins and activate mitophagy.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay (IKKα on AMBRA1-S1014), AMBRA1 phospho-mutant analysis, LC3 interaction assays, mitophagy flux assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay, site-specific phospho-mutagenesis, Co-IP, functional mitophagy readout; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30217973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HPV-E7 protein interacts with AMBRA1 and competes with its binding to BECLIN1, triggering calpain-dependent AMBRA1 degradation, thereby reducing autophagy activity in HPV-positive oropharyngeal squamous cell carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, calpain inhibitor assays, competition binding experiments, autophagy flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP of HPV-E7 with AMBRA1, competition assay with BECLIN1, calpain inhibition rescue; single lab\",\n      \"pmids\": [\"33172332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MCL-1 inhibits AMBRA1-mediated mitophagy by blocking recruitment of HUWE1 E3 ligase to mitochondria. During AMBRA1-mediated mitophagy, GSK-3β phosphorylates MCL1 at S159, leading to HUWE1-dependent MCL1 degradation. GSK-3β inhibition sustains MCL1 levels and delays mitophagy.\",\n      \"method\": \"Co-immunoprecipitation, GSK-3β kinase assay, MCL1 phospho-mutant (S159A), HUWE1 knockdown, mitophagy flux assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, kinase assay with mutagenesis, functional mitophagy readout; single lab\",\n      \"pmids\": [\"31434979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM32 E3 ubiquitin ligase is conveyed to ULK1 by AMBRA1 in muscle cells, stimulating ULK1 kinase activity through unanchored K63-linked polyubiquitin chains to induce autophagy upon atrophy. Limb-girdle muscular dystrophy 2H mutations in TRIM32 disrupt its ability to bind ULK1 and to induce autophagy.\",\n      \"method\": \"Co-immunoprecipitation, ULK1 kinase activity assay, ubiquitin linkage analysis, TRIM32 disease-mutation functional assays in muscle cells\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, kinase assay, specific ubiquitin chain analysis, disease mutant validation; single lab\",\n      \"pmids\": [\"31234693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AMBRA1 interacts with ERLIN1 at mitochondria-associated membrane (MAM) raft-like microdomains, and this interaction is required for autophagosome formation upon nutrient starvation. The interaction depends on ganglioside GD3 and MFN2 integrity; knockdown of ERLIN1, ST8SIA1, or MFN2 impairs AMBRA1–ERLIN1 interaction and autophagy.\",\n      \"method\": \"Co-immunoprecipitation, FRET microscopy, subcellular fractionation to MAMs, ERLIN1/GD3-synthase/MFN2 knockdown, autophagy flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — FRET and Co-IP for interaction, functional knockdowns, MAM fractionation; single lab\",\n      \"pmids\": [\"33034545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AMBRA1 serves as the substrate receptor for the CUL4-RING E3 ubiquitin ligase complex (CRL4AMBRA1/CRL4DCAF3) that ubiquitylates all three D-type cyclins (D1, D2, D3) for proteasomal degradation. Loss of AMBRA1 leads to cyclin D accumulation, RB hyperphosphorylation, and hyperproliferation. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and stabilize them.\",\n      \"method\": \"Biochemical reconstitution of ubiquitylation, genome-wide CRISPR-Cas9 screen, Co-immunoprecipitation, mouse genetic models, in vitro ubiquitination assay, cancer mutation binding assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro ubiquitination, CRISPR screen, genetic mouse models, multiple labs reporting same finding (PMIDs 33854235, 33854239, 33854232)\",\n      \"pmids\": [\"33854235\", \"33854239\", \"33854232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AMBRA1 regulates the G1-to-S phase transition and prevents replication stress by mediating proteasomal degradation of D-type cyclins and by controlling MYC/MYCN-mediated transcription of cyclin genes. AMBRA1-deficient cells show replication stress and CHK1 kinase dependency as a synthetic lethal vulnerability.\",\n      \"method\": \"Cyclin D stability assays, cell cycle analysis, DNA fiber assays (replication stress), CHK1 inhibitor synthetic lethality screen, in vivo neural stem cell and mouse models\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (protein stability, replication stress, in vivo models), convergent with simultaneous publications\",\n      \"pmids\": [\"33854232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Upon mitochondrial depolarization, AMBRA1 is recruited to the outer mitochondrial membrane and interacts with PINK1 and ATAD3A (a transmembrane protein mediating PINK1 import and degradation). AMBRA1 deficiency reduces PINK1 stability by enhancing its degradation via the mitochondrial protease LONP1, decreasing PINK1-mediated ubiquitin phosphorylation and PARKIN recruitment. ATAD3A silencing rescues defective PINK1 accumulation in AMBRA1-deficient cells.\",\n      \"method\": \"Co-immunoprecipitation (AMBRA1–PINK1, AMBRA1–ATAD3A), AMBRA1 knockdown/ATAD3A knockdown epistasis, LONP1-dependent PINK1 degradation assays, mitophagy flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, epistasis between AMBRA1 and ATAD3A, protease inhibition experiments, multiple orthogonal approaches; single lab\",\n      \"pmids\": [\"34798798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AMBRA1 (as the CRL4 substrate receptor) targets Elongin C (ELOC), the shared adapter of CRL5 ubiquitin ligase complexes, for polyubiquitination and degradation, thereby attenuating CRL5SOCS3 and CRL5VIF ligase activity. This cross-regulation modulates IL-6/STAT3 signaling and HIV-1 infectivity.\",\n      \"method\": \"Proteomics (affinity purification-MS), in vitro ubiquitination assay, Co-immunoprecipitation, CRL5 assembly assays, IL-6/STAT3 reporter assays, HIV infectivity assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — proteomics-validated substrate, in vitro ubiquitination reconstitution, functional downstream assays; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30166453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AMBRA1 promotes TGFβ signaling as the substrate receptor of the CRL4 ubiquitin ligase complex, mediating nonproteolytic polyubiquitylation of Smad4 to enhance its transcriptional activity, thereby promoting TGFβ-induced EMT, migration, and invasion of breast cancer cells and metastasis in mouse models.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, reporter gene assays, AMBRA1 knockdown/overexpression, mouse metastasis models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro ubiquitination, Co-IP, functional migration and in vivo assays; single lab\",\n      \"pmids\": [\"34362797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AMBRA1 negatively regulates ALDH1B1 (a cancer stem cell marker) by mediating its K27- and K33-linked non-canonical ubiquitination in cooperation with TRAF6. Ubiquitination sites K506, K511, and K515 are important; ubiquitination-defective ALDH1B1 shows increased self-association, suggesting ubiquitination suppresses ALDH1B1 oligomerization.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay with ubiquitin linkage mapping (K27, K33), site-directed mutagenesis, self-association assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Weak — ubiquitination assay with linkage mapping and mutagenesis, but single lab, single study\",\n      \"pmids\": [\"34769507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ambra1 deficiency in skeletal muscle (muscle-specific knockout) impairs mitophagic flux without affecting bulk autophagy, causing accumulation of TOMM20, swollen mitochondria, reduced mitochondrial membrane potential, decreased complex I activity, reduced recruitment of DRP1 and Parkin to mitochondria, and lysosomal accumulation. Overexpression of mitochondria-targeted Ambra1 in wild-type muscle is sufficient to enhance mitochondria clearance.\",\n      \"method\": \"Muscle-specific conditional Ambra1 knockout mice (Ambra1fl/fl:Mlc1f-Cre), in vivo Ambra1 overexpression, mitochondrial fractionation, electron microscopy, respiratory complex activity assay, mitophagy flux assays\",\n      \"journal\": \"Journal of cachexia, sarcopenia and muscle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific conditional KO with multiple orthogonal phenotypic readouts and gain-of-function complementation; single lab\",\n      \"pmids\": [\"35593053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AMBRA1 is phosphorylated during mitosis by CDK1 and PLK1 on multiple sites. This phosphorylation is required for proper mitotic spindle orientation, mediated through NUMA1. AMBRA1 localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation, and binding ability.\",\n      \"method\": \"Phosphorylation mapping during mitotic arrest, CDK1/PLK1 kinase assays, AMBRA1 phospho-mutant analysis, NUMA1 localization by imaging, spindle orientation assays\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay, phospho-mutagenesis, functional spindle assays; single lab\",\n      \"pmids\": [\"37584777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structure of AMBRA1 in complex with DDB1 resolved at 3.08 Å. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1, creating a scaffold for substrate recruitment. DDB1-binding-defective AMBRA1 mutants prevent Cyclin D1 ubiquitination in vitro and increase cell cycle progression.\",\n      \"method\": \"Cryo-EM structure determination, HDX-MS, in vitro ubiquitination assay, DDB1-binding mutagenesis, cell cycle assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure at 3.08 Å with in vitro functional validation by mutagenesis and reconstituted ubiquitination assay\",\n      \"pmids\": [\"37993427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AMBRA1 interacts with MAVS (mitochondrial antiviral signaling protein) at mitochondria and stabilizes MAVS by preventing its dsRNA-induced proteasomal degradation, thereby promoting dsRNA- and virus-induced apoptosis through a caspase-8-dependent pathway.\",\n      \"method\": \"Co-immunoprecipitation (AMBRA1–MAVS), AMBRA1 gene editing (knockout), proteasome inhibitor assays, apoptosis assays, Semliki Forest virus infection model\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, genetic KO with rescue, functional apoptosis assays; single lab\",\n      \"pmids\": [\"34859815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BAG2, an effector of the ULK1 complex, regulates AMBRA1 subcellular localization. In nutrient-replete conditions, unphosphorylated BAG2 sequesters AMBRA1, attenuating autophagy. Upon starvation, ULK1 phosphorylates BAG2 at Ser31, which promotes AMBRA1 recruitment to the ER membrane and autophagy induction.\",\n      \"method\": \"Affinity purification-MS and proximity labeling-MS interactome of ULK1 complex subunits, Co-immunoprecipitation, BAG2 phospho-mutant (S31A), AMBRA1 localization imaging\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics-validated interaction, Co-IP, phospho-mutagenesis, localization imaging; single lab\",\n      \"pmids\": [\"39207901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AMBRA1 promotes intestinal NF-κB-driven inflammation in an autophagy-independent manner by antagonizing the PP4R1/PP4c phosphatase complex: the N-terminal F1 domain of AMBRA1 competitively binds PP4R1, disrupting PP4R1/PP4c interaction with IKK and preventing IKK dephosphorylation. Upon TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043, impairing its CUL4A-mediated K48-linked ubiquitination and stabilizing AMBRA1.\",\n      \"method\": \"Co-immunoprecipitation (AMBRA1–PP4R1, AMBRA1–PP4c, AMBRA1–IKK), AMBRA1 domain deletion/mutagenesis (F1 domain, S1043), IKKα kinase assay, CUL4A ubiquitination assay, AMBRA1 conditional knockout mice\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — kinase assay, ubiquitination assay, domain mutagenesis, Co-IP, in vivo mouse model; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"38424148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In intestinal epithelial cells, AMBRA1 acts as a pro-oxidative factor by competitively binding NRF2 through its N-terminal F1 domain, antagonizing DUB3-mediated NRF2 deubiquitination and promoting NRF2 degradation. Under oxidative stress (H2O2), USP7 interaction with AMBRA1 is enhanced, leading to USP7-mediated deubiquitination of AMBRA1 at K83 and K86, stabilizing AMBRA1.\",\n      \"method\": \"Co-immunoprecipitation (AMBRA1–NRF2, AMBRA1–DUB3, AMBRA1–USP7), domain mutagenesis (F1 domain, K83/K86 sites), in vitro deubiquitination assay, AMBRA1 conditional knockout mice, IBD patient tissue analysis\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, site-specific mutagenesis, in vitro deubiquitination, in vivo mouse model; single lab\",\n      \"pmids\": [\"39887666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AMBRA1 binds α-synuclein phosphorylated at serine 129 with ~9-fold higher affinity than non-phosphorylated α-synuclein (in vitro and in vivo analyses). Silencing AMBRA1 function causes α-synuclein aggregates in the cytoplasm of mouse primary neurons, similar to the effect of the autophagy inhibitor Bafilomycin.\",\n      \"method\": \"Binding affinity assays (in vitro and in vivo), AMBRA1 knockdown in primary neurons, immunohistochemistry of human MSA brain samples\",\n      \"journal\": \"Brain pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — quantified binding assay, functional knockdown, human tissue validation; single lab\",\n      \"pmids\": [\"27875637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AMBRA1 suppresses SOCS3 in medulloblastoma Group 3 stem cells, enabling STAT3 activation. AMBRA1 expression depends on c-MYC levels, and AMBRA1 knockdown reduces stem potential, growth, and migration of MBGroup3 cells. Combined anti-autophagy and anti-STAT3 treatment impacts MBGroup3 outcome.\",\n      \"method\": \"AMBRA1 knockdown/overexpression, SOCS3 expression assays, STAT3 activation assays, tumor sphere formation, migration assays\",\n      \"journal\": \"Acta neuropathologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — functional knockdown with defined pathway placement (AMBRA1→SOCS3→STAT3), but mechanistic basis of SOCS3 suppression not fully biochemically characterized; single lab\",\n      \"pmids\": [\"34302498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of Ambra1 in melanoma promotes aggressiveness and metastasis by increasing cell motility/invasion, activating an EMT-like process, remodeling the extracellular matrix, and inducing hyperactivation of FAK1 signaling. FAK1 inhibition reduces cell invasion and melanoma growth in Ambra1-deficient cells.\",\n      \"method\": \"Ambra1-depleted Braf/Pten mouse melanoma model, invasion and migration assays, EMT marker analysis, FAK1 phosphorylation assays, FAK1 pharmacological inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with defined signaling pathway (Ambra1→FAK1 hyperactivation), pharmacological rescue; single lab\",\n      \"pmids\": [\"33953176\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AMBRA1 is a multifunctional scaffold protein that (1) positively regulates autophagy initiation by interacting with BECLIN1-VPS34; under basal conditions it is tethered to dynein via DLC1/2 and is released upon ULK1-mediated phosphorylation to relocalize to the ER for autophagosome nucleation; (2) serves as the substrate receptor of the CRL4 (CUL4-DDB1) ubiquitin E3 ligase to drive proteasomal degradation of all D-type cyclins, thereby restraining G1/S progression; (3) regulates mitophagy via interactions with Parkin, PINK1/ATAD3A, and HUWE1/IKKα-mediated phosphorylation at S1014; (4) is itself regulated by mTOR phosphorylation, Cullin-4/5 ubiquitylation, RNF2/WASH-mediated K48-ubiquitylation, USP7-mediated deubiquitylation at K83/K86, and caspase/calpain cleavage during apoptosis; (5) controls c-Myc levels through PP2A interaction; and (6) performs additional autophagy-independent functions including NF-κB/IKK regulation, TGFβ-Smad4 polyubiquitylation, mitotic spindle orientation via CDK1/PLK1 phosphorylation and NUMA1 regulation, and spatial control of Src/FAK signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AMBRA1 is a WD40-domain scaffold protein that couples autophagy initiation to cell-cycle control, signaling, and cell-fate decisions [#0, #17]. In its founding role it positively regulates BECLIN1-dependent autophagy, and its loss in mouse embryos impairs autophagic flux and produces neural tube defects with unbalanced proliferation and apoptosis [#0]. Mechanistically, the BECLIN1-VPS34 complex is held inactive at the dynein motor through AMBRA1's interaction with dynein light chains; ULK1-mediated phosphorylation releases AMBRA1, allowing relocalization to the endoplasmic reticulum and mitochondria-associated membranes for autophagosome nucleation [#1, #16]. AMBRA1 sits within a dense network of E3 ligases and kinases that tune its abundance and activity: it directs TRAF6-dependent K63 ubiquitylation of ULK1 in a positive feedback loop opposed by mTOR phosphorylation, while its own levels are restrained by Cullin-4 and RNF2/WASH-mediated K48 ubiquitylation and reset by USP7 deubiquitylation at K83/K86 [#5, #7, #8, #29]. Independently of autophagosome formation, AMBRA1 serves as the substrate receptor of a CRL4 (CUL4-DDB1) ubiquitin ligase that drives proteasomal degradation of all three D-type cyclins, thereby restraining the G1-to-S transition and limiting replication stress; a 3.08 Å cryo-EM structure shows its N-terminal helix-loop-helix and WD40 domains clamping the DDB1 double-propeller to build the substrate-recruitment platform [#17, #18, #25]. As a CRL4 receptor AMBRA1 also ubiquitylates Elongin C and Smad4, linking it to CRL5 cross-regulation and TGFβ-driven EMT [#20, #21]. In mitochondrial quality control, AMBRA1 drives both PINK1/PARKIN-dependent and -independent mitophagy, acting through PINK1/ATAD3A to stabilize PINK1 and through HUWE1/IKKα-mediated phosphorylation at S1014 to engage LC3/GABARAP [#3, #12, #19, #23]. Through PP2A it promotes dephosphorylation and degradation of c-Myc, establishing AMBRA1 as a haploinsufficient tumor suppressor [#6]. Additional autophagy-independent functions include NF-κB/IKK regulation via the PP4R1/PP4c phosphatase complex and mitotic spindle orientation through CDK1/PLK1 phosphorylation and NUMA1 [#28, #24].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established AMBRA1 as a positive regulator of autophagy and an essential developmental gene, answering whether it acts in the BECLIN1 pathway and what its loss does in vivo.\",\n      \"evidence\": \"RNAi and overexpression in vitro plus mouse knockout with phenotypic analysis\",\n      \"pmids\": [\"17589504\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular trigger linking AMBRA1 to autophagosome nucleation\", \"Mechanism of the neural tube/proliferation phenotype unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved how AMBRA1 is held inactive and activated, showing dynein tethering of BECLIN1-VPS34 is released by ULK1 phosphorylation to permit ER relocalization.\",\n      \"evidence\": \"Co-IP, live-cell imaging, ULK1 kinase assays and DLC1-binding-site mutagenesis\",\n      \"pmids\": [\"20921139\", \"21079415\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise ER docking partners not identified at this stage\", \"ULK1 phosphosites on AMBRA1 not fully mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed AMBRA1 within mitochondrial autophagy control through BCL-2 inhibition and Parkin-dependent recruitment to depolarized mitochondria, connecting it to mitophagy.\",\n      \"evidence\": \"Reciprocal Co-IP, fractionation, TAP-MS and endogenous Co-IP across HEK293, SH-SY5Y and mouse brain with knockdown readouts\",\n      \"pmids\": [\"21358617\", \"21753002\", \"21921694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct AMBRA1-driven steps in mitophagosome formation not biochemically reconstituted\", \"BCL-2 binding interface not mapped at this point\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed AMBRA1 is dismantled during apoptosis, defining a caspase/calpain switch (D482) that couples autophagy shutdown to cell death rate.\",\n      \"evidence\": \"In vitro cleavage assays, inhibitor treatment, D482 mutagenesis and cell death assays\",\n      \"pmids\": [\"22441670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where this cleavage dominates unclear\", \"Fate of cleavage fragments not yet characterized (addressed later)\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the kinase/ligase feedback architecture: mTOR inhibits AMBRA1, while dephosphorylated AMBRA1 recruits TRAF6 to K63-ubiquitylate and stabilize ULK1, with ULK1 reciprocally activating AMBRA1.\",\n      \"evidence\": \"Co-IP, K63-linkage-specific ubiquitylation assays, phospho-mapping and mTOR inhibition\",\n      \"pmids\": [\"23524951\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mTOR target residues on AMBRA1 not exhaustively defined\", \"Stoichiometry of the feedback loop not quantified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified the ubiquitin machinery controlling AMBRA1 abundance (Cullin-4, RNF2/WASH at K45) and a proliferation-restraining role via PP2A-mediated c-Myc degradation, establishing tumor-suppressor function.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro ubiquitination with linkage/site mapping, PP2A activity assays and mouse tumor models\",\n      \"pmids\": [\"25499913\", \"24980959\", \"25438055\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How multiple ligases are coordinated temporally not resolved\", \"Direct enzymatic basis of AMBRA1-stimulated PP2A activity unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed the caspase-generated AMBRA1 C-terminal fragment carries a BH3-like domain that binds and inhibits BCL-2, defining a proapoptotic feedback role for the cleavage product.\",\n      \"evidence\": \"Co-IP of AMBRA1-CT with BCL-2 and BH3-domain mutagenesis with apoptosis assays\",\n      \"pmids\": [\"27123694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding without reconstituted structural confirmation\", \"Contribution relative to canonical BH3 proteins not quantified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked AMBRA1 to focal-adhesion signaling and to α-synuclein clearance, broadening its roles beyond canonical autophagy into Src/FAK trafficking and neurodegeneration-relevant cargo recognition.\",\n      \"evidence\": \"AP-MS interactomics, Co-IP, live imaging and invasion assays; phospho-S129 α-synuclein binding affinity assays with neuronal knockdown and MSA tissue\",\n      \"pmids\": [\"28362576\", \"27875637\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs scaffold-mediated nature of FAK/Src interactions not fully dissected\", \"Structural basis of phospho-α-synuclein preference unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined a PINK1/PARKIN-independent mitophagy route (HUWE1/IKKα phosphorylating AMBRA1-S1014 to engage LC3/GABARAP) and immune/Treg functions via PP2A-FOXO3-FOXP3.\",\n      \"evidence\": \"In vitro IKKα kinase assay, S1014 phospho-mutant, LC3 interaction and mitophagy assays; Treg Co-IP and in vivo mouse models\",\n      \"pmids\": [\"30217973\", \"30513302\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural change induced by S1014 phosphorylation inferred, not resolved\", \"Interplay between PINK1-dependent and -independent routes not reconciled\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Refined mitophagy regulation (GSK-3β/MCL-1 gating of HUWE1 recruitment) and revealed a muscle TRIM32-ULK1 axis, connecting AMBRA1 to disease-linked autophagy in skeletal muscle.\",\n      \"evidence\": \"Co-IP, GSK-3β kinase assays, phospho-mutants, HUWE1 knockdown, ULK1 activity assays and TRIM32 disease-mutation tests\",\n      \"pmids\": [\"31434979\", \"31234693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab mechanisms not independently reconstituted\", \"Tissue specificity of these regulatory arms unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Localized AMBRA1's nucleation function to MAM raft-like microdomains via ERLIN1, GD3 and MFN2, defining the ER membrane platform for starvation-induced autophagosome formation.\",\n      \"evidence\": \"Co-IP, FRET, MAM fractionation and ERLIN1/ST8SIA1/MFN2 knockdowns\",\n      \"pmids\": [\"33034545\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs lipid-dependent nature of the ERLIN1 interaction not separated\", \"Single-lab without structural validation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established the major autophagy-independent identity of AMBRA1 as the CRL4 substrate receptor degrading D-type cyclins, controlling G1/S and replication stress, and extended this receptor role to ELOC, Smad4 and ALDH1B1 substrates.\",\n      \"evidence\": \"Reconstituted in vitro ubiquitination, genome-wide CRISPR screens, mouse genetic models, DNA fiber/CHK1 synthetic-lethality assays, and substrate-specific ubiquitination/Co-IP assays\",\n      \"pmids\": [\"33854235\", \"33854239\", \"33854232\", \"30166453\", \"34362797\", \"34769507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How substrate selection among cyclins, ELOC and Smad4 is regulated unknown\", \"Crosstalk between CRL4-receptor and autophagy roles not integrated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Detailed AMBRA1's PINK1/ATAD3A mitophagy axis and disease-context roles in melanoma (FAK1) and medulloblastoma (SOCS3/STAT3), linking its loss to tumor aggressiveness.\",\n      \"evidence\": \"Co-IP, ATAD3A epistasis, LONP1-dependent PINK1 degradation assays; in vivo melanoma and medulloblastoma models with pathway inhibition\",\n      \"pmids\": [\"34798798\", \"33953176\", \"34302498\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical basis of SOCS3 suppression not established\", \"Whether FAK1 hyperactivation is direct or indirect not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated muscle-specific mitophagy dependence and a MAVS-stabilizing antiviral apoptosis role, separating AMBRA1's mitophagic and innate-immune functions.\",\n      \"evidence\": \"Muscle-specific conditional knockout with mitochondrial/respiratory phenotyping and mito-targeted rescue; AMBRA1-MAVS Co-IP, knockout and viral infection apoptosis assays\",\n      \"pmids\": [\"35593053\", \"34859815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity of muscle mitophagy versus bulk autophagy mechanism unclear\", \"MAVS stabilization mechanism (ligase competition) not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided the structural basis for CRL4 substrate recruitment (cryo-EM AMBRA1-DDB1) and a mitotic function (CDK1/PLK1 phosphorylation controlling NUMA1-dependent spindle orientation).\",\n      \"evidence\": \"3.08 Å cryo-EM, HDX-MS, DDB1-binding mutagenesis and reconstituted ubiquitination; mitotic phospho-mapping, CDK1/PLK1 assays and spindle orientation assays\",\n      \"pmids\": [\"37993427\", \"37584777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate-engaging surface of the WD40 domain not resolved with cyclin bound\", \"How mitotic phosphorylation reroutes AMBRA1 to NUMA1 not mechanistically detailed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined autophagy-independent NF-κB inflammation control via PP4R1/PP4c antagonism and an IKKα-S1043/CUL4A stabilization switch, plus BAG2-mediated localization control of AMBRA1.\",\n      \"evidence\": \"Co-IP, F1-domain/S1043 mutagenesis, IKKα kinase and CUL4A ubiquitination assays, conditional knockout mice; ULK1-complex interactomics with BAG2-S31A phospho-mutant\",\n      \"pmids\": [\"38424148\", \"39207901\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of S1014 vs S1043 phosphorylation signals not resolved\", \"Whether BAG2 sequestration competes with dynein tethering unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a pro-oxidative function in intestinal epithelium where AMBRA1 antagonizes DUB3-mediated NRF2 deubiquitination, with USP7 stabilizing AMBRA1 at K83/K86 under oxidative stress.\",\n      \"evidence\": \"Co-IP, F1-domain and K83/K86 mutagenesis, in vitro deubiquitination, conditional knockout mice and IBD tissue analysis\",\n      \"pmids\": [\"39887666\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without independent confirmation\", \"Relationship between USP7-AMBRA1 stabilization and the CRL4 receptor pool unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AMBRA1's distinct functions—autophagy scaffold, CRL4 substrate receptor, phosphatase regulator, and mitotic/spindle factor—are partitioned and switched within a cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of how post-translational marks route AMBRA1 between its competing complexes\", \"Substrate-binding surface of the WD40 domain not structurally defined with a bound substrate\", \"Quantitative partitioning of cytosolic, ER, and mitochondrial AMBRA1 pools unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 17, 25]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [17, 20, 21, 22, 25]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 11, 28, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 16, 27]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [3, 12, 19, 23, 26]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0, 1, 5, 7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [17, 18, 24, 25]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [8, 17, 20, 21]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 9, 26]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [28, 32, 6]}\n    ],\n    \"complexes\": [\n      \"CRL4 (CUL4-DDB1) E3 ubiquitin ligase\",\n      \"BECLIN1-VPS34 (class III PI3K) complex\",\n      \"ULK1 complex\"\n    ],\n    \"partners\": [\n      \"BECLIN1\",\n      \"ULK1\",\n      \"DDB1\",\n      \"TRAF6\",\n      \"PP2A\",\n      \"PINK1\",\n      \"HUWE1\",\n      \"DLC1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}