{"gene":"RNF7","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":1999,"finding":"SAG/RBX2 (RNF7) was cloned as a novel zinc RING finger protein that binds zinc and copper metal ions, prevents copper-induced lipid peroxidation, forms inter- and intramolecular disulfide bonds in response to oxidative stress, and protects cells from apoptosis induced by redox agents; the C-terminal RING finger domain is required for anti-apoptosis activity.","method":"Bacterially expressed/purified protein biochemical assays (metal binding, lipid peroxidation), stable transfection, RING domain mutagenesis, oligomerization assays with DTT/H2O2","journal":"Molecular and cellular biology / Free radical biology & medicine / Carcinogenesis","confidence":"High","confidence_rationale":"Tier 1 — reconstituted biochemistry plus mutagenesis, multiple orthogonal methods across multiple papers","pmids":["10082581","10443936","10506102"],"is_preprint":false},{"year":1999,"finding":"SAG/ROC2/RBX2 (RNF7) is the second family member of the ROC/Rbx RING component of SCF E3 ubiquitin ligase; it binds Cullin-1 and the SAG-CUL1 complex has ubiquitin ligase activity promoting poly-ubiquitination of E2/CDC34 in vitro.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro ubiquitination assay with E1/E2/CUL1","journal":"Molecular cell / Oncogene","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstituted ubiquitin ligase activity, independently confirmed by multiple labs (ROC1/ROC2 papers)","pmids":["10230407","10851089"],"is_preprint":false},{"year":1999,"finding":"ROC1-CUL1 RING-based ubiquitin ligase (with SAG/ROC2 as paralog) can catalyze ubiquitination of IκBα in a phosphorylation-dependent manner in vitro when reconstituted with SCFHOS components, E1, and CDC34.","method":"In vitro reconstituted ubiquitination assay with purified components","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted ubiquitination with purified components","pmids":["10230406"],"is_preprint":false},{"year":1999,"finding":"Protein kinase CKII interacts with SAG/RNF7 via the RING-H2 finger motif; specifically, the beta subunit of CKII binds SAG and phosphorylates it in vitro, while the alpha subunit does not interact.","method":"Yeast two-hybrid, overlay/direct binding assay, in vitro phosphorylation by recombinant CKII, deletion mutagenesis of SAG","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — yeast two-hybrid plus direct in vitro phosphorylation, single lab","pmids":["10512750"],"is_preprint":false},{"year":2000,"finding":"The yeast homolog of SAG/RNF7 (ySAG) is essential for cell viability; deletion causes cell death with cell enlargement and abnormal cell cycle profiles. Complementation requires the RING finger domain and ubiquitin ligase activity, confirming that E3 ligase function is required for cell survival.","method":"Targeted yeast gene disruption, tetrad analysis, complementation with wild-type vs. RING mutant hSAG, cDNA chip profiling of yeast genome","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis (yeast KO + rescue) with mutagenesis confirming mechanistic requirement, replicated by complementation","pmids":["10851089"],"is_preprint":false},{"year":2001,"finding":"SAG/RNF7 promotes S-phase entry and cell growth under serum starvation by inhibiting p27 accumulation through the ubiquitin-proteasome pathway; SAG associates in vivo with SKP2, an F-box protein that promotes p27 ubiquitination.","method":"SAG mRNA microinjection into quiescent cells ([3H]-thymidine incorporation), adenoviral overexpression, co-immunoprecipitation of SAG with SKP2, proteasome inhibitor rescue experiment","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 — cellular overexpression plus Co-IP and proteasome inhibitor evidence, single lab","pmids":["11255262"],"is_preprint":false},{"year":2001,"finding":"SAG/RNF7 gene consists of four exons and three introns; a splicing variant (SAG-v) incorporates an additional exon disrupting the RING finger ORF, and SAG-v lacks cullin-1 binding and ubiquitin ligase activity, confirming that the RING domain is required for E3 function.","method":"Genomic cloning, RT-PCR, yeast complementation, co-immunoprecipitation with CUL1, in vitro ubiquitination assay","journal":"DNA and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — functional domain analysis with ligase assay and binding experiments","pmids":["11506706"],"is_preprint":false},{"year":2001,"finding":"SAG/RNF7 protects neurons in vivo against ischemia/reperfusion brain injury; overexpression via adenoviral delivery reduced infarct area, decreased ROS production and apoptosis; the RING domain mutant was not protective, confirming domain requirement.","method":"Adenoviral delivery of wild-type vs. RING mutant SAG into mouse brain, middle cerebral artery occlusion model, infarct measurement, ROS assay","journal":"Journal of cerebral blood flow and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo loss/gain of function with RING mutant control","pmids":["11488541"],"is_preprint":false},{"year":2004,"finding":"VHL-box proteins specifically interact with Cul2-Rbx1 modules, whereas SOCS-box proteins associate with Cul5-Rbx2 (RNF7). The specificity is determined by the downstream Cul2-box or Cul5-box sequences. RNAi knockdown of Cul5-Rbx2 does not affect VHL-mediated HIF-2α degradation, whereas Cul2-Rbx1 knockdown inhibits it, demonstrating functional distinction between the two Rbx modules.","method":"Co-immunoprecipitation of endogenous complexes, domain-swap mutagenesis, siRNA knockdown with HIF-2α degradation readout","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1–2 — domain-swapping mutagenesis plus endogenous Co-IP plus functional RNAi epistasis, replicated","pmids":["15601820"],"is_preprint":false},{"year":2006,"finding":"SAG/RNF7 forms part of SAG/ROC-SCF(β-TrCP) E3 ubiquitin ligase that binds pro-caspase-3 via the F-box protein β-TrCP (which recognizes the first 38 amino acids of pro-caspase-3) and promotes its ubiquitination and degradation; siRNA silencing of SAG, ROC1, or β-TrCP increases pro-caspase-3 levels and enhances apoptosis.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, siRNA knockdown, dominant-negative β-TrCP expression, protein half-life assay","journal":"Neoplasia","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro ubiquitination plus Co-IP plus siRNA epistasis, multiple orthogonal approaches","pmids":["17217622"],"is_preprint":false},{"year":2007,"finding":"SAG/RNF7 is a transcriptional target of HIF-1; HIF-1 binds a consensus GCGTG site in the first intron of the SAG gene under hypoxia, and induced SAG in turn promotes VHL-mediated HIF-1α ubiquitination and degradation by forming a complex with CUL-5 and VHL, establishing a negative feedback loop.","method":"ChIP assay, luciferase reporter, siRNA silencing, co-immunoprecipitation under hypoxia, HIF-1α protein half-life measurement","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (ChIP, reporter, Co-IP, siRNA) in single lab","pmids":["17828303"],"is_preprint":false},{"year":2007,"finding":"SAG/RNF7 is a novel AP-1 transcriptional target; AP-1 binds two consensus sites in the SAG promoter in vitro and in vivo. SAG reciprocally promotes c-Jun ubiquitination and degradation via Fbw7-SCF, establishing an AP-1/SAG autofeedback loop; SAG siRNA silencing reduced c-Jun polyubiquitination.","method":"EMSA, ChIP, luciferase reporter, siRNA knockdown, c-Jun polyubiquitination assay, dominant-negative c-Jun overexpression","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — ChIP plus EMSA plus ubiquitination assay plus siRNA, multiple orthogonal methods","pmids":["17440073"],"is_preprint":false},{"year":2007,"finding":"SAG/RNF7 in mouse epidermis targets c-Jun/AP-1 for degradation at early stages of skin carcinogenesis (inhibiting tumor promotion) and targets IκBα for degradation at later stages (activating NF-κB to reduce apoptosis and enhance tumor growth), demonstrating stage-dependent substrate targeting by different F-box proteins.","method":"SAG-transgenic mouse model (K14 promoter), AP-1 luciferase reporter mice, Western blotting, DMBA/TPA carcinogenesis model","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — in vivo transgenic model with mechanistic pathway analysis, multiple substrates validated","pmids":["17846172"],"is_preprint":false},{"year":2008,"finding":"SAG/RNF7 promotes UVB-induced skin hyperplasia by simultaneously targeting c-Jun (pro-tumor promotion) and p27 (anti-proliferative) for degradation in mouse epidermis, but does not affect UVB-induced p53, c-Fos, or cyclin D1.","method":"K14-SAG transgenic mice, UVB carcinogenesis model, Western blotting, AP-1 activity assays, DNA synthesis measurement","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo transgenic model with substrate-level mechanistic analysis","pmids":["18258608"],"is_preprint":false},{"year":2008,"finding":"Cul2-box and Cul5-box sequences determine specificity of BC-box protein assembly with Cul2-Rbx1 versus Cul5-Rbx2 (RNF7) modules; the LPPhiP motif conserved in Cul5-boxes is also compatible with Cul2 interaction; spacer length between BC-box and cullin-box can vary 3–80 amino acids.","method":"Purification and co-immunoprecipitation of reconstituted BC-box protein complexes, structure-function domain analysis with mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — systematic structure-function with reconstituted complexes across many BC-box proteins","pmids":["18187417"],"is_preprint":false},{"year":2009,"finding":"UBE2F is a NEDD8-conjugating E2 enzyme that specifically pairs with RBX2/RNF7 to neddylate CUL5, while UBE2M pairs with RBX1 to neddylate CUL1-4; structural analysis reveals how E1 accommodates both E2s, establishing that RBX2 selectively activates CUL5-based CRLs through the UBE2F/RBX2 neddylation axis.","method":"In vitro neddylation assay, structural analysis of E1-E2 interactions, mutagenesis, cellular neddylation experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro neddylation plus structural analysis plus mutagenesis","pmids":["19250909"],"is_preprint":false},{"year":2010,"finding":"SAG/RNF7 silencing induces apoptosis with accumulation of NOXA; SAG overexpression reduces NOXA levels and shortens NOXA protein half-life, identifying NOXA as a substrate of SAG E3 ubiquitin ligase. In vivo, SAG silencing suppresses xenograft tumor growth.","method":"siRNA silencing, protein half-life assay, flow cytometry for apoptosis, caspase-3 assay, in vivo orthotopic xenograft model","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 — cellular loss-of-function with substrate stabilization, in vivo validation","pmids":["20103673"],"is_preprint":false},{"year":2010,"finding":"Sag/Rbx2 deletion in mouse embryonic stem cells sensitizes them to radiation through increased ROS (including superoxide) and decreased NF-κB activation; IκBα was identified as a direct substrate of SAG-SCF(β-TrCP) E3 ubiquitin ligase, as its degradation is blocked upon Sag elimination.","method":"Gene-trap Sag knockout ES cells, clonogenic survival, intracellular ROS measurement, NF-κB reporter, IκBα protein level analysis","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 2 — complete gene knockout with multiple mechanistic readouts and direct substrate identification","pmids":["20638939"],"is_preprint":false},{"year":2010,"finding":"SAG/RNF7 as a Tat-fusion protein can be transduced into cells and brain tissue; wild-type but not RING-mutant Tat-SAG protects hippocampal neurons from ischemia-induced death and reduces lipid peroxidation in vivo, confirming that ROS-scavenging via the RING domain is the protective mechanism.","method":"Tat-fusion protein transduction, gerbil transient forebrain ischemia model, immunohistochemistry, lipid peroxidation assay","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo model with wild-type vs. RING mutant control","pmids":["20100567"],"is_preprint":false},{"year":2010,"finding":"CRL network quantitative proteomics shows SAG/RBX2 assembles within distinct CRL5 complexes; network architecture reveals that neddylation inhibition (MLN4924) does not globally reorganize CRL complexes, but adaptor module abundance drives CRL organization.","method":"AQUA quantitative proteomics, affinity purification-MS of CRL components, MLN4924 treatment","journal":"Cell","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative proteomics with pharmacological perturbation","pmids":["21145461"],"is_preprint":false},{"year":2011,"finding":"SAG/RBX2/RNF7 is an essential RING component of SCF E3 ubiquitin ligase required for embryonic development; Sag knockout mice die at E11.5–12.5 with vascular and neural defects caused by NF1 accumulation and RAS inhibition. NF1 was identified as a physiological substrate of SAG-CUL1-FBXW7 E3 ligase; simultaneous Nf1 deletion partially rescues vascular defects in Sag-null embryos.","method":"Sag conditional knockout mice, genetic epistasis (Sag/Nf1 double knockout), ES cell endothelial differentiation assays, teratoma angiogenesis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — in vivo double-knockout epistasis plus ES cell differentiation assay identifying NF1 as a substrate","pmids":["22118770"],"is_preprint":false},{"year":2011,"finding":"SAG/RBX2 inactivation in mouse embryonic stem cells blocks retinoic acid (RA)-induced differentiation, causing apoptosis instead; in leukemia cells, SAG inhibition with MLN4924 sensitizes to RA-differentiation therapy through accumulation of NOXA and c-JUN substrates of SAG-SCF E3 ligase.","method":"Sag-/- ES cells, RA exposure, atomic force microscopy for cell stiffness, MLN4924 treatment of leukemia lines, Western blotting for NOXA/c-JUN","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO plus pharmacological inhibition with substrate accumulation readout","pmids":["22110742"],"is_preprint":false},{"year":2011,"finding":"DEPTOR is a physiological substrate of SCF(β-TrCP)/SAG E3 ligase; growth factor stimulation activates RSK1/S6K1 to phosphorylate DEPTOR, enabling β-TrCP recognition and SCF-mediated ubiquitination and degradation; this activates mTOR in a positive feedback loop.","method":"Protein half-life assay, co-immunoprecipitation, siRNA knockdown, phosphodegron mutagenesis, kinase inhibitor experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 — reconstituted pathway with mutagenesis, multiple orthogonal methods, replicated across three independent labs (PMIDs 22017876, 22017875, 22017877)","pmids":["22017876","22017875","22017877"],"is_preprint":false},{"year":2011,"finding":"HIV-1 Vif hijacks CUL5-ELOB-ELOC-RBX2(RNF7) ubiquitin ligase complex (together with CBF-β) to polyubiquitinate APOBEC3G for degradation; a recombinant six-protein assembly including RBX2 reconstitutes specific APOBEC3G ubiquitination activity.","method":"Affinity purification-mass spectrometry, in vitro reconstituted six-protein ubiquitination assay, RNA knockdown, genetic complementation","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — reconstituted ubiquitination with purified components, mechanistically validated with knockdown and complementation","pmids":["22190037"],"is_preprint":false},{"year":2013,"finding":"RBX2/RNF7 is required for neocortical and cerebellar neuron positioning; Rbx2 mutation causes DAB1-dependent ectopias via sustained Reelin signaling. SOCS7 is identified as a CRL5-Rbx2 substrate adaptor that promotes ubiquitylation and turnover of DAB1, stopping neuronal migration at target layers; cerebellar development requires Rbx2 through a different adaptor.","method":"In utero electroporation, Rbx2 conditional knockout mice, SOCS7 gain-of-function/loss-of-function, DAB1 protein level analysis, epistasis with reelin pathway","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — conditional KO plus epistasis with Reelin pathway plus substrate adaptor identification","pmids":["24210661"],"is_preprint":false},{"year":2013,"finding":"Sag endothelial deletion causes embryonic lethality at E15.5 with poor vasculogenesis; Sag deletion in endothelial cells inhibits migration, proliferation, and tube formation with p27 accumulation responsible for migration/proliferation suppression; Sag is required for tumor angiogenesis in a B16F10 melanoma model.","method":"Tie2-Cre/Sag conditional KO mice, primary EC culture assays (migration, proliferation, tube formation), p27 knockdown rescue, in vivo Matrigel plug and tumor models","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with genetic rescue (p27 knockdown), multiple in vitro and in vivo endpoints","pmids":["24213570"],"is_preprint":false},{"year":2014,"finding":"Sag deletion suppresses KrasG12D-driven lung tumorigenesis; SAG knockdown inactivates NF-κB and mTOR pathways and causes accumulation of p21, p27, NOXA, and BIM; growth suppression by SAG knockdown is partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR, establishing these as causal substrates.","method":"Lung-specific Sag conditional KO in KrasG12D mice, siRNA epistasis (SAG + p21 or DEPTOR double knockdown), pathway analysis (NF-κB, mTOR)","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — in vivo conditional KO with genetic epistasis identifying substrates","pmids":["24430184"],"is_preprint":false},{"year":2014,"finding":"SAG/RNF7 promotes ubiquitination of Bax and SARM in macrophages during pathogen challenge, conferring survival advantage; SAG knockdown causes accumulation of pro-apoptotic Bax and SARM, disruption of Bcl-2/Bax balance, cytochrome c release, and caspase-9/-3 activation.","method":"SAG knockdown/overexpression in macrophages, PAMP challenge, flow cytometry, Western blotting, cytochrome c release assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2–3 — cellular loss/gain of function with multiple substrate and pathway readouts, single lab","pmids":["24786833"],"is_preprint":false},{"year":2014,"finding":"SOCS2-EloBC-CUL5-RBX2 (RNF7) complex can be reconstituted in vitro in neddylated and unneddylated forms; the complex exists as a monomer (unlike other E3 ligases); affinities of protein-protein interactions within the complex were determined by isothermal titration calorimetry; structural model supported by ion mobility mass spectrometry.","method":"Pull-down from human cell lysates, recombinant expression in E. coli and insect cells, SEC-MALS, native MS, ITC, traveling wave ion mobility MS","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of full CRL5 complex with multiple biophysical methods","pmids":["25247507"],"is_preprint":false},{"year":2014,"finding":"NEDD4-1 E3 ubiquitin ligase binds directly via its HECT domain to SAG's C-terminal RING domain and ubiquitylates SAG for proteasomal degradation; SAG bridges NEDD4-1 (via C-terminus) and CUL-5 (via N-terminus) forming a NEDD4-1/SAG/CUL-5 tri-complex; NEDD4-1 overexpression sensitizes cancer cells to apoptosis by reducing SAG levels.","method":"Co-immunoprecipitation, protein half-life assay (cycloheximide chase), in vitro binding assay with recombinant proteins, NEDD4-1 overexpression/silencing","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding with recombinant proteins plus cellular half-life assay, single lab","pmids":["25216516"],"is_preprint":false},{"year":2015,"finding":"RNF7 (SAG/RBX2) interacts with PCNA in living human cells; this interaction was validated by co-immunoprecipitation from human cell extracts and by interaction analysis using recombinant proteins, suggesting a role for RNF7 in DNA replication or repair.","method":"Bimolecular fluorescence complementation (BiFC) screen with human cDNA library, co-immunoprecipitation from cell extracts, recombinant protein interaction analysis","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2–3 — BiFC screen validated by Co-IP and recombinant protein assay, single study","pmids":["26030842"],"is_preprint":false},{"year":2015,"finding":"Erbin is a novel substrate of SAG-β-TrCP E3 ligase; Sag deletion in skin keratinocytes causes Erbin accumulation, which blocks Ras activation of Raf and prevents Nrf2-driven ROS scavenging; knockdown of the Erbin-encoding gene Erbb2ip partially rescues the phenotypes, establishing Erbin as a causal substrate.","method":"Sag/Kras double conditional KO keratinocytes, in vivo ubiquitylation assay for Erbin, Erbb2ip genetic rescue, Ras-Raf pathway analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — substrate identification with in vivo ubiquitylation assay plus genetic rescue","pmids":["26056141"],"is_preprint":false},{"year":2016,"finding":"SAG/RBX2 (RNF7) promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis in prostate cancer; growth suppression by SAG knockdown is partially rescued by simultaneous knockdown of PHLPP1 or DEPTOR; in vivo ubiquitylation assays confirm both as SAG substrates.","method":"siRNA knockdown epistasis, in vivo and in vitro ubiquitylation assays, Sag/Pten double conditional KO prostate mouse model","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro ubiquitylation assays plus in vivo genetic epistasis with double KO mice","pmids":["27955654"],"is_preprint":false},{"year":2016,"finding":"SAG/RBX2 E3 ligase complex specifically binds E2 enzymes UBCH10 and UBE2S (which mediate K11-linked ubiquitin chains) to ubiquitylate β-TrCP1 via K11-linkage, leading to its degradation; in contrast, RBX1 exclusively binds CDC34 and UBCH5C (K48 linkage). Silencing UBCH10 or UBE2S, but not UBCH5C, causes accumulation of β-TrCP1, establishing it as a physiological SAG-CUL5 substrate.","method":"Co-immunoprecipitation of E2 binding, protein half-life assay, siRNA silencing of E2s, SAG-CUL5-βTrCP1 complex co-IP, chemical inducers of SAG","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches distinguishing SAG from RBX1 biochemically via E2 binding specificity","pmids":["27910872"],"is_preprint":false},{"year":2016,"finding":"SAG/RBX2-dependent neddylation regulates T-cell activation; T-cell-specific Sag knockout mice show phenotypically normal T-cell development but dramatically decreased T-cell activation, proliferation, and effector cytokine release; SAG effects in T cells are associated with increased SOCS expression but not NF-κB translocation.","method":"T-cell-specific conditional Sag KO mice, in vitro T-cell stimulation, allogeneic bone marrow transplantation model, MLN4924 pharmacological inhibition, SOCS and NF-κB pathway analysis","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with pharmacological validation and pathway mechanistic analysis","pmids":["27543965"],"is_preprint":false},{"year":2017,"finding":"RNF7 negatively regulates CARMA2sh (CARD14) signaling by regulating the ubiquitination state of MALT1 and NEMO; RNF7 interacts with CARMA2 (identified by yeast two-hybrid) and suppresses NF-κB activation; psoriasis-associated CARMA2sh mutants escape RNF7 negative regulation.","method":"Yeast two-hybrid screen, co-immunoprecipitation, NF-κB reporter assay, ubiquitination assay for MALT1 and NEMO, gain/loss of function with mutants","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 — yeast two-hybrid validated by Co-IP plus functional ubiquitination assay for downstream substrates","pmids":["29194363"],"is_preprint":false},{"year":2017,"finding":"RNF7 knockdown in castration-resistant prostate cancer cells suppresses proliferation, invasiveness, and sensitizes to cisplatin; mechanistically, RNF7 knockdown causes accumulation of p21, p27, and NOXA and inactivates ERK1/2 signaling, suggesting these are functional substrates or downstream effectors.","method":"shRNA knockdown, proliferation/invasion assays, clonogenic survival, Western blotting for p21/p27/NOXA/ERK1/2","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 — cellular KD with pathway readouts, single lab","pmids":["28252001"],"is_preprint":false},{"year":2018,"finding":"RBX2/RNF7 is required for proper retinal lamination and function; RBX2 depletion causes misposition of rod bipolar cells (via sustained DAB1/Reelin signaling), cone photoreceptors, and Muller glia; SOCS7-CRL5 controls rod bipolar and Muller glia positioning but not cone localization, indicating that RBX2 controls additional CRL5-dependent pathways for cone positioning; RBX2 depletion also reduces ribbon synapses and disrupts cone photoreceptor function.","method":"In vivo RBX2 knockdown (shRNA), SOCS7 knockdown, retinal electrophysiology (ERG), immunohistochemistry for cell-layer markers and DAB1 levels","journal":"Development","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo knockdown with functional electrophysiology and molecular pathway validation","pmids":["29361558"],"is_preprint":false},{"year":2018,"finding":"SAG/RBX2 differentially regulates inflammatory responses of macrophages versus neutrophils; in vivo LysM-Cre/Sag myeloid-specific KO mice show increased LPS-induced mortality with decreased cytokine release from Sag-null macrophages but increased cytokine release from Sag-null neutrophils; myeloperoxidase (Mpo) and elastase (Elane) induction by LPS is significantly decreased in Sag-null bone marrow.","method":"LysM-Cre conditional Sag KO mice, in vivo LPS challenge, cytokine measurement, gene expression profiling of bone marrow cells","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO in vivo with cell-type specific mechanistic analysis","pmids":["30574150"],"is_preprint":false},{"year":2020,"finding":"Gossypol inhibits cullin neddylation by directly binding to SAG-CUL5 and RBX1-CUL1 complexes; CUL5-H572 is a key residue for gossypol binding; cellular treatment with gossypol selectively causes accumulation of NOXA (CUL5 substrate) and MCL1 (CUL1 substrate).","method":"AlphaScreen HTS for CUL5 neddylation, biochemical binding assays, mutagenesis of CUL5-H572, cellular substrate accumulation assays","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical binding assay with mutagenesis plus cellular substrate readout","pmids":["32145688"],"is_preprint":false},{"year":2022,"finding":"RNF7 inhibits apoptosis and promotes glycolysis in renal cell carcinoma via ubiquitination of SOCS1, activating JAK/STAT3 signaling; STAT3 activation in turn transcriptionally induces RNF7, creating a feedback loop; RNF7 overexpression also reduces sunitinib sensitivity.","method":"siRNA knockdown and overexpression, in vitro apoptosis/glycolysis assays, in vivo xenograft tumor model, STAT3 pathway analysis, ubiquitination of SOCS1","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 2–3 — cellular loss/gain-of-function with substrate (SOCS1) ubiquitination and pathway validation","pmids":["35562668"],"is_preprint":false}],"current_model":"RNF7 (SAG/RBX2/ROC2) is a dual-function RING finger protein that acts as an antioxidant when alone (scavenging ROS via disulfide bond formation) and as the RING component of CUL5-based (and CUL1-based) Cullin-RING E3 ubiquitin ligase complexes, where it selectively partners with the E2 enzyme UBE2F for CUL5 neddylation and with UBCH10/UBE2S for K11-linked ubiquitylation of substrates including NOXA, p27, IκBα, DEPTOR, PHLPP1, NF1, c-Jun, pro-caspase-3, β-TrCP1, Erbin, SOCS1, Bax, and SARM, thereby regulating apoptosis, NF-κB and mTOR signaling, cell cycle progression, neuronal migration, vascular development, and immune responses."},"narrative":{"teleology":[{"year":1999,"claim":"Identification of SAG/RNF7 as a zinc/copper-binding RING finger protein with intrinsic antioxidant activity established the gene's initial functional identity as an anti-apoptotic, redox-protective factor independent of its later-discovered E3 ligase role.","evidence":"Bacterially purified protein biochemistry (metal binding, lipid peroxidation), stable transfection, RING domain mutagenesis","pmids":["10082581","10443936","10506102"],"confidence":"High","gaps":["Whether antioxidant activity operates independently of ubiquitin ligase activity in vivo remains unclear","No structural model of the disulfide-based ROS-scavenging mechanism"]},{"year":1999,"claim":"Demonstrating that SAG/ROC2 binds CUL1 and reconstitutes SCF-type E3 ubiquitin ligase activity repositioned RNF7 from a standalone antioxidant to a core component of the Cullin-RING ligase machinery, with IκBα as an early validated substrate.","evidence":"Yeast two-hybrid, co-immunoprecipitation, in vitro reconstituted ubiquitination with E1/E2/CUL1 and SCF components","pmids":["10230407","10230406","10851089"],"confidence":"High","gaps":["Whether SAG preferentially functions with CUL1 or CUL5 was not resolved","Substrate specificity determinants were unknown"]},{"year":2000,"claim":"Yeast knockout and complementation showed that the SAG homolog is essential for viability and that RING domain-dependent E3 ligase activity — not antioxidant activity alone — is required for cell survival.","evidence":"Yeast gene disruption, tetrad analysis, complementation with wild-type vs. RING mutant human SAG","pmids":["10851089"],"confidence":"High","gaps":["Essential substrates in yeast were not identified","Not tested whether the yeast requirement reflects CUL5 vs. CUL1 function"]},{"year":2004,"claim":"Domain-swap and RNAi experiments resolved the longstanding question of CRL specificity: SOCS-box proteins assemble with CUL5-RBX2/RNF7 while VHL-box proteins assemble with CUL2-RBX1, establishing RNF7 as the dedicated RING for CRL5 complexes.","evidence":"Endogenous co-immunoprecipitation, Cul-box domain-swap mutagenesis, siRNA knockdown with HIF-2α degradation readout","pmids":["15601820"],"confidence":"High","gaps":["Whether RNF7 can functionally substitute for RBX1 in any CUL1 context remained debated","Structural basis of CUL5-RBX2 selectivity was not determined"]},{"year":2006,"claim":"Identification of pro-caspase-3 as a substrate of SAG-SCF(β-TrCP) demonstrated that RNF7-containing CRLs directly regulate apoptosis execution machinery, expanding the substrate repertoire beyond cell cycle regulators.","evidence":"Co-immunoprecipitation, in vitro ubiquitination, siRNA knockdown of SAG/ROC1/β-TrCP, dominant-negative β-TrCP","pmids":["17217622"],"confidence":"High","gaps":["Phosphodegron on pro-caspase-3 not fully mapped","Relative contributions of SAG vs. ROC1 to caspase-3 regulation not quantified"]},{"year":2007,"claim":"Discovery of transcriptional feedback loops — HIF-1 induces SAG under hypoxia and AP-1 induces SAG constitutively, while SAG reciprocally degrades HIF-1α (via CUL5-VHL) and c-Jun (via Fbw7-SCF) — revealed autoregulatory circuits that integrate SAG expression with its substrate turnover.","evidence":"ChIP, EMSA, luciferase reporters, siRNA knockdown, c-Jun and HIF-1α ubiquitination/half-life assays","pmids":["17828303","17440073"],"confidence":"High","gaps":["Quantitative dynamics of these feedback loops are uncharacterized","Whether the HIF-1/SAG loop operates through CUL5 exclusively or also CUL2 was not resolved"]},{"year":2007,"claim":"Transgenic mouse skin carcinogenesis studies revealed that SAG exhibits stage-dependent substrate switching: targeting c-Jun/AP-1 at early stages (tumor-suppressive) and IκBα at late stages (tumor-promoting via NF-κB activation), explaining paradoxical roles in cancer.","evidence":"K14-SAG transgenic mice, AP-1 reporter mice, DMBA/TPA two-stage carcinogenesis model","pmids":["17846172"],"confidence":"High","gaps":["Molecular signal that triggers the adaptor/substrate switch is unknown","Whether stage-dependent switching occurs in non-skin tissues is untested"]},{"year":2009,"claim":"Biochemical reconstitution and structural analysis established that UBE2F is the dedicated NEDD8-conjugating E2 for RBX2/RNF7, selectively neddylating CUL5, while UBE2M neddylates CUL1-4 via RBX1 — defining a fundamental axis of CRL activation specificity.","evidence":"In vitro neddylation assay with purified components, structural analysis of E1-E2 interfaces, mutagenesis","pmids":["19250909"],"confidence":"High","gaps":["Crystal structure of the UBE2F-RBX2 catalytic complex not available","Regulation of UBE2F expression/stability not addressed"]},{"year":2011,"claim":"Sag knockout in mice proved embryonic lethality (E11.5–12.5) and identified NF1 as a physiological SAG-CUL1-FBXW7 substrate; genetic epistasis (Sag/Nf1 double KO) partially rescued vascular defects, demonstrating that RAS pathway hyperactivation via NF1 accumulation causes the lethal phenotype.","evidence":"Conditional Sag knockout mice, Sag/Nf1 double knockout epistasis, ES cell differentiation, teratoma angiogenesis","pmids":["22118770"],"confidence":"High","gaps":["Neural defects in Sag-null embryos not fully rescued by Nf1 deletion, suggesting additional substrates","Contribution of CUL5-based complexes to embryonic phenotype not dissected"]},{"year":2011,"claim":"DEPTOR was identified as a substrate of SCF(β-TrCP)/SAG through phosphodegron-mediated recognition, linking SAG directly to mTOR pathway activation via a positive feedback loop (growth factor → RSK1/S6K1 → DEPTOR phosphorylation → SCF-mediated degradation → mTOR activation).","evidence":"Protein half-life assays, co-immunoprecipitation, phosphodegron mutagenesis, kinase inhibitors — independently replicated by three labs","pmids":["22017876","22017875","22017877"],"confidence":"High","gaps":["Relative roles of SAG vs. RBX1 in DEPTOR degradation not quantified","Whether other phosphodegrons in DEPTOR contribute is unclear"]},{"year":2011,"claim":"Reconstitution of a six-protein HIV-1 Vif/CUL5/ELOB/ELOC/CBF-β/RBX2 complex demonstrated that viral hijacking of the CRL5 machinery requires RNF7 for APOBEC3G polyubiquitination, establishing a direct role for RNF7 in viral immune evasion.","evidence":"Affinity purification-mass spectrometry, reconstituted six-protein in vitro ubiquitination assay, RNA knockdown, genetic complementation","pmids":["22190037"],"confidence":"High","gaps":["Structural basis of Vif preferring RBX2 over RBX1 not determined at atomic resolution","Whether other viral proteins exploit CRL5-RBX2 similarly is unexplored"]},{"year":2013,"claim":"Conditional Rbx2 knockout in the brain revealed that CRL5-RBX2 controls neuronal migration by promoting DAB1 degradation via the SOCS7 adaptor, establishing that proper cortical and cerebellar lamination requires RBX2-dependent Reelin signal termination.","evidence":"Rbx2 conditional knockout mice, in utero electroporation, SOCS7 gain/loss-of-function, DAB1 protein accumulation, epistasis with Reelin pathway","pmids":["24210661"],"confidence":"High","gaps":["The CRL5 adaptor controlling cerebellar (non-SOCS7) neuronal positioning is unknown","Whether RBX2 loss affects adult neuronal plasticity is untested"]},{"year":2013,"claim":"Endothelial-specific Sag deletion (E15.5 lethal) and p27 knockdown rescue showed that SAG is required for endothelial migration, proliferation, and tube formation through p27 degradation, explaining the vascular defects in global Sag knockouts.","evidence":"Tie2-Cre conditional KO, primary endothelial cell culture assays, p27 knockdown rescue, in vivo Matrigel plug and B16F10 tumor angiogenesis","pmids":["24213570"],"confidence":"High","gaps":["Which F-box protein mediates p27 ubiquitination in the SAG complex in endothelial cells is not defined","Contribution of CUL5 vs. CUL1 in vascular SAG function not distinguished"]},{"year":2014,"claim":"Biophysical reconstitution of the complete SOCS2-EloBC-CUL5-RBX2 complex demonstrated it exists as a monomer and established interaction affinities within the complex, providing the first complete structural framework for a CRL5 module.","evidence":"Recombinant expression, SEC-MALS, native MS, ITC, ion mobility MS","pmids":["25247507"],"confidence":"High","gaps":["High-resolution crystal or cryo-EM structure of the full complex not available","How neddylation changes conformational dynamics not resolved at structural level"]},{"year":2016,"claim":"Distinguishing SAG from RBX1 at the E2-binding level showed that SAG specifically recruits UBCH10/UBE2S to build K11-linked ubiquitin chains (targeting β-TrCP1), while RBX1 uses CDC34/UBCH5C for K48 chains — establishing a fundamental biochemical distinction between the two RING-box paralogs.","evidence":"E2 co-immunoprecipitation, protein half-life assays, E2-specific siRNA with substrate accumulation readouts","pmids":["27910872"],"confidence":"High","gaps":["Structural basis for SAG preferring UBCH10/UBE2S is unknown","Whether all SAG substrates are modified with K11 chains or some use K48 is not resolved"]},{"year":2016,"claim":"T-cell-specific Sag knockout revealed that SAG is dispensable for T-cell development but required for T-cell activation and proliferation, with SOCS accumulation as the likely mechanism, extending SAG function to adaptive immunity.","evidence":"T-cell-specific conditional Sag KO mice, in vitro stimulation, allogeneic BMT model, MLN4924 treatment","pmids":["27543965"],"confidence":"Medium","gaps":["Specific SOCS family member(s) responsible not identified","Whether CRL5 or CRL1 mediates SOCS degradation in T cells is unclear","Single lab study without independent replication"]},{"year":2016,"claim":"Lung-specific Sag deletion suppressed KrasG12D-driven tumorigenesis with accumulation of p21, p27, NOXA, BIM, and DEPTOR; epistatic rescue by p21 or DEPTOR knockdown confirmed their causal roles, solidifying SAG as a multi-substrate oncogenic E3 ligase in Kras-driven cancer.","evidence":"Lung-specific Sag conditional KO in KrasG12D mice, double-knockdown rescue experiments","pmids":["24430184"],"confidence":"High","gaps":["Relative contribution of each substrate to tumor suppression not quantified individually","Whether therapeutic SAG inhibition is tolerated in normal lung tissue is unknown"]},{"year":2018,"claim":"RBX2 was shown to control retinal lamination through DAB1/SOCS7-dependent and SOCS7-independent pathways, extending the neuronal migration paradigm from cortex/cerebellum to the retina and revealing that cone photoreceptor positioning requires an as-yet-unidentified CRL5 adaptor.","evidence":"In vivo shRNA knockdown of RBX2 and SOCS7, retinal electrophysiology (ERG), immunohistochemistry","pmids":["29361558"],"confidence":"Medium","gaps":["Identity of the CRL5 adaptor for cone photoreceptor positioning unknown","shRNA-based approach; conditional KO confirmation pending"]},{"year":null,"claim":"Key unresolved questions include the structural basis for RBX2-specific E2 (UBE2F, UBCH10) recruitment, the signals that determine substrate-adaptor switching in different tissue/disease contexts, and whether the intrinsic antioxidant activity of RNF7 operates independently of its E3 ligase function in physiological settings.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of RBX2 in complex with UBE2F or UBCH10","Mechanism controlling stage-dependent adaptor switching remains unknown","In vivo separation of antioxidant vs. E3 ligase functions not achieved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,2,4,9,15,23,28,33]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,9,16,22,31,32,33]},{"term_id":"GO:0016209","term_label":"antioxidant activity","supporting_discovery_ids":[0,18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[15,33]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,19,28]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[30]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,9,15,22,23,28,33]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[12,17,22,26,40]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,9,16,27]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5,25,26]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[23,34,38]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[20,24,25,37]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[12,26,32,40]}],"complexes":["CRL5 (CUL5-RBX2-ElonginB/C-SOCS-box)","SCF (CUL1-RBX2-SKP1-F-box)","Vif-CUL5-ElonginBC-CBFβ-RBX2"],"partners":["CUL5","CUL1","UBE2F","UBCH10","UBE2S","BTRC","FBXW7","NEDD4"],"other_free_text":[]},"mechanistic_narrative":"RNF7 (SAG/RBX2/ROC2) is the RING-box component of CUL5-based Cullin-RING E3 ubiquitin ligase (CRL5) complexes and functions as a critical regulator of protein turnover governing apoptosis, NF-κB and mTOR signaling, cell cycle progression, vascular development, neuronal migration, and immune cell activation. RNF7 selectively pairs with the E2 neddylation enzyme UBE2F to activate CUL5 by neddylation and with UBCH10/UBE2S to catalyze K11-linked polyubiquitination, distinguishing it biochemically from its paralog RBX1 [PMID:19250909, PMID:27910872]. Through association with different substrate adaptors (β-TrCP, FBXW7, SOCS-box proteins), RNF7-containing CRL complexes target a broad substrate repertoire — including IκBα, NOXA, p27, NF1, DEPTOR, PHLPP1, c-Jun, pro-caspase-3, β-TrCP1, Erbin, SOCS1, Bax, SARM, and DAB1 — for ubiquitin-dependent degradation, thereby controlling context-dependent oncogenic and tumor-suppressive outputs [PMID:17846172, PMID:24430184, PMID:24210661, PMID:27955654]. Sag/Rbx2 knockout in mice is embryonic lethal with severe vascular and neural tube defects caused by accumulation of NF1 and impaired RAS-MAPK signaling, and tissue-specific deletions reveal essential roles in endothelial angiogenesis, cortical/cerebellar neuron positioning, retinal lamination, and T-cell activation [PMID:22118770, PMID:24213570, PMID:24210661, PMID:27543965]."},"prefetch_data":{"uniprot":{"accession":"Q9UBF6","full_name":"RING-box protein 2","aliases":["CKII beta-binding protein 1","CKBBP1","RING finger protein 7","Regulator of cullins 2","Sensitive to apoptosis gene protein"],"length_aa":113,"mass_kda":12.7,"function":"Catalytic component of multiple cullin-5-RING E3 ubiquitin-protein ligase complexes (ECS complexes), which mediate the ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:21980433, PubMed:33268465, PubMed:38418882, PubMed:38574733, PubMed:35512830, PubMed:40440427). It is thereby involved in various biological processes, such as cell cycle progression, signal transduction and transcription (PubMed:21980433, PubMed:33268465, PubMed:38418882, PubMed:38574733). The functional specificity of the E3 ubiquitin-protein ligase ECS complexes depend on the variable SOCS box-containing substrate recognition component (PubMed:21980433, PubMed:33268465). Within ECS complexes, RNF7/RBX2 recruits the E2 ubiquitination enzyme to the complex via its RING-type and brings it into close proximity to the substrate (PubMed:34518685). Catalytic subunit of various SOCS-containing ECS complexes, such as the ECS(SOCS7) complex, that regulate reelin signaling by mediating ubiquitination and degradation of DAB1 (By similarity). The ECS(SOCS2) complex mediates the ubiquitination and subsequent proteasomal degradation of phosphorylated EPOR and GHR (PubMed:21980433, PubMed:25505247). Promotes ubiquitination and degradation of NF1, thereby regulating Ras protein signal transduction (By similarity). As part of the ECS(ASB9) complex, catalyzes ubiquitination and degradation of CKB (PubMed:33268465). The ECS(SPSB3) complex catalyzes ubiquitination of nuclear CGAS (PubMed:38418882). As part of the ECS(RAB40C) complex, mediates ANKRD28 ubiquitination and degradation, thereby inhibiting protein phosphatase 6 (PP6) complex activity and focal adhesion assembly during cell migration (PubMed:35512830). The ECS(ASB7) complex acts a negative regulator of H3K9me3 histone mark by mediating ubiquitination and degradation of SUV39H1 (PubMed:40440427). As part of some ECS complex, catalyzes 'Lys-11'-linked ubiquitination and degradation of BTRC (PubMed:27910872). ECS complexes and ARIH2 collaborate in tandem to mediate ubiquitination of target proteins; ARIH2 mediating addition of the first ubiquitin on CRLs targets (PubMed:34518685, PubMed:38418882). Specifically catalyzes the neddylation of CUL5 via its interaction with UBE2F (PubMed:19250909). Does not catalyze neddylation of other cullins (CUL1, CUL2, CUL3, CUL4A or CUL4B) (PubMed:19250909). May play a role in protecting cells from apoptosis induced by redox agents (PubMed:10082581) Inactive (Microbial infection) Following infection by HIV-1 virus, catalytic component of a cullin-5-RING E3 ubiquitin-protein ligase complex (ECS complex) hijacked by the HIV-1 Vif protein, which catalyzes ubiquitination and degradation of APOBEC3F and APOBEC3G","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UBF6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF7","classification":"Not Classified","n_dependent_lines":20,"n_total_lines":1208,"dependency_fraction":0.016556291390728478},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RNF7","total_profiled":1310},"omim":[{"mim_id":"620077","title":"PROTEIN-L-ISOASPARTATE (D-ASPARTATE) O-METHYLTRANSFERASE DOMAIN-CONTAINING PROTEIN 2; PCMTD2","url":"https://www.omim.org/entry/620077"},{"mim_id":"617700","title":"UBIQUITIN-CONJUGATING ENZYME E2 F; UBE2F","url":"https://www.omim.org/entry/617700"},{"mim_id":"615052","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 7; ASB7","url":"https://www.omim.org/entry/615052"},{"mim_id":"615051","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 6; ASB6","url":"https://www.omim.org/entry/615051"},{"mim_id":"605759","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 2; ASB2","url":"https://www.omim.org/entry/605759"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF7"},"hgnc":{"alias_symbol":["SAG","ROC2","CKBBP1","RBX2"],"prev_symbol":[]},"alphafold":{"accession":"Q9UBF6","domains":[{"cath_id":"3.30.40.10","chopping":"61-111","consensus_level":"high","plddt":94.0553,"start":61,"end":111}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBF6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBF6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBF6-F1-predicted_aligned_error_v6.png","plddt_mean":81.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF7","jax_strain_url":"https://www.jax.org/strain/search?query=RNF7"},"sequence":{"accession":"Q9UBF6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UBF6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UBF6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBF6"}},"corpus_meta":[{"pmid":"15601820","id":"PMC_15601820","title":"VHL-box and 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antioxidant/anti-apoptotic activity, and it binds zinc and copper metal ions to prevent lipid peroxidation.\",\n      \"method\": \"Bacterially expressed and purified recombinant protein; H2O2-induced disulfide bond formation assay; RING finger mutants; copper-induced lipid peroxidation assay\",\n      \"journal\": \"Molecular and cellular biology / Free radical biology & medicine / Carcinogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical reconstitution with mutagenesis, replicated across multiple papers\",\n      \"pmids\": [\"10082581\", \"10443936\", \"10506102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SAG/RNF7 binds to Cullin-1 (Cul1) and the SAG-Cul1 complex has E3 ubiquitin ligase activity promoting poly-ubiquitination of E2/Cdc34; this ligase activity is essential for rescuing lethality caused by deletion of the yeast SAG homolog (ySAG).\",\n      \"method\": \"Yeast genetics (targeted gene disruption, complementation with wild-type and mutant SAG); in vitro ubiquitin ligase assay; binding assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ligase assay plus genetic complementation with mutagenesis\",\n      \"pmids\": [\"10851089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SAG/RNF7 promotes S-phase entry and cell growth under serum starvation by inhibiting p27 accumulation through ubiquitin-proteasome degradation; SAG binds Skp2 (F-box protein that promotes p27 ubiquitination) in vivo, and the growth-promoting effect is abolished by proteasome inhibitors.\",\n      \"method\": \"Microinjection of SAG mRNA; adenovirus/transfection overexpression; co-immunoprecipitation of SAG with Skp2; proteasome inhibitor rescue\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus functional rescue by proteasome inhibitor, multiple cell lines\",\n      \"pmids\": [\"11255262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SAG/RBX2 (RNF7) specifically assembles with Cul5-Rbx2, while RBX1 associates with Cul2-Rbx1; the specificity of Cullin-Rbx module interaction is determined by the Cul2 or Cul5 box sequences located downstream of the BC box in substrate-recognition subunits.\",\n      \"method\": \"Co-immunoprecipitation with endogenous proteins; domain-swapping/mutagenesis analysis; RNAi knockdown of Cul2-Rbx1 vs. Cul5-Rbx2\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — domain-swapping mutagenesis plus reciprocal co-IP plus RNAi epistasis, highly cited foundational study\",\n      \"pmids\": [\"15601820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SAG/RBX2 (RNF7) as part of the SAG/ROC-SCF(β-TrCP) E3 ubiquitin ligase complex binds pro-caspase-3 (mediated through F-box protein β-TrCP that binds the first 38 amino acids of pro-caspase-3) and promotes its ubiquitination and degradation, thereby raising the apoptotic threshold.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; siRNA silencing of SAG, ROC1, or β-TrCPs; dominant-negative β-TrCP1 (F-box deleted); protein half-life measurement\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ubiquitination assay plus Co-IP plus siRNA with defined functional readout\",\n      \"pmids\": [\"17217622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAG/RBX2 (RNF7) is a transcriptional target of AP-1/c-Jun; on induction, SAG promotes c-Jun ubiquitination and degradation via SCF-Fbw7 E3 ligase, establishing a negative-feedback loop that inhibits AP-1 transcriptional activity.\",\n      \"method\": \"Luciferase reporter assay; AP-1 binding site mutagenesis; SAG siRNA silencing; c-Jun polyubiquitination assay; dominant-negative c-Jun overexpression\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (reporter, siRNA, ubiquitination assay) in one study\",\n      \"pmids\": [\"17440073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAG/RBX2 (RNF7) is a HIF-1 target gene that participates in a negative-feedback loop: hypoxia/HIF-1 transactivates SAG via a consensus HIF-1-binding site in the first SAG intron; SAG in turn promotes VHL-mediated HIF-1α ubiquitination and degradation. SAG forms a complex in vivo with Cul-5 and VHL under hypoxia.\",\n      \"method\": \"HIF-1 binding site mutagenesis; luciferase reporter; ChIP; SAG/ROC1 siRNA silencing; in vivo co-immunoprecipitation under hypoxia; HIF-1α protein-level measurements\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP, Co-IP, siRNA, and reporter mutagenesis in single study\",\n      \"pmids\": [\"17828303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SAG/RBX2 (RNF7) silencing induces apoptosis by accumulation of NOXA and shortens NOXA protein half-life when overexpressed, identifying NOXA as a substrate of SAG E3 ubiquitin ligase.\",\n      \"method\": \"SAG siRNA silencing; Western blotting for NOXA accumulation; protein half-life measurement by cycloheximide chase; cancer cell proliferation and apoptosis assays\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA with defined substrate accumulation, single lab\",\n      \"pmids\": [\"20103673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SAG/RBX2 (RNF7) gene disruption in mouse embryonic stem cells sensitizes them to radiation by increasing steady-state ROS levels and blocking NF-κB activation; IκBα is a direct substrate of SAG-SCF(β-TrCP) E3 ubiquitin ligase.\",\n      \"method\": \"Gene-trap Sag knockout in ES cells; clonogenic survival assay; intracellular ROS measurement; NF-κB reporter; IκBα degradation assay\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic knockout with multiple orthogonal mechanistic readouts\",\n      \"pmids\": [\"20638939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SAG/RBX2 (RNF7) is an essential RING component of SCF E3 ligase required for vascular and neural development; it promotes degradation of NF1 (neurofibromatosis type 1) as part of a SAG-CUL1-FBXW7 complex. Sag knockout mice die at E11.5-12.5 with vascular and neural defects, and simultaneous Nf1 deletion partially rescues these defects.\",\n      \"method\": \"Sag knockout mouse embryos; genetic epistasis (double knockout Sag/Nf1); ES cell endothelial differentiation assay; teratoma angiogenesis assay; NF1 protein accumulation upon Sag loss\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with conditional KO plus substrate accumulation, multiple orthogonal in vivo and in vitro readouts\",\n      \"pmids\": [\"22118770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RBX2/RNF7, as the RING component of CRL5, stops neocortical projection neurons at their target layers by ubiquitylating and promoting turnover of Dab1 (a key Reelin-pathway signaling protein) via the CRL5 adaptor SOCS7; RBX2 mutation causes neocortical and cerebellar ectopias dependent on sustained Dab1 signaling.\",\n      \"method\": \"In utero electroporation knockdown; genetic epistasis (Rbx2 vs. Dab1 double mutant); SOCS7-CRL5 reconstitution; ubiquitylation assay; unscheduled SOCS7 expression to prematurely stop migration\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitylation assay, epistasis, and gain-of-function/loss-of-function in vivo\",\n      \"pmids\": [\"24210661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Protein kinase CKII physically interacts with SAG/RNF7 through its β subunit; the RING-H2 finger motif of SAG is necessary and sufficient for this interaction, and CKII phosphorylates SAG in vitro.\",\n      \"method\": \"Yeast two-hybrid; co-immunoprecipitation; in vitro kinase assay; SAG deletion mutant analysis; overlay assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by in vitro kinase assay and overlay, single lab\",\n      \"pmids\": [\"10512750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SAG/RBX2 (RNF7) deletion in endothelial cells causes embryonic lethality at E15.5 with defective vasculogenesis; SAG controls endothelial migration, proliferation, and tube formation, with p27 accumulation responsible for suppression of migration and proliferation.\",\n      \"method\": \"Endothelial-specific Sag conditional knockout; primary endothelial cell Sag deletion/knockdown; Matrigel plug assay; B16F10 melanoma tumor angiogenesis model; p27 Western blotting\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with defined substrate accumulation and multiple in vivo/in vitro readouts\",\n      \"pmids\": [\"24213570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SAG/RBX2 (RNF7) promotes ubiquitylation and degradation of Bax and SARM in macrophages, conferring survival advantage against pathogen-associated molecular patterns (PAMPs); SAG knockdown causes accumulation of proapoptotic Bax and SARM, imbalance of Bcl-2/Bax, and caspase-9/-3 activation.\",\n      \"method\": \"SAG knockdown by siRNA; Western blotting for Bax, SARM, Bcl-2; co-immunoprecipitation; caspase activation assay; cytochrome c release assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — siRNA plus Co-IP plus mechanistic readouts, single lab\",\n      \"pmids\": [\"24786833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Inactivation of SAG/RBX2 (RNF7) suppresses KrasG12D-driven lung tumorigenesis; SAG knockdown inactivates both NF-κB and mTOR pathways, accumulating tumor suppressor substrates p21, p27, NOXA, and BIM; growth suppression by SAG knockdown is partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR (identifying DEPTOR as a SAG substrate).\",\n      \"method\": \"Conditional Sag/KrasG12D mouse model; SAG siRNA in cultured lung cancer cells; genetic rescue (double siRNA); substrate accumulation by Western blot; NF-κB activity assay\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic in vivo model plus epistasis rescue plus substrate accumulation, multiple orthogonal methods\",\n      \"pmids\": [\"24430184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NEDD4-1 (a HECT E3 ubiquitin ligase) binds via its HECT domain directly to the C-terminal RING domain of SAG/RNF7 and ubiquitylates SAG for proteasome-mediated degradation; SAG bridges NEDD4-1 via its C-terminus and CUL-5 via its N-terminus to form a NEDD4-1/SAG/CUL-5 tri-complex.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitylation assay; protein half-life measurement (cycloheximide chase); domain-mapping of interaction\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitylation reconstitution plus co-IP plus half-life measurement\",\n      \"pmids\": [\"25216516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SAG/RBX2 (RNF7) in a complex with β-TrCP E3 ligase promotes ubiquitination and degradation of Erbin (encoded by Erbb2ip); accumulation of Erbin upon Sag deletion blocks Ras activation of Raf and inhibits autophagy/senescence, accelerating KrasG12D-induced papillomagenesis in skin.\",\n      \"method\": \"Skin-targeted Sag/KrasG12D conditional knockout; in vitro ubiquitylation assay; Erbb2ip one-allele genetic rescue; Western blotting; ROS measurement\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitylation plus genetic epistasis plus substrate accumulation\",\n      \"pmids\": [\"26056141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF7 interacts with PCNA in human cells; this interaction was identified by bimolecular fluorescence complementation (BiFC) screen and validated by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC) screen; co-immunoprecipitation from cell extracts; recombinant protein interaction assay\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — BiFC confirmed by co-IP and recombinant protein assay, single lab\",\n      \"pmids\": [\"26030842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG/RBX2 (RNF7) preferentially binds E2 enzymes UBCH10 and UBE2S (which mediate K11-linked ubiquitin chains), while RBX1 exclusively binds CDC34 and UBCH5C (K48 linkage); SAG-CUL5 promotes K11-linked ubiquitylation and degradation of β-TrCP1, establishing a cross-talk between CRL5 and CRL1.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitylation assay with defined E2s; siRNA silencing of UBCH10, UBE2S, or UBCH5C; protein half-life measurement; mass spectrometry for ubiquitin chain type\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with defined E2s plus siRNA epistasis plus chain-type determination\",\n      \"pmids\": [\"27910872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG/RBX2 (RNF7) promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis in prostate cancer cells; growth suppression triggered by SAG knockdown is partially rescued by simultaneous knockdown of PHLPP1 or DEPTOR.\",\n      \"method\": \"In vivo and in vitro ubiquitylation assays; SAG knockdown in prostate cancer cells; double-knockdown genetic rescue; Sag/Pten double conditional knockout mouse model; IHC and Western blotting\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitylation assay plus genetic epistasis in mouse model\",\n      \"pmids\": [\"27955654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG/RBX2 (RNF7)-dependent neddylation regulates T-cell activation; T-cell-specific SAG knockout reduces T-cell activation, proliferation, and effector cytokine release; mechanistically, SAG-mediated effects correlate with accumulation of suppressors of cytokine signaling (SOCS), but not NF-κB translocation.\",\n      \"method\": \"T-cell-specific Sag genetic knockout; in vitro T-cell stimulation; in vivo bone marrow transplantation; MLN4924 chemical inhibition; SOCS protein level measurement\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with mechanistic readout, single lab\",\n      \"pmids\": [\"27543965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF7 functions as a negative regulator of CARMA2sh (CARD14)-mediated NF-κB activation; RNF7 was identified as a CARMA2 interactor and regulates the ubiquitination state of MALT1 and NEMO; CARMA2sh psoriasis-associated mutants escape this RNF7-mediated negative control.\",\n      \"method\": \"Yeast two-hybrid screen; co-immunoprecipitation; ubiquitination assay; NF-κB reporter assay; CARMA2sh mutant analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — yeast two-hybrid confirmed by co-IP plus functional NF-κB reporter assay, single lab\",\n      \"pmids\": [\"29194363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RBX2/RNF7 (as CRL5 RING component) is essential for retinal lamination and function; RBX2 regulates final cell position of rod bipolar cells, cone photoreceptors, and Müller glia; SOCS7-CRL5 controls rod bipolar cell and Müller glia positioning via Dab1 ubiquitylation, while cone photoreceptor positioning requires other CRL5 adaptors; RBX2 depletion reduces ribbon synapses and disrupts cone photoreceptor function.\",\n      \"method\": \"Rbx2 conditional knockout; in vivo retinal layering analysis; SOCS7 knockout; electrophysiology (ERG); synapse counting\",\n      \"journal\": \"Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with multiple functional readouts and epistasis\",\n      \"pmids\": [\"29361558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Gossypol inhibits cullin neddylation by directly binding to SAG-CUL5 or RBX1-CUL1 complexes; CUL5-H572 plays a key role for gossypol binding, blocking neddylation of both CUL5 and CUL1.\",\n      \"method\": \"Alpha-Screen-based high-throughput in vitro CUL5 neddylation assay; biochemical binding studies; site-directed mutagenesis of CUL5-H572; substrate accumulation in cancer cell lines\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro neddylation assay with mutagenesis, single lab\",\n      \"pmids\": [\"32145688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF7 promotes ubiquitination of SOCS1, activating the JAK/STAT3 signaling pathway in renal cell carcinoma; a feedback loop exists where STAT3 activation transcriptionally induces RNF7 expression.\",\n      \"method\": \"RNF7 knockdown and overexpression; ubiquitination assay for SOCS1; JAK/STAT3 pathway activity measurement; STAT3 transcriptional reporter; xenograft tumor model\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — ubiquitination assay plus functional pathway assays, single lab\",\n      \"pmids\": [\"35562668\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF7 (SAG/RBX2/ROC2) is a dual-function protein: when acting alone it scavenges reactive oxygen species via RING-domain-mediated disulfide bond formation, and when assembled into Cullin-RING E3 ubiquitin ligase complexes (preferentially CRL5 via CUL5, and CRL1 via CUL1) it promotes K11- and K48-linked ubiquitylation and proteasomal degradation of substrates including NF1, p27, NOXA, BIM, IκBα, HIF-1α, pro-caspase-3, c-Jun, DEPTOR, PHLPP1, β-TrCP1, Erbin, Bax, SARM, and SOCS1, thereby regulating apoptosis suppression, NF-κB and mTOR pathway activation, cell cycle progression, angiogenesis, neuronal migration, and immune responses; its activity is regulated by NEDD4-1-mediated ubiquitination/degradation of SAG itself, by CKII-mediated phosphorylation, and by HIF-1α and AP-1 transcriptional induction.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll systematically classify each paper before extracting discoveries.\n\n**Classification:**\n\nPapers clearly about RNF7/SAG/RBX2/ROC2 (the canonical RING finger E3 ubiquitin ligase protein, also known as RNF7):\n- KEEP: [4], [8], [10], [11], [12], [13], [14], [15], [16], [18], [21], [22], [23], [29], [30], [32], [33], [36], [44], [45], [48], [49], [50], [51], [52], [53], [54], [55], [59], [60], [61], [65], [66], [67], [76], [78], [81], [85], [91], [96], [99]\n- KEEP from additional: [13_add (ROC1/ROC2)], [16_add], [18_add], [19_add], [22_add], [23_add], [25_add], [29_add], [30_add]\n\nPapers about other SAG entities (Toxoplasma SAG antigens, Drosophila SAG neurons, Streptococcus sag operon, SAG blood storage, arrestin SAG, plant SAG, Sonic hedgehog SAG agonist, Leishmania SAG drug, salivary agglutinin SAG, MMTV sag superantigen, algae SAG, etc.):\n- EXCLUDE: [1_retrieved] (about Cul2-Rbx1 vs Cul5-Rbx2 — actually KEEP, directly about RNF7/RBX2)\n- KEEP: [1], [2], [7_add... wait let me recheck]\n\nLet me re-examine more carefully:\n\n- [1] KEEP - directly about Cul2-Rbx1 and Cul5-Rbx2 specificity, relevant to RNF7/RBX2\n- [2] KEEP - about Cullin-box sequences for Cul5-Rbx2, relevant\n- [3] EXCLUDE - Toxoplasma SAG-1/P30\n- [5] EXCLUDE - Toxoplasma SAG-1\n- [6] EXCLUDE - Drosophila SAG neurons\n- [7] EXCLUDE - DMBT1/SAG salivary agglutinin\n- [8] KEEP - ASB proteins with Cul5-Rbx2\n- [9] EXCLUDE - SAG-2 rabies vaccine\n- [17] EXCLUDE - sag currents in neurons\n- [19] EXCLUDE - Toxoplasma SAG\n- [20] EXCLUDE - algae SAG\n- [24] EXCLUDE - Toxoplasma SAG-1 vaccine\n- [25] EXCLUDE - algae SAG\n- [26] EXCLUDE - Leishmania SAG drug resistance\n- [27] EXCLUDE - Toxoplasma SAG1\n- [28] EXCLUDE - Streptococcus sag operon\n- [31] EXCLUDE - Toxoplasma SAG superfamily\n- [34] EXCLUDE - HBsAg\n- [35] EXCLUDE - salivary agglutinin SAG\n- [37] EXCLUDE - arrestin SAG gene (different protein - retinal arrestin)\n- [38] EXCLUDE - MMTV sag superantigen\n- [39] EXCLUDE - SAG-M blood storage\n- [40] EXCLUDE - senescence-associated SAG (different gene)\n- [41] EXCLUDE - microbial dark matter SAG\n- [42] EXCLUDE - Sarcocystis SAG antigens\n- [43] EXCLUDE - Toxoplasma SAG-1 vaccine\n- [46] EXCLUDE - Arabidopsis SAG promoters (plant)\n- [47] EXCLUDE - Drosophila SAG neurons\n- [56] EXCLUDE - salivary agglutinin gp340/SAG\n- [57] EXCLUDE - sag in muscle motor units\n- [58] EXCLUDE - Leishmania SAG drug\n- [62] EXCLUDE - arrestin SAG gene (retinal disease)\n- [63] EXCLUDE - Streptococcus sag operon\n- [64] EXCLUDE - SAG-M blood storage\n- [68] EXCLUDE - Ag2S nanoprisms\n- [69] KEEP - genomic structure of SAG/RNF7\n- [70] EXCLUDE - MMTV sag superantigen\n- [71] EXCLUDE - arrestin S-antigen gene\n- [72] EXCLUDE - Arabidopsis SAG (plant, different protein)\n- [73] EXCLUDE - SAG-M blood storage\n- [74] EXCLUDE - Leishmania SAG drug resistance\n- [75] EXCLUDE - SAG Sonic hedgehog agonist\n- [77] EXCLUDE - SAG-M blood storage\n- [79] EXCLUDE - staphylococcal superantigens\n- [80] EXCLUDE - SA/G hydrogel\n- [82] EXCLUDE - SAG TCCS testicular cancer cohort\n- [83] EXCLUDE - Leishmania SAG drug\n- [84] - lncRNA RNF7 - EXCLUDE (case B, alt-locus product)\n- [86] EXCLUDE - Schwann cell SAG glycoprotein (different protein)\n- [87] EXCLUDE - GWAS/SNP association only\n- [88] EXCLUDE - Sonic hedgehog SAG agonist\n- [89] EXCLUDE - Leishmania SAG drug resistance\n- [90] EXCLUDE - SAG-M blood storage\n- [92] EXCLUDE - arrestin SAG gene\n- [93] EXCLUDE - Sarcocystis SAG antigens\n- [94] EXCLUDE - Streptococcus sag operon\n- [95] EXCLUDE - SAG-M blood storage\n- [97] EXCLUDE - Leishmania SAG drug\n- [98] EXCLUDE - algae Chlamydomonas SAG\n- [100] EXCLUDE - Sonic hedgehog SAG agonist\n\nAdditional papers:\n- [1_add] KEEP - protein interactome (RNF7 detected)\n- [2_add] EXCLUDE - growth hormone receptor\n- [3_add] KEEP - cDNA sequences (marginal)\n- [4_add] KEEP - BioPlex interactome\n- [5_add] EXCLUDE - cGAS/STING\n- [6_add] KEEP - human interactome\n- [7_add] KEEP - HuRI interactome\n- [8_add] KEEP - BioPlex 3.0\n- [9_add] EXCLUDE - cGAS\n- [10_add] EXCLUDE - cGAS\n- [11_add] KEEP - HIV interactome\n- [12_add] EXCLUDE - MGC cDNA\n- [13_add] KEEP - ROC1/ROC2 ubiquitin ligase activity\n- [14_add] EXCLUDE - promoters\n- [15_add] EXCLUDE - LOXL2/Snail\n- [16_add] KEEP - CRL network proteomics\n- [17_add] KEEP - Vif/CBF-β/CUL5/RBX2\n- [18_add] KEEP - ROC1-CUL1 ubiquitin ligase\n- [19_add] KEEP - NEDD8/SCF E3 ligase\n- [20_add] KEEP - heterotypic ubiquitin chains\n- [21_add] EXCLUDE - erythropoietin receptor\n- [22_add] KEEP - DEPTOR/SCFβTrCP\n- [23_add] KEEP - NEDD8/RBX2/UBE2F\n- [24_add] EXCLUDE - cGAS/nucleosome\n- [25_add] KEEP - mTOR/DEPTOR/βTrCP\n- [26_add] KEEP - Stitch-seq interactome\n- [27_add] KEEP - SOCS2/CRL5\n- [28_add] EXCLUDE - cGAS/nucleosome\n- [29_add] KEEP - DEPTOR/βTrCP/CK1α\n- [30_add] KEEP - estrogen/SOCS-2/GH signaling\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"SAG/RBX2 (RNF7) was cloned as a novel zinc RING finger protein that binds zinc and copper metal ions, prevents copper-induced lipid peroxidation, forms inter- and intramolecular disulfide bonds in response to oxidative stress, and protects cells from apoptosis induced by redox agents; the C-terminal RING finger domain is required for anti-apoptosis activity.\",\n      \"method\": \"Bacterially expressed/purified protein biochemical assays (metal binding, lipid peroxidation), stable transfection, RING domain mutagenesis, oligomerization assays with DTT/H2O2\",\n      \"journal\": \"Molecular and cellular biology / Free radical biology & medicine / Carcinogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted biochemistry plus mutagenesis, multiple orthogonal methods across multiple papers\",\n      \"pmids\": [\"10082581\", \"10443936\", \"10506102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"SAG/ROC2/RBX2 (RNF7) is the second family member of the ROC/Rbx RING component of SCF E3 ubiquitin ligase; it binds Cullin-1 and the SAG-CUL1 complex has ubiquitin ligase activity promoting poly-ubiquitination of E2/CDC34 in vitro.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro ubiquitination assay with E1/E2/CUL1\",\n      \"journal\": \"Molecular cell / Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstituted ubiquitin ligase activity, independently confirmed by multiple labs (ROC1/ROC2 papers)\",\n      \"pmids\": [\"10230407\", \"10851089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ROC1-CUL1 RING-based ubiquitin ligase (with SAG/ROC2 as paralog) can catalyze ubiquitination of IκBα in a phosphorylation-dependent manner in vitro when reconstituted with SCFHOS components, E1, and CDC34.\",\n      \"method\": \"In vitro reconstituted ubiquitination assay with purified components\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted ubiquitination with purified components\",\n      \"pmids\": [\"10230406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Protein kinase CKII interacts with SAG/RNF7 via the RING-H2 finger motif; specifically, the beta subunit of CKII binds SAG and phosphorylates it in vitro, while the alpha subunit does not interact.\",\n      \"method\": \"Yeast two-hybrid, overlay/direct binding assay, in vitro phosphorylation by recombinant CKII, deletion mutagenesis of SAG\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid plus direct in vitro phosphorylation, single lab\",\n      \"pmids\": [\"10512750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The yeast homolog of SAG/RNF7 (ySAG) is essential for cell viability; deletion causes cell death with cell enlargement and abnormal cell cycle profiles. Complementation requires the RING finger domain and ubiquitin ligase activity, confirming that E3 ligase function is required for cell survival.\",\n      \"method\": \"Targeted yeast gene disruption, tetrad analysis, complementation with wild-type vs. RING mutant hSAG, cDNA chip profiling of yeast genome\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis (yeast KO + rescue) with mutagenesis confirming mechanistic requirement, replicated by complementation\",\n      \"pmids\": [\"10851089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SAG/RNF7 promotes S-phase entry and cell growth under serum starvation by inhibiting p27 accumulation through the ubiquitin-proteasome pathway; SAG associates in vivo with SKP2, an F-box protein that promotes p27 ubiquitination.\",\n      \"method\": \"SAG mRNA microinjection into quiescent cells ([3H]-thymidine incorporation), adenoviral overexpression, co-immunoprecipitation of SAG with SKP2, proteasome inhibitor rescue experiment\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — cellular overexpression plus Co-IP and proteasome inhibitor evidence, single lab\",\n      \"pmids\": [\"11255262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SAG/RNF7 gene consists of four exons and three introns; a splicing variant (SAG-v) incorporates an additional exon disrupting the RING finger ORF, and SAG-v lacks cullin-1 binding and ubiquitin ligase activity, confirming that the RING domain is required for E3 function.\",\n      \"method\": \"Genomic cloning, RT-PCR, yeast complementation, co-immunoprecipitation with CUL1, in vitro ubiquitination assay\",\n      \"journal\": \"DNA and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional domain analysis with ligase assay and binding experiments\",\n      \"pmids\": [\"11506706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SAG/RNF7 protects neurons in vivo against ischemia/reperfusion brain injury; overexpression via adenoviral delivery reduced infarct area, decreased ROS production and apoptosis; the RING domain mutant was not protective, confirming domain requirement.\",\n      \"method\": \"Adenoviral delivery of wild-type vs. RING mutant SAG into mouse brain, middle cerebral artery occlusion model, infarct measurement, ROS assay\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo loss/gain of function with RING mutant control\",\n      \"pmids\": [\"11488541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"VHL-box proteins specifically interact with Cul2-Rbx1 modules, whereas SOCS-box proteins associate with Cul5-Rbx2 (RNF7). The specificity is determined by the downstream Cul2-box or Cul5-box sequences. RNAi knockdown of Cul5-Rbx2 does not affect VHL-mediated HIF-2α degradation, whereas Cul2-Rbx1 knockdown inhibits it, demonstrating functional distinction between the two Rbx modules.\",\n      \"method\": \"Co-immunoprecipitation of endogenous complexes, domain-swap mutagenesis, siRNA knockdown with HIF-2α degradation readout\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — domain-swapping mutagenesis plus endogenous Co-IP plus functional RNAi epistasis, replicated\",\n      \"pmids\": [\"15601820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SAG/RNF7 forms part of SAG/ROC-SCF(β-TrCP) E3 ubiquitin ligase that binds pro-caspase-3 via the F-box protein β-TrCP (which recognizes the first 38 amino acids of pro-caspase-3) and promotes its ubiquitination and degradation; siRNA silencing of SAG, ROC1, or β-TrCP increases pro-caspase-3 levels and enhances apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, siRNA knockdown, dominant-negative β-TrCP expression, protein half-life assay\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro ubiquitination plus Co-IP plus siRNA epistasis, multiple orthogonal approaches\",\n      \"pmids\": [\"17217622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAG/RNF7 is a transcriptional target of HIF-1; HIF-1 binds a consensus GCGTG site in the first intron of the SAG gene under hypoxia, and induced SAG in turn promotes VHL-mediated HIF-1α ubiquitination and degradation by forming a complex with CUL-5 and VHL, establishing a negative feedback loop.\",\n      \"method\": \"ChIP assay, luciferase reporter, siRNA silencing, co-immunoprecipitation under hypoxia, HIF-1α protein half-life measurement\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (ChIP, reporter, Co-IP, siRNA) in single lab\",\n      \"pmids\": [\"17828303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAG/RNF7 is a novel AP-1 transcriptional target; AP-1 binds two consensus sites in the SAG promoter in vitro and in vivo. SAG reciprocally promotes c-Jun ubiquitination and degradation via Fbw7-SCF, establishing an AP-1/SAG autofeedback loop; SAG siRNA silencing reduced c-Jun polyubiquitination.\",\n      \"method\": \"EMSA, ChIP, luciferase reporter, siRNA knockdown, c-Jun polyubiquitination assay, dominant-negative c-Jun overexpression\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus EMSA plus ubiquitination assay plus siRNA, multiple orthogonal methods\",\n      \"pmids\": [\"17440073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAG/RNF7 in mouse epidermis targets c-Jun/AP-1 for degradation at early stages of skin carcinogenesis (inhibiting tumor promotion) and targets IκBα for degradation at later stages (activating NF-κB to reduce apoptosis and enhance tumor growth), demonstrating stage-dependent substrate targeting by different F-box proteins.\",\n      \"method\": \"SAG-transgenic mouse model (K14 promoter), AP-1 luciferase reporter mice, Western blotting, DMBA/TPA carcinogenesis model\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo transgenic model with mechanistic pathway analysis, multiple substrates validated\",\n      \"pmids\": [\"17846172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SAG/RNF7 promotes UVB-induced skin hyperplasia by simultaneously targeting c-Jun (pro-tumor promotion) and p27 (anti-proliferative) for degradation in mouse epidermis, but does not affect UVB-induced p53, c-Fos, or cyclin D1.\",\n      \"method\": \"K14-SAG transgenic mice, UVB carcinogenesis model, Western blotting, AP-1 activity assays, DNA synthesis measurement\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo transgenic model with substrate-level mechanistic analysis\",\n      \"pmids\": [\"18258608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Cul2-box and Cul5-box sequences determine specificity of BC-box protein assembly with Cul2-Rbx1 versus Cul5-Rbx2 (RNF7) modules; the LPPhiP motif conserved in Cul5-boxes is also compatible with Cul2 interaction; spacer length between BC-box and cullin-box can vary 3–80 amino acids.\",\n      \"method\": \"Purification and co-immunoprecipitation of reconstituted BC-box protein complexes, structure-function domain analysis with mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — systematic structure-function with reconstituted complexes across many BC-box proteins\",\n      \"pmids\": [\"18187417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"UBE2F is a NEDD8-conjugating E2 enzyme that specifically pairs with RBX2/RNF7 to neddylate CUL5, while UBE2M pairs with RBX1 to neddylate CUL1-4; structural analysis reveals how E1 accommodates both E2s, establishing that RBX2 selectively activates CUL5-based CRLs through the UBE2F/RBX2 neddylation axis.\",\n      \"method\": \"In vitro neddylation assay, structural analysis of E1-E2 interactions, mutagenesis, cellular neddylation experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro neddylation plus structural analysis plus mutagenesis\",\n      \"pmids\": [\"19250909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SAG/RNF7 silencing induces apoptosis with accumulation of NOXA; SAG overexpression reduces NOXA levels and shortens NOXA protein half-life, identifying NOXA as a substrate of SAG E3 ubiquitin ligase. In vivo, SAG silencing suppresses xenograft tumor growth.\",\n      \"method\": \"siRNA silencing, protein half-life assay, flow cytometry for apoptosis, caspase-3 assay, in vivo orthotopic xenograft model\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — cellular loss-of-function with substrate stabilization, in vivo validation\",\n      \"pmids\": [\"20103673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Sag/Rbx2 deletion in mouse embryonic stem cells sensitizes them to radiation through increased ROS (including superoxide) and decreased NF-κB activation; IκBα was identified as a direct substrate of SAG-SCF(β-TrCP) E3 ubiquitin ligase, as its degradation is blocked upon Sag elimination.\",\n      \"method\": \"Gene-trap Sag knockout ES cells, clonogenic survival, intracellular ROS measurement, NF-κB reporter, IκBα protein level analysis\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — complete gene knockout with multiple mechanistic readouts and direct substrate identification\",\n      \"pmids\": [\"20638939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SAG/RNF7 as a Tat-fusion protein can be transduced into cells and brain tissue; wild-type but not RING-mutant Tat-SAG protects hippocampal neurons from ischemia-induced death and reduces lipid peroxidation in vivo, confirming that ROS-scavenging via the RING domain is the protective mechanism.\",\n      \"method\": \"Tat-fusion protein transduction, gerbil transient forebrain ischemia model, immunohistochemistry, lipid peroxidation assay\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo model with wild-type vs. RING mutant control\",\n      \"pmids\": [\"20100567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CRL network quantitative proteomics shows SAG/RBX2 assembles within distinct CRL5 complexes; network architecture reveals that neddylation inhibition (MLN4924) does not globally reorganize CRL complexes, but adaptor module abundance drives CRL organization.\",\n      \"method\": \"AQUA quantitative proteomics, affinity purification-MS of CRL components, MLN4924 treatment\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative proteomics with pharmacological perturbation\",\n      \"pmids\": [\"21145461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SAG/RBX2/RNF7 is an essential RING component of SCF E3 ubiquitin ligase required for embryonic development; Sag knockout mice die at E11.5–12.5 with vascular and neural defects caused by NF1 accumulation and RAS inhibition. NF1 was identified as a physiological substrate of SAG-CUL1-FBXW7 E3 ligase; simultaneous Nf1 deletion partially rescues vascular defects in Sag-null embryos.\",\n      \"method\": \"Sag conditional knockout mice, genetic epistasis (Sag/Nf1 double knockout), ES cell endothelial differentiation assays, teratoma angiogenesis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo double-knockout epistasis plus ES cell differentiation assay identifying NF1 as a substrate\",\n      \"pmids\": [\"22118770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SAG/RBX2 inactivation in mouse embryonic stem cells blocks retinoic acid (RA)-induced differentiation, causing apoptosis instead; in leukemia cells, SAG inhibition with MLN4924 sensitizes to RA-differentiation therapy through accumulation of NOXA and c-JUN substrates of SAG-SCF E3 ligase.\",\n      \"method\": \"Sag-/- ES cells, RA exposure, atomic force microscopy for cell stiffness, MLN4924 treatment of leukemia lines, Western blotting for NOXA/c-JUN\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus pharmacological inhibition with substrate accumulation readout\",\n      \"pmids\": [\"22110742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"DEPTOR is a physiological substrate of SCF(β-TrCP)/SAG E3 ligase; growth factor stimulation activates RSK1/S6K1 to phosphorylate DEPTOR, enabling β-TrCP recognition and SCF-mediated ubiquitination and degradation; this activates mTOR in a positive feedback loop.\",\n      \"method\": \"Protein half-life assay, co-immunoprecipitation, siRNA knockdown, phosphodegron mutagenesis, kinase inhibitor experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstituted pathway with mutagenesis, multiple orthogonal methods, replicated across three independent labs (PMIDs 22017876, 22017875, 22017877)\",\n      \"pmids\": [\"22017876\", \"22017875\", \"22017877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HIV-1 Vif hijacks CUL5-ELOB-ELOC-RBX2(RNF7) ubiquitin ligase complex (together with CBF-β) to polyubiquitinate APOBEC3G for degradation; a recombinant six-protein assembly including RBX2 reconstitutes specific APOBEC3G ubiquitination activity.\",\n      \"method\": \"Affinity purification-mass spectrometry, in vitro reconstituted six-protein ubiquitination assay, RNA knockdown, genetic complementation\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted ubiquitination with purified components, mechanistically validated with knockdown and complementation\",\n      \"pmids\": [\"22190037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RBX2/RNF7 is required for neocortical and cerebellar neuron positioning; Rbx2 mutation causes DAB1-dependent ectopias via sustained Reelin signaling. SOCS7 is identified as a CRL5-Rbx2 substrate adaptor that promotes ubiquitylation and turnover of DAB1, stopping neuronal migration at target layers; cerebellar development requires Rbx2 through a different adaptor.\",\n      \"method\": \"In utero electroporation, Rbx2 conditional knockout mice, SOCS7 gain-of-function/loss-of-function, DAB1 protein level analysis, epistasis with reelin pathway\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO plus epistasis with Reelin pathway plus substrate adaptor identification\",\n      \"pmids\": [\"24210661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Sag endothelial deletion causes embryonic lethality at E15.5 with poor vasculogenesis; Sag deletion in endothelial cells inhibits migration, proliferation, and tube formation with p27 accumulation responsible for migration/proliferation suppression; Sag is required for tumor angiogenesis in a B16F10 melanoma model.\",\n      \"method\": \"Tie2-Cre/Sag conditional KO mice, primary EC culture assays (migration, proliferation, tube formation), p27 knockdown rescue, in vivo Matrigel plug and tumor models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with genetic rescue (p27 knockdown), multiple in vitro and in vivo endpoints\",\n      \"pmids\": [\"24213570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Sag deletion suppresses KrasG12D-driven lung tumorigenesis; SAG knockdown inactivates NF-κB and mTOR pathways and causes accumulation of p21, p27, NOXA, and BIM; growth suppression by SAG knockdown is partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR, establishing these as causal substrates.\",\n      \"method\": \"Lung-specific Sag conditional KO in KrasG12D mice, siRNA epistasis (SAG + p21 or DEPTOR double knockdown), pathway analysis (NF-κB, mTOR)\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo conditional KO with genetic epistasis identifying substrates\",\n      \"pmids\": [\"24430184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SAG/RNF7 promotes ubiquitination of Bax and SARM in macrophages during pathogen challenge, conferring survival advantage; SAG knockdown causes accumulation of pro-apoptotic Bax and SARM, disruption of Bcl-2/Bax balance, cytochrome c release, and caspase-9/-3 activation.\",\n      \"method\": \"SAG knockdown/overexpression in macrophages, PAMP challenge, flow cytometry, Western blotting, cytochrome c release assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — cellular loss/gain of function with multiple substrate and pathway readouts, single lab\",\n      \"pmids\": [\"24786833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SOCS2-EloBC-CUL5-RBX2 (RNF7) complex can be reconstituted in vitro in neddylated and unneddylated forms; the complex exists as a monomer (unlike other E3 ligases); affinities of protein-protein interactions within the complex were determined by isothermal titration calorimetry; structural model supported by ion mobility mass spectrometry.\",\n      \"method\": \"Pull-down from human cell lysates, recombinant expression in E. coli and insect cells, SEC-MALS, native MS, ITC, traveling wave ion mobility MS\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of full CRL5 complex with multiple biophysical methods\",\n      \"pmids\": [\"25247507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NEDD4-1 E3 ubiquitin ligase binds directly via its HECT domain to SAG's C-terminal RING domain and ubiquitylates SAG for proteasomal degradation; SAG bridges NEDD4-1 (via C-terminus) and CUL-5 (via N-terminus) forming a NEDD4-1/SAG/CUL-5 tri-complex; NEDD4-1 overexpression sensitizes cancer cells to apoptosis by reducing SAG levels.\",\n      \"method\": \"Co-immunoprecipitation, protein half-life assay (cycloheximide chase), in vitro binding assay with recombinant proteins, NEDD4-1 overexpression/silencing\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding with recombinant proteins plus cellular half-life assay, single lab\",\n      \"pmids\": [\"25216516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNF7 (SAG/RBX2) interacts with PCNA in living human cells; this interaction was validated by co-immunoprecipitation from human cell extracts and by interaction analysis using recombinant proteins, suggesting a role for RNF7 in DNA replication or repair.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC) screen with human cDNA library, co-immunoprecipitation from cell extracts, recombinant protein interaction analysis\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — BiFC screen validated by Co-IP and recombinant protein assay, single study\",\n      \"pmids\": [\"26030842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Erbin is a novel substrate of SAG-β-TrCP E3 ligase; Sag deletion in skin keratinocytes causes Erbin accumulation, which blocks Ras activation of Raf and prevents Nrf2-driven ROS scavenging; knockdown of the Erbin-encoding gene Erbb2ip partially rescues the phenotypes, establishing Erbin as a causal substrate.\",\n      \"method\": \"Sag/Kras double conditional KO keratinocytes, in vivo ubiquitylation assay for Erbin, Erbb2ip genetic rescue, Ras-Raf pathway analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — substrate identification with in vivo ubiquitylation assay plus genetic rescue\",\n      \"pmids\": [\"26056141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG/RBX2 (RNF7) promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis in prostate cancer; growth suppression by SAG knockdown is partially rescued by simultaneous knockdown of PHLPP1 or DEPTOR; in vivo ubiquitylation assays confirm both as SAG substrates.\",\n      \"method\": \"siRNA knockdown epistasis, in vivo and in vitro ubiquitylation assays, Sag/Pten double conditional KO prostate mouse model\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro ubiquitylation assays plus in vivo genetic epistasis with double KO mice\",\n      \"pmids\": [\"27955654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG/RBX2 E3 ligase complex specifically binds E2 enzymes UBCH10 and UBE2S (which mediate K11-linked ubiquitin chains) to ubiquitylate β-TrCP1 via K11-linkage, leading to its degradation; in contrast, RBX1 exclusively binds CDC34 and UBCH5C (K48 linkage). Silencing UBCH10 or UBE2S, but not UBCH5C, causes accumulation of β-TrCP1, establishing it as a physiological SAG-CUL5 substrate.\",\n      \"method\": \"Co-immunoprecipitation of E2 binding, protein half-life assay, siRNA silencing of E2s, SAG-CUL5-βTrCP1 complex co-IP, chemical inducers of SAG\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches distinguishing SAG from RBX1 biochemically via E2 binding specificity\",\n      \"pmids\": [\"27910872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SAG/RBX2-dependent neddylation regulates T-cell activation; T-cell-specific Sag knockout mice show phenotypically normal T-cell development but dramatically decreased T-cell activation, proliferation, and effector cytokine release; SAG effects in T cells are associated with increased SOCS expression but not NF-κB translocation.\",\n      \"method\": \"T-cell-specific conditional Sag KO mice, in vitro T-cell stimulation, allogeneic bone marrow transplantation model, MLN4924 pharmacological inhibition, SOCS and NF-κB pathway analysis\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with pharmacological validation and pathway mechanistic analysis\",\n      \"pmids\": [\"27543965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF7 negatively regulates CARMA2sh (CARD14) signaling by regulating the ubiquitination state of MALT1 and NEMO; RNF7 interacts with CARMA2 (identified by yeast two-hybrid) and suppresses NF-κB activation; psoriasis-associated CARMA2sh mutants escape RNF7 negative regulation.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, NF-κB reporter assay, ubiquitination assay for MALT1 and NEMO, gain/loss of function with mutants\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — yeast two-hybrid validated by Co-IP plus functional ubiquitination assay for downstream substrates\",\n      \"pmids\": [\"29194363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RNF7 knockdown in castration-resistant prostate cancer cells suppresses proliferation, invasiveness, and sensitizes to cisplatin; mechanistically, RNF7 knockdown causes accumulation of p21, p27, and NOXA and inactivates ERK1/2 signaling, suggesting these are functional substrates or downstream effectors.\",\n      \"method\": \"shRNA knockdown, proliferation/invasion assays, clonogenic survival, Western blotting for p21/p27/NOXA/ERK1/2\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — cellular KD with pathway readouts, single lab\",\n      \"pmids\": [\"28252001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RBX2/RNF7 is required for proper retinal lamination and function; RBX2 depletion causes misposition of rod bipolar cells (via sustained DAB1/Reelin signaling), cone photoreceptors, and Muller glia; SOCS7-CRL5 controls rod bipolar and Muller glia positioning but not cone localization, indicating that RBX2 controls additional CRL5-dependent pathways for cone positioning; RBX2 depletion also reduces ribbon synapses and disrupts cone photoreceptor function.\",\n      \"method\": \"In vivo RBX2 knockdown (shRNA), SOCS7 knockdown, retinal electrophysiology (ERG), immunohistochemistry for cell-layer markers and DAB1 levels\",\n      \"journal\": \"Development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo knockdown with functional electrophysiology and molecular pathway validation\",\n      \"pmids\": [\"29361558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SAG/RBX2 differentially regulates inflammatory responses of macrophages versus neutrophils; in vivo LysM-Cre/Sag myeloid-specific KO mice show increased LPS-induced mortality with decreased cytokine release from Sag-null macrophages but increased cytokine release from Sag-null neutrophils; myeloperoxidase (Mpo) and elastase (Elane) induction by LPS is significantly decreased in Sag-null bone marrow.\",\n      \"method\": \"LysM-Cre conditional Sag KO mice, in vivo LPS challenge, cytokine measurement, gene expression profiling of bone marrow cells\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO in vivo with cell-type specific mechanistic analysis\",\n      \"pmids\": [\"30574150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Gossypol inhibits cullin neddylation by directly binding to SAG-CUL5 and RBX1-CUL1 complexes; CUL5-H572 is a key residue for gossypol binding; cellular treatment with gossypol selectively causes accumulation of NOXA (CUL5 substrate) and MCL1 (CUL1 substrate).\",\n      \"method\": \"AlphaScreen HTS for CUL5 neddylation, biochemical binding assays, mutagenesis of CUL5-H572, cellular substrate accumulation assays\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical binding assay with mutagenesis plus cellular substrate readout\",\n      \"pmids\": [\"32145688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF7 inhibits apoptosis and promotes glycolysis in renal cell carcinoma via ubiquitination of SOCS1, activating JAK/STAT3 signaling; STAT3 activation in turn transcriptionally induces RNF7, creating a feedback loop; RNF7 overexpression also reduces sunitinib sensitivity.\",\n      \"method\": \"siRNA knockdown and overexpression, in vitro apoptosis/glycolysis assays, in vivo xenograft tumor model, STAT3 pathway analysis, ubiquitination of SOCS1\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — cellular loss/gain-of-function with substrate (SOCS1) ubiquitination and pathway validation\",\n      \"pmids\": [\"35562668\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF7 (SAG/RBX2/ROC2) is a dual-function RING finger protein that acts as an antioxidant when alone (scavenging ROS via disulfide bond formation) and as the RING component of CUL5-based (and CUL1-based) Cullin-RING E3 ubiquitin ligase complexes, where it selectively partners with the E2 enzyme UBE2F for CUL5 neddylation and with UBCH10/UBE2S for K11-linked ubiquitylation of substrates including NOXA, p27, IκBα, DEPTOR, PHLPP1, NF1, c-Jun, pro-caspase-3, β-TrCP1, Erbin, SOCS1, Bax, and SARM, thereby regulating apoptosis, NF-κB and mTOR signaling, cell cycle progression, neuronal migration, vascular development, and immune responses.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF7 (also known as SAG/RBX2/ROC2) is the RING-finger component of Cullin-RING E3 ubiquitin ligase complexes—preferentially CRL5 (via CUL5) and also CRL1 (via CUL1)—that controls the stability of a broad spectrum of substrates to regulate apoptosis, cell cycle progression, vascular and neural development, and immune signaling. As the catalytic RING subunit, RNF7 preferentially engages E2 conjugating enzymes UBE2S and UBCH10 to assemble K11-linked ubiquitin chains, while also supporting K48-linked ubiquitylation, and targets substrates including p27, NF1, NOXA, BIM, IκBα, HIF-1α, pro-caspase-3, DEPTOR, PHLPP1, Erbin, Dab1, β-TrCP1, Bax, SARM, and SOCS1 for proteasomal degradation [PMID:10851089, PMID:15601820, PMID:27910872, PMID:22118770, PMID:24430184, PMID:26056141, PMID:35562668]. RNF7 also possesses a Cullin-independent antioxidant function: its RING-H2 domain scavenges reactive oxygen species through reversible disulfide bond formation and chelates redox-active metal ions to prevent lipid peroxidation [PMID:10082581, PMID:10443936]. RNF7 expression is transcriptionally induced by HIF-1 and AP-1/c-Jun, generating negative-feedback loops in which RNF7-containing ligases degrade HIF-1α (via VHL-CUL5) and c-Jun (via SCF-Fbw7), and RNF7 protein turnover is controlled by NEDD4-1-mediated ubiquitylation of its RING domain [PMID:17828303, PMID:17440073, PMID:25216516].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"The initial characterization established RNF7 as a RING-finger metalloprotein with an intrinsic antioxidant activity independent of any ligase function, answering how a small RING protein could protect cells from oxidative damage.\",\n      \"evidence\": \"Recombinant protein biochemistry with H₂O₂-induced disulfide bond assays, RING mutants, and lipid peroxidation inhibition assays\",\n      \"pmids\": [\"10082581\", \"10443936\", \"10506102\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the antioxidant function operates independently of Cullin complexes in vivo was not resolved\",\n        \"The stoichiometry and reversibility of disulfide bond formation in a cellular context remained unclear\"\n      ]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of CKII as a physical interactor and kinase of SAG raised the question of whether phosphorylation regulates SAG ligase or antioxidant activity.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, and in vitro kinase assay with domain-deletion mutants\",\n      \"pmids\": [\"10512750\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequence of CKII-mediated phosphorylation on SAG activity was not determined\",\n        \"Phosphorylation sites were not mapped\",\n        \"Awaits in vivo validation of the kinase–substrate relationship\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrating that SAG binds Cullin-1 and the SAG-Cul1 complex possesses E3 ubiquitin ligase activity established RNF7 as a bona fide catalytic RING component of SCF-type CRLs, not merely an antioxidant protein.\",\n      \"evidence\": \"In vitro ubiquitin ligase reconstitution with Cdc34; genetic complementation of yeast SAG deletion\",\n      \"pmids\": [\"10851089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Substrate specificity of the SAG-containing ligase was not yet defined\",\n        \"Distinction between SAG and RBX1 Cullin preference was not addressed\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of p27 as a functionally relevant target of SAG-containing SCF ligase connected RNF7 to cell cycle control, explaining how SAG overexpression promotes S-phase entry.\",\n      \"evidence\": \"SAG mRNA microinjection; co-immunoprecipitation with Skp2; proteasome inhibitor rescue of p27 levels\",\n      \"pmids\": [\"11255262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct ubiquitylation of p27 by a SAG-containing complex was not reconstituted in vitro\",\n        \"Relative contributions of SAG vs. RBX1 to p27 turnover were not delineated\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The discovery that RBX2/SAG preferentially pairs with CUL5 while RBX1 pairs with CUL2 resolved how two homologous RING proteins achieve functional specificity within the CRL family.\",\n      \"evidence\": \"Endogenous co-immunoprecipitation, domain-swapping mutagenesis of Cul2/Cul5 box sequences, and RNAi knockdown\",\n      \"pmids\": [\"15601820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of CUL5-RBX2 selectivity was not determined at atomic resolution\",\n        \"Whether this specificity is absolute or context-dependent in all tissues was not tested\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identification of pro-caspase-3 as a SAG-SCF(β-TrCP) substrate linked RNF7 directly to apoptosis regulation by showing how its ligase activity sets the apoptotic threshold.\",\n      \"evidence\": \"In vitro ubiquitination assay; co-IP of SAG/β-TrCP/pro-caspase-3; siRNA silencing with caspase activation readout\",\n      \"pmids\": [\"17217622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The ubiquitin chain type on pro-caspase-3 was not identified\",\n        \"In vivo relevance in a genetic model was not shown\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Two parallel studies uncovered transcriptional feedback loops: HIF-1 and AP-1 induce SAG expression, and SAG-containing ligases then degrade HIF-1α and c-Jun, respectively, establishing RNF7 as a node in signal-dependent negative feedback.\",\n      \"evidence\": \"ChIP, reporter mutagenesis, siRNA, co-IP of SAG-CUL5-VHL under hypoxia; c-Jun ubiquitination via SCF-Fbw7\",\n      \"pmids\": [\"17828303\", \"17440073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the two feedback loops interact was not explored\",\n        \"Kinetics of feedback-loop engagement in physiological contexts were not measured\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Genetic knockout of Sag in mouse ES cells demonstrated that SAG protects cells from radiation by maintaining ROS homeostasis and sustaining NF-κB activity through IκBα degradation, providing the first loss-of-function genetic evidence for SAG's dual antioxidant and ligase roles.\",\n      \"evidence\": \"Gene-trap Sag knockout ES cells; clonogenic survival; ROS measurement; NF-κB reporter; IκBα degradation assay\",\n      \"pmids\": [\"20638939\", \"20103673\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contribution of antioxidant vs. ligase function to radioprotection was not separated\",\n        \"NOXA as a substrate was based on half-life changes without full in vitro reconstitution\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Whole-body Sag knockout lethality at E11.5-12.5 with vascular and neural defects—partially rescued by Nf1 co-deletion—established NF1 as a key SAG-CUL1-FBXW7 substrate controlling developmental angiogenesis and vasculogenesis.\",\n      \"evidence\": \"Sag knockout mouse; Sag/Nf1 double knockout epistasis; ES cell endothelial differentiation; teratoma angiogenesis\",\n      \"pmids\": [\"22118770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether NF1 is the sole critical substrate for the vascular phenotype was not fully resolved\",\n        \"The mechanism connecting NF1 accumulation to impaired vasculogenesis was not detailed\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of Dab1 as a CRL5(SOCS7)-RBX2 substrate in cortical neurons resolved how Reelin signaling is terminated to halt neuronal migration at target cortical layers.\",\n      \"evidence\": \"In utero electroporation; Rbx2/Dab1 double-mutant epistasis; SOCS7-CRL5 ubiquitylation reconstitution\",\n      \"pmids\": [\"24210661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether RBX2 uses additional CRL5 adaptors beyond SOCS7 for other neuronal substrates was unresolved\",\n        \"Mechanism by which RBX2 loss causes cerebellar ectopia distinct from cortical ectopia was not detailed\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Endothelial-specific Sag deletion causing lethality at E15.5 with defective vasculogenesis confirmed p27 accumulation as a key driver of impaired endothelial proliferation and migration.\",\n      \"evidence\": \"Endothelial-specific Sag conditional knockout; Matrigel plug assay; tumor angiogenesis model\",\n      \"pmids\": [\"24213570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether p27 is the sole substrate responsible or additional endothelial-specific substrates contribute was not tested genetically\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Multiple studies expanded the substrate repertoire of SAG-containing CRLs to include Bax, SARM, DEPTOR, and BIM, and demonstrated that Sag loss suppresses KrasG12D-driven lung tumorigenesis by inactivating both NF-κB and mTOR pathways.\",\n      \"evidence\": \"Conditional Sag/KrasG12D mouse model; double-siRNA epistasis rescue; macrophage siRNA with Bax/SARM accumulation; in vivo/in vitro ubiquitylation assays\",\n      \"pmids\": [\"24430184\", \"24786833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct in vitro ubiquitylation of Bax and SARM by purified SAG-CRL complexes was not shown\",\n        \"Which specific F-box/SOCS adaptors mediate DEPTOR and BIM targeting was not identified\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that NEDD4-1 ubiquitylates SAG via its RING domain for proteasomal degradation revealed a HECT-RING E3 cross-regulation mechanism controlling SAG protein levels.\",\n      \"evidence\": \"In vitro ubiquitylation reconstitution; co-IP; cycloheximide chase; domain mapping of NEDD4-1 HECT/SAG RING interaction\",\n      \"pmids\": [\"25216516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological contexts in which NEDD4-1-mediated SAG turnover is rate-limiting were not identified\",\n        \"Whether the NEDD4-1/SAG/CUL-5 tri-complex has a function beyond SAG degradation is unknown\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of Erbin as a SAG-β-TrCP substrate explained why Sag deletion paradoxically accelerates KrasG12D-driven skin papillomagenesis: Erbin accumulation blocks Ras-Raf signaling and suppresses protective autophagy/senescence.\",\n      \"evidence\": \"Skin-targeted Sag/KrasG12D conditional knockout; in vitro ubiquitylation; Erbb2ip one-allele genetic rescue\",\n      \"pmids\": [\"26056141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How tissue context determines whether SAG loss is tumor-suppressive (lung) or tumor-promoting (skin) is mechanistically unresolved\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Biochemical demonstration that SAG preferentially engages UBCH10 and UBE2S to build K11-linked ubiquitin chains—and that β-TrCP1 is a CRL5-SAG substrate—established the E2 selectivity principle distinguishing SAG from RBX1 and revealed CRL5-CRL1 cross-talk.\",\n      \"evidence\": \"In vitro ubiquitylation with defined E2s; mass spectrometry for ubiquitin chain linkage; siRNA epistasis; half-life measurements\",\n      \"pmids\": [\"27910872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether K11-linked chains serve as a general degradation signal or require heterotypic K11/K48 branching was not addressed\",\n        \"Structural basis for UBCH10/UBE2S preference by SAG over RBX1 is unknown\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"T-cell-specific Sag knockout impaired T-cell activation and cytokine production, with accumulation of SOCS proteins, extending SAG's role to adaptive immune regulation.\",\n      \"evidence\": \"T-cell-specific Sag knockout; in vitro stimulation assays; bone marrow transplantation; MLN4924 pharmacological confirmation\",\n      \"pmids\": [\"27543965\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Which specific SOCS family member is the direct SAG-CRL5 substrate in T cells was not determined\",\n        \"In vivo immune challenge models were not reported\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Conditional Rbx2 knockout in retina demonstrated that CRL5-RBX2 controls laminar positioning of multiple retinal cell types and synaptic organization, with SOCS7-dependent Dab1 ubiquitylation responsible for rod bipolar and Müller glia positioning.\",\n      \"evidence\": \"Retina-specific Rbx2 conditional knockout; SOCS7 knockout comparison; electroretinography; synapse quantification\",\n      \"pmids\": [\"29361558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"CRL5 adaptors responsible for cone photoreceptor positioning were not identified\",\n        \"Whether ribbon synapse loss is cell-autonomous was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of SOCS1 as an RNF7 ubiquitylation substrate linked SAG to JAK/STAT3 activation and a STAT3-RNF7 positive feedback loop in renal cell carcinoma.\",\n      \"evidence\": \"RNF7 knockdown/overexpression; SOCS1 ubiquitination assay; STAT3 reporter; xenograft model\",\n      \"pmids\": [\"35562668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The specific CRL complex (CRL5 vs. CRL1) and adaptor mediating SOCS1 ubiquitylation was not defined\",\n        \"Findings from a single lab await independent validation\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of RBX2-CUL5 selectivity, the tissue-specific logic determining whether SAG loss is tumor-suppressive or tumor-promoting, and whether the antioxidant and E3 ligase functions of SAG are mechanistically coupled in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No atomic-resolution structure of a SAG-CUL5 complex exists\",\n        \"Systematic identification of tissue-specific SAG-CRL substrate repertoires has not been performed\",\n        \"The relative in vivo contribution of antioxidant versus ligase activity remains unseparated\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 18, 19, 24]},\n      {\"term_id\": \"GO:0016209\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 4, 17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 4, 7, 9, 10, 14, 15, 16, 18, 19, 24]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 7, 8, 13, 14]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [14, 19, 21, 24]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9, 10, 12, 22]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"complexes\": [\n      \"CRL5 (CUL5-RBX2)\",\n      \"CRL1/SCF (CUL1-RBX2)\",\n      \"SAG-SCF-β-TrCP\",\n      \"SAG-CUL1-FBXW7\"\n    ],\n    \"partners\": [\n      \"CUL5\",\n      \"CUL1\",\n      \"BTRC\",\n      \"FBXW7\",\n      \"NEDD4\",\n      \"SKP2\",\n      \"SOCS7\",\n      \"PCNA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"RNF7 (SAG/RBX2/ROC2) is the RING-box component of CUL5-based Cullin-RING E3 ubiquitin ligase (CRL5) complexes and functions as a critical regulator of protein turnover governing apoptosis, NF-κB and mTOR signaling, cell cycle progression, vascular development, neuronal migration, and immune cell activation. RNF7 selectively pairs with the E2 neddylation enzyme UBE2F to activate CUL5 by neddylation and with UBCH10/UBE2S to catalyze K11-linked polyubiquitination, distinguishing it biochemically from its paralog RBX1 [PMID:19250909, PMID:27910872]. Through association with different substrate adaptors (β-TrCP, FBXW7, SOCS-box proteins), RNF7-containing CRL complexes target a broad substrate repertoire — including IκBα, NOXA, p27, NF1, DEPTOR, PHLPP1, c-Jun, pro-caspase-3, β-TrCP1, Erbin, SOCS1, Bax, SARM, and DAB1 — for ubiquitin-dependent degradation, thereby controlling context-dependent oncogenic and tumor-suppressive outputs [PMID:17846172, PMID:24430184, PMID:24210661, PMID:27955654]. Sag/Rbx2 knockout in mice is embryonic lethal with severe vascular and neural tube defects caused by accumulation of NF1 and impaired RAS-MAPK signaling, and tissue-specific deletions reveal essential roles in endothelial angiogenesis, cortical/cerebellar neuron positioning, retinal lamination, and T-cell activation [PMID:22118770, PMID:24213570, PMID:24210661, PMID:27543965].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of SAG/RNF7 as a zinc/copper-binding RING finger protein with intrinsic antioxidant activity established the gene's initial functional identity as an anti-apoptotic, redox-protective factor independent of its later-discovered E3 ligase role.\",\n      \"evidence\": \"Bacterially purified protein biochemistry (metal binding, lipid peroxidation), stable transfection, RING domain mutagenesis\",\n      \"pmids\": [\"10082581\", \"10443936\", \"10506102\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether antioxidant activity operates independently of ubiquitin ligase activity in vivo remains unclear\",\n        \"No structural model of the disulfide-based ROS-scavenging mechanism\"\n      ]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrating that SAG/ROC2 binds CUL1 and reconstitutes SCF-type E3 ubiquitin ligase activity repositioned RNF7 from a standalone antioxidant to a core component of the Cullin-RING ligase machinery, with IκBα as an early validated substrate.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro reconstituted ubiquitination with E1/E2/CUL1 and SCF components\",\n      \"pmids\": [\"10230407\", \"10230406\", \"10851089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether SAG preferentially functions with CUL1 or CUL5 was not resolved\",\n        \"Substrate specificity determinants were unknown\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Yeast knockout and complementation showed that the SAG homolog is essential for viability and that RING domain-dependent E3 ligase activity — not antioxidant activity alone — is required for cell survival.\",\n      \"evidence\": \"Yeast gene disruption, tetrad analysis, complementation with wild-type vs. RING mutant human SAG\",\n      \"pmids\": [\"10851089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Essential substrates in yeast were not identified\",\n        \"Not tested whether the yeast requirement reflects CUL5 vs. CUL1 function\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Domain-swap and RNAi experiments resolved the longstanding question of CRL specificity: SOCS-box proteins assemble with CUL5-RBX2/RNF7 while VHL-box proteins assemble with CUL2-RBX1, establishing RNF7 as the dedicated RING for CRL5 complexes.\",\n      \"evidence\": \"Endogenous co-immunoprecipitation, Cul-box domain-swap mutagenesis, siRNA knockdown with HIF-2α degradation readout\",\n      \"pmids\": [\"15601820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether RNF7 can functionally substitute for RBX1 in any CUL1 context remained debated\",\n        \"Structural basis of CUL5-RBX2 selectivity was not determined\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identification of pro-caspase-3 as a substrate of SAG-SCF(β-TrCP) demonstrated that RNF7-containing CRLs directly regulate apoptosis execution machinery, expanding the substrate repertoire beyond cell cycle regulators.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro ubiquitination, siRNA knockdown of SAG/ROC1/β-TrCP, dominant-negative β-TrCP\",\n      \"pmids\": [\"17217622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Phosphodegron on pro-caspase-3 not fully mapped\",\n        \"Relative contributions of SAG vs. ROC1 to caspase-3 regulation not quantified\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Discovery of transcriptional feedback loops — HIF-1 induces SAG under hypoxia and AP-1 induces SAG constitutively, while SAG reciprocally degrades HIF-1α (via CUL5-VHL) and c-Jun (via Fbw7-SCF) — revealed autoregulatory circuits that integrate SAG expression with its substrate turnover.\",\n      \"evidence\": \"ChIP, EMSA, luciferase reporters, siRNA knockdown, c-Jun and HIF-1α ubiquitination/half-life assays\",\n      \"pmids\": [\"17828303\", \"17440073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Quantitative dynamics of these feedback loops are uncharacterized\",\n        \"Whether the HIF-1/SAG loop operates through CUL5 exclusively or also CUL2 was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Transgenic mouse skin carcinogenesis studies revealed that SAG exhibits stage-dependent substrate switching: targeting c-Jun/AP-1 at early stages (tumor-suppressive) and IκBα at late stages (tumor-promoting via NF-κB activation), explaining paradoxical roles in cancer.\",\n      \"evidence\": \"K14-SAG transgenic mice, AP-1 reporter mice, DMBA/TPA two-stage carcinogenesis model\",\n      \"pmids\": [\"17846172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular signal that triggers the adaptor/substrate switch is unknown\",\n        \"Whether stage-dependent switching occurs in non-skin tissues is untested\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Biochemical reconstitution and structural analysis established that UBE2F is the dedicated NEDD8-conjugating E2 for RBX2/RNF7, selectively neddylating CUL5, while UBE2M neddylates CUL1-4 via RBX1 — defining a fundamental axis of CRL activation specificity.\",\n      \"evidence\": \"In vitro neddylation assay with purified components, structural analysis of E1-E2 interfaces, mutagenesis\",\n      \"pmids\": [\"19250909\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Crystal structure of the UBE2F-RBX2 catalytic complex not available\",\n        \"Regulation of UBE2F expression/stability not addressed\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Sag knockout in mice proved embryonic lethality (E11.5–12.5) and identified NF1 as a physiological SAG-CUL1-FBXW7 substrate; genetic epistasis (Sag/Nf1 double KO) partially rescued vascular defects, demonstrating that RAS pathway hyperactivation via NF1 accumulation causes the lethal phenotype.\",\n      \"evidence\": \"Conditional Sag knockout mice, Sag/Nf1 double knockout epistasis, ES cell differentiation, teratoma angiogenesis\",\n      \"pmids\": [\"22118770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Neural defects in Sag-null embryos not fully rescued by Nf1 deletion, suggesting additional substrates\",\n        \"Contribution of CUL5-based complexes to embryonic phenotype not dissected\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"DEPTOR was identified as a substrate of SCF(β-TrCP)/SAG through phosphodegron-mediated recognition, linking SAG directly to mTOR pathway activation via a positive feedback loop (growth factor → RSK1/S6K1 → DEPTOR phosphorylation → SCF-mediated degradation → mTOR activation).\",\n      \"evidence\": \"Protein half-life assays, co-immunoprecipitation, phosphodegron mutagenesis, kinase inhibitors — independently replicated by three labs\",\n      \"pmids\": [\"22017876\", \"22017875\", \"22017877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative roles of SAG vs. RBX1 in DEPTOR degradation not quantified\",\n        \"Whether other phosphodegrons in DEPTOR contribute is unclear\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Reconstitution of a six-protein HIV-1 Vif/CUL5/ELOB/ELOC/CBF-β/RBX2 complex demonstrated that viral hijacking of the CRL5 machinery requires RNF7 for APOBEC3G polyubiquitination, establishing a direct role for RNF7 in viral immune evasion.\",\n      \"evidence\": \"Affinity purification-mass spectrometry, reconstituted six-protein in vitro ubiquitination assay, RNA knockdown, genetic complementation\",\n      \"pmids\": [\"22190037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of Vif preferring RBX2 over RBX1 not determined at atomic resolution\",\n        \"Whether other viral proteins exploit CRL5-RBX2 similarly is unexplored\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Conditional Rbx2 knockout in the brain revealed that CRL5-RBX2 controls neuronal migration by promoting DAB1 degradation via the SOCS7 adaptor, establishing that proper cortical and cerebellar lamination requires RBX2-dependent Reelin signal termination.\",\n      \"evidence\": \"Rbx2 conditional knockout mice, in utero electroporation, SOCS7 gain/loss-of-function, DAB1 protein accumulation, epistasis with Reelin pathway\",\n      \"pmids\": [\"24210661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The CRL5 adaptor controlling cerebellar (non-SOCS7) neuronal positioning is unknown\",\n        \"Whether RBX2 loss affects adult neuronal plasticity is untested\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Endothelial-specific Sag deletion (E15.5 lethal) and p27 knockdown rescue showed that SAG is required for endothelial migration, proliferation, and tube formation through p27 degradation, explaining the vascular defects in global Sag knockouts.\",\n      \"evidence\": \"Tie2-Cre conditional KO, primary endothelial cell culture assays, p27 knockdown rescue, in vivo Matrigel plug and B16F10 tumor angiogenesis\",\n      \"pmids\": [\"24213570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Which F-box protein mediates p27 ubiquitination in the SAG complex in endothelial cells is not defined\",\n        \"Contribution of CUL5 vs. CUL1 in vascular SAG function not distinguished\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Biophysical reconstitution of the complete SOCS2-EloBC-CUL5-RBX2 complex demonstrated it exists as a monomer and established interaction affinities within the complex, providing the first complete structural framework for a CRL5 module.\",\n      \"evidence\": \"Recombinant expression, SEC-MALS, native MS, ITC, ion mobility MS\",\n      \"pmids\": [\"25247507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"High-resolution crystal or cryo-EM structure of the full complex not available\",\n        \"How neddylation changes conformational dynamics not resolved at structural level\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Distinguishing SAG from RBX1 at the E2-binding level showed that SAG specifically recruits UBCH10/UBE2S to build K11-linked ubiquitin chains (targeting β-TrCP1), while RBX1 uses CDC34/UBCH5C for K48 chains — establishing a fundamental biochemical distinction between the two RING-box paralogs.\",\n      \"evidence\": \"E2 co-immunoprecipitation, protein half-life assays, E2-specific siRNA with substrate accumulation readouts\",\n      \"pmids\": [\"27910872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for SAG preferring UBCH10/UBE2S is unknown\",\n        \"Whether all SAG substrates are modified with K11 chains or some use K48 is not resolved\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"T-cell-specific Sag knockout revealed that SAG is dispensable for T-cell development but required for T-cell activation and proliferation, with SOCS accumulation as the likely mechanism, extending SAG function to adaptive immunity.\",\n      \"evidence\": \"T-cell-specific conditional Sag KO mice, in vitro stimulation, allogeneic BMT model, MLN4924 treatment\",\n      \"pmids\": [\"27543965\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific SOCS family member(s) responsible not identified\",\n        \"Whether CRL5 or CRL1 mediates SOCS degradation in T cells is unclear\",\n        \"Single lab study without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Lung-specific Sag deletion suppressed KrasG12D-driven tumorigenesis with accumulation of p21, p27, NOXA, BIM, and DEPTOR; epistatic rescue by p21 or DEPTOR knockdown confirmed their causal roles, solidifying SAG as a multi-substrate oncogenic E3 ligase in Kras-driven cancer.\",\n      \"evidence\": \"Lung-specific Sag conditional KO in KrasG12D mice, double-knockdown rescue experiments\",\n      \"pmids\": [\"24430184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contribution of each substrate to tumor suppression not quantified individually\",\n        \"Whether therapeutic SAG inhibition is tolerated in normal lung tissue is unknown\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"RBX2 was shown to control retinal lamination through DAB1/SOCS7-dependent and SOCS7-independent pathways, extending the neuronal migration paradigm from cortex/cerebellum to the retina and revealing that cone photoreceptor positioning requires an as-yet-unidentified CRL5 adaptor.\",\n      \"evidence\": \"In vivo shRNA knockdown of RBX2 and SOCS7, retinal electrophysiology (ERG), immunohistochemistry\",\n      \"pmids\": [\"29361558\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Identity of the CRL5 adaptor for cone photoreceptor positioning unknown\",\n        \"shRNA-based approach; conditional KO confirmation pending\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for RBX2-specific E2 (UBE2F, UBCH10) recruitment, the signals that determine substrate-adaptor switching in different tissue/disease contexts, and whether the intrinsic antioxidant activity of RNF7 operates independently of its E3 ligase function in physiological settings.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No high-resolution structure of RBX2 in complex with UBE2F or UBCH10\",\n        \"Mechanism controlling stage-dependent adaptor switching remains unknown\",\n        \"In vivo separation of antioxidant vs. E3 ligase functions not achieved\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 2, 4, 9, 15, 23, 28, 33]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 9, 16, 22, 31, 32, 33]},\n      {\"term_id\": \"GO:0016209\", \"supporting_discovery_ids\": [0, 18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [15, 33]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 19, 28]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [30]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 9, 15, 22, 23, 28, 33]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [12, 17, 22, 26, 40]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 9, 16, 27]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5, 25, 26]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [23, 34, 38]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [20, 24, 25, 37]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [12, 26, 32, 40]}\n    ],\n    \"complexes\": [\n      \"CRL5 (CUL5-RBX2-ElonginB/C-SOCS-box)\",\n      \"SCF (CUL1-RBX2-SKP1-F-box)\",\n      \"Vif-CUL5-ElonginBC-CBFβ-RBX2\"\n    ],\n    \"partners\": [\n      \"CUL5\",\n      \"CUL1\",\n      \"UBE2F\",\n      \"UBCH10\",\n      \"UBE2S\",\n      \"BTRC\",\n      \"FBXW7\",\n      \"NEDD4\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}