{"gene":"FBXO31","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2005,"finding":"FBXO31 is a component of an SCF ubiquitin ligase complex, associating with Skp1, Roc-1, and Cullin-1, and its ectopic expression induces G1 cell cycle arrest in breast cancer cell lines.","method":"Co-immunoprecipitation (association with Skp1, Roc-1, Cullin-1); ectopic expression with cell cycle analysis by flow cytometry","journal":"Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP showing SCF complex membership, plus functional cell cycle readout, single lab","pmids":["16357137"],"is_preprint":false},{"year":2009,"finding":"FBXO31 directly binds cyclin D1 and mediates its proteasomal degradation, requiring the F-box motif of FBXO31 and phosphorylation of cyclin D1 at Thr286, resulting in G1 arrest. DNA damage (gamma-irradiation) induces FBXO31 accumulation through ATM-mediated phosphorylation of FBXO31, and FBXO31 knockdown prevents efficient G1 arrest after DNA damage.","method":"Co-immunoprecipitation, RNAi knockdown, ectopic expression, proteasome inhibitor assays, domain mutagenesis (F-box deletion), ATM kinase epistasis, gamma-irradiation DNA damage model","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, mutagenesis, RNAi epistasis, in vivo kinase dependency), replicated in subsequent studies","pmids":["19412162"],"is_preprint":false},{"year":2013,"finding":"FBXO31-SCF localizes to the centrosome and regulates neuronal morphogenesis, axonal identity, dendrite growth, and neuronal migration in developing cerebellar cortex. FBXO31 interacts with and targets the polarity protein Par6c for proteasomal degradation to control axon (but not dendrite) growth.","method":"Centrosomal localization (immunofluorescence), Co-immunoprecipitation, RNAi knockdown in cerebellar neurons, in vivo migration assays in cerebellar cortex","journal":"PLoS One","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization tied to functional consequence, Co-IP of substrate, in vivo neuronal migration assay, single lab","pmids":["23469015"],"is_preprint":false},{"year":2014,"finding":"FBXO31 interacts with Cdt1 (DNA replication licensing factor) and mediates its ubiquitylation and proteasomal degradation specifically during G2 phase, independently of previously described Cdt1 proteolysis pathways (CRL4-Cdt2 and SCF-Skp2), preventing re-replication. Targeting is mediated through the N-terminus of Cdt1.","method":"Co-immunoprecipitation, ubiquitylation assay, cell cycle-synchronized degradation assay, RNAi knockdown with re-replication readout (flow cytometry)","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — Co-IP, in-cell ubiquitylation assay, domain mapping, functional re-replication phenotype with KD, multiple orthogonal methods","pmids":["24828503"],"is_preprint":false},{"year":2014,"finding":"FBXO31 (as part of SCF complex) binds MKK6 and mediates its Lys48-linked polyubiquitination and proteasomal degradation, thereby negatively regulating p38 MAPK signaling and protecting cells from stress-induced apoptosis.","method":"Co-immunoprecipitation, ubiquitin linkage-specific assay (Lys48), RNAi knockdown, ectopic expression with p38 activation readout","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, Lys48 linkage specificity assay, and functional p38 signaling readout, single lab","pmids":["24936062"],"is_preprint":false},{"year":2015,"finding":"FBXO31 directs MDM2 degradation following genotoxic stress: ATM phosphorylates both FBXO31 (increasing its levels) and MDM2 (enabling recognition); FBXO31 then interacts with MDM2 and promotes its proteasomal degradation, resulting in elevated p53 levels and growth arrest. FBXO31 depletion prevents MDM2 degradation and p53 accumulation after DNA damage.","method":"Co-immunoprecipitation, RNAi knockdown, ATM kinase inhibition/epistasis, proteasome inhibitor assays, Western blot for p53/MDM2 levels","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ATM epistasis, RNAi phenotype with multiple molecular readouts, multiple orthogonal methods","pmids":["26124108"],"is_preprint":false},{"year":2016,"finding":"FBXO31 interacts with and ubiquitinates FOXM1 specifically during the G2/M transition, promoting its degradation. Loss of FBXO31 leads to increased FOXM1 levels, spindle checkpoint activation, lagging chromosomes, and anaphase bridges; co-depletion of FOXM1 rescues genomic instability but not the mitotic delay, indicating FBXO31 has additional mitotic substrates.","method":"Co-immunoprecipitation, ubiquitination assay, RNAi double knockdown epistasis, flow cytometry, live-cell imaging for mitotic progression","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitination assay, genetic epistasis by double KD, multiple functional readouts, single lab with multiple orthogonal methods","pmids":["27568981"],"is_preprint":false},{"year":2017,"finding":"Crystal structures of Skp1-FBXO31 complex alone and bound to phosphorylated cyclin D1 C-terminal peptide revealed that FBXO31 possesses a unique substrate-binding domain with two β-barrel motifs, and cyclin D1 binds by inserting its free C-terminal carboxylate tail into a cavity of the C-terminal β-barrel. Biophysical and functional studies showed SCFFBXO31 can ubiquitinate cyclin D1 in a phosphorylation-independent manner.","method":"X-ray crystallography, biophysical binding assays, in vitro ubiquitination assay, mutagenesis","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by in vitro ubiquitination and mutagenesis, multiple orthogonal methods in a single rigorous study","pmids":["29279382"],"is_preprint":false},{"year":2017,"finding":"FBXO31 interacts with Snail1 (SNAI1) and mediates its ubiquitin- and proteasome-dependent degradation in gastric cancer cells, suppressing EMT. The F-box domain of FBXO31 and phosphorylation of Snail1 are required for the interaction.","method":"Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown with EMT markers, domain/phosphorylation mutagenesis, mouse xenograft model","journal":"Molecular Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, mutagenesis of interaction determinants, in vivo confirmation, single lab","pmids":["29117943"],"is_preprint":false},{"year":2018,"finding":"APC/C (with coactivators CDH1 and CDC20) degrades FBXO31 via a D-box motif in a cell-cycle-regulated manner, maintaining low basal FBXO31 levels. AKT phosphorylates FBXO31 at Ser33 to enable APC/C-mediated degradation, while ATM phosphorylation of FBXO31 at Ser278 following DNA damage disrupts interaction with CDH1/CDC20, preventing FBXO31 degradation and allowing its levels to rise.","method":"Co-immunoprecipitation, site-directed mutagenesis (Ser33, Ser278, D-box), kinase inhibition, RNAi knockdown, cell-cycle synchronization, ubiquitination assay","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP, mutagenesis of multiple phosphorylation sites and D-box, kinase inhibitor epistasis, multiple orthogonal methods","pmids":["29343641"],"is_preprint":false},{"year":2018,"finding":"The SCF-E3 ligase FBXO46 recognizes an RXXR motif at the C-terminus of FBXO31 and directs its polyubiquitination and proteasomal degradation to maintain basal FBXO31 levels in unstressed cells, preventing premature senescence. Following DNA damage, ATM phosphorylates FBXO46 at Ser21/Ser67, leading to FBXO46 degradation via FBXO31 (negative feedback loop).","method":"Co-immunoprecipitation, molecular docking, RXXR motif mutagenesis, RNAi knockdown, ubiquitination assay, ATM kinase inhibitor","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — Co-IP, mutagenesis of recognition motif, ubiquitination assay, ATM epistasis, negative feedback experimentally demonstrated","pmids":["30171069"],"is_preprint":false},{"year":2019,"finding":"FBXO31 interacts with Smad7 (negative regulator of TGF-β/Smad signaling) and enhances its ubiquitination, promoting fibrogenesis in hepatic stellate cells. FBXO31 and Smad7 co-localize in HSC-T6 cells and in mouse liver fibrosis tissues.","method":"Co-immunoprecipitation, immunofluorescence co-localization, ubiquitination assay, ectopic expression/knockdown with α-SMA and Col-1 readouts","journal":"Journal of Cellular Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, co-localization, functional phenotype, single lab","pmids":["31680332"],"is_preprint":false},{"year":2019,"finding":"FBXO31 is essential for maintaining low cyclin A levels during G1 phase. Stable FBXO31 knockdown causes atypical cyclin A accumulation in G1, leading to premature DNA replication, compromised MCM loading, replication from fewer origins, and DNA double-strand breaks, resulting in genomic instability.","method":"Flow cytometry, Western blot, immunofluorescence, RNAi stable knockdown, DNA combing for replication origin analysis, γH2AX foci for DSBs","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (flow cytometry, DNA combing, immunofluorescence), single lab","pmids":["31413110"],"is_preprint":false},{"year":2021,"finding":"CRL1FBXO31 promotes ubiquitylation-mediated degradation of DUSP6, a dual-specificity phosphatase that inactivates ERK1/2. FBXO31 depletion stabilizes DUSP6, suppresses ERK signaling, and activates PI3K-AKT signaling, promoting prostate tumor development. Pharmacological inhibition of DUSP6 rescues the tumor-promoting effects of FBXO31 loss.","method":"Co-immunoprecipitation, ubiquitylation assay, RNAi knockdown, mouse orthotopic tumor model, DUSP6 inhibitor (BCI) treatment","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitylation assay, in vivo tumor model, pharmacological rescue, multiple orthogonal methods","pmids":["34686346"],"is_preprint":false},{"year":2022,"finding":"FBXO31 interacts with and promotes ubiquitination and proteasomal degradation of GPX4, sensitizing cholangiocarcinoma cancer stem cell-like cells to cisplatin-induced ferroptosis. GPX4 overexpression reverses FBXO31-promoted ferroptosis.","method":"Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown, ferroptosis assay, GPX4 rescue experiment, in vivo tumor model","journal":"Liver International","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, rescue experiment, functional ferroptosis readout, single lab","pmids":["36269678"],"is_preprint":false},{"year":2022,"finding":"c-Myc suppresses FBXO31 transcription by binding its promoter (shown by ChIP), while FBXO31 reciprocally interacts with c-Myc and directs its polyubiquitination via the SCF complex and proteasomal degradation in a phosphorylation-independent manner, inhibiting ovarian cancer growth.","method":"Chromatin immunoprecipitation (c-Myc at FBXO31 promoter), Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown, in vitro and in vivo tumor growth assays","journal":"International Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for transcriptional regulation, Co-IP and ubiquitination for degradation mechanism, multiple orthogonal methods, single lab","pmids":["34706096"],"is_preprint":false},{"year":2024,"finding":"FBXO31 promotes proteasome-dependent degradation of SIRT2 by interacting with its sirtuin-type domain, and this is regulated upstream by METTL3-mediated m6A modification of FBXO31 mRNA, enhancing FBXO31 translation in a YTHDF1-dependent manner.","method":"Co-immunoprecipitation, ubiquitination assay, protein half-life assay, domain mapping (sirtuin-type domain of SIRT2), m6A RNA modification analysis, YTHDF1 knockdown","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, domain mapping, m6A mechanism with YTHDF1 epistasis, single lab multiple orthogonal methods","pmids":["38216561"],"is_preprint":false},{"year":2025,"finding":"FBXO31 is a reader of C-terminal amide-bearing proteins (CTAPs), recognizing their C-terminal amide modification via a conserved binding pocket in its β-barrel domain. FBXO31 recruits CTAPs to the SCF ubiquitin ligase for ubiquitylation and proteasomal degradation, enabling surveillance of chemically damaged proteins. A dominant neurodevelopmental disorder mutation reverses substrate specificity such that non-amidated neo-substrates are now degraded.","method":"CRISPR screen to identify FBXO31, semi-synthetic chemical biology approach, crystal structure-informed binding pocket analysis, in vitro ubiquitylation, mutagenesis of binding pocket residues, cellular degradation assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — CRISPR screen, structural basis of recognition, in vitro ubiquitylation, mutagenesis, disease mutation functional characterization — multiple orthogonal methods in single rigorous study","pmids":["39880951"],"is_preprint":false},{"year":2025,"finding":"FBXO31 mediates ubiquitination and proteasomal degradation of OGT (O-GlcNAc transferase), thereby regulating O-GlcNAcylation homeostasis in endometrial cancer cells.","method":"CRISPR screen, Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown, endometrial organoid models, mouse tumor model","journal":"Nature Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen identification, Co-IP, ubiquitination assay, in vivo model, single lab","pmids":["39894887"],"is_preprint":false},{"year":2025,"finding":"FBXO31 phosphorylation at Thr37 and Ser523 contributes to FBXO31 protein stabilization, as shown by identification of five phosphorylation sites (Thr28, Thr37, Ser33, Ser400, Ser523) by LC-MS/MS in HEK293T cells, with mutagenesis demonstrating differential effects on protein turnover.","method":"LC-MS/MS phosphoproteomics, site-directed mutagenesis, cycloheximide chase assay, Western blot, flow cytometry","journal":"Advanced Biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — mass spectrometry identification plus mutagenesis and cycloheximide chase, single lab","pmids":["40847744"],"is_preprint":false},{"year":2026,"finding":"FBXO31 can be exploited as a TPD-competent E3 ligase using C-terminal amide-bearing degrons (amidated Ala-Phe motif as chemical recruiter), forming ternary complexes with neo-substrates (FKBP12, multiple kinases, BRD2/3/4) for targeted degradation. Key residues in FBXO31 required for recruiter engagement were identified.","method":"Ternary complex formation assay, targeted protein degradation assays, mutagenesis of FBXO31 residues, biochemical degradation assays for multiple substrates","journal":"Journal of the American Chemical Society","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ternary complex confirmation, mutagenesis, degradation of multiple neo-substrates, single lab","pmids":["41963263"],"is_preprint":false},{"year":2026,"finding":"FBXO31 interacts with ABL2 via its F-box motif, promotes ABL2 ubiquitination and proteasomal degradation, and thereby induces xCT-mediated ferroptosis and inhibits progression in triple-negative breast cancer.","method":"Co-immunoprecipitation, ubiquitination assay, F-box domain mutagenesis, ectopic expression/knockdown, ferroptosis assay, rescue experiments, mouse tumor model","journal":"American Journal of Translational Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, F-box mutagenesis, rescue experiment with functional ferroptosis readout, single lab","pmids":["42170439"],"is_preprint":false},{"year":2021,"finding":"FBXO31 binds cofilin-1 (shown by mass spectrometry and co-immunoprecipitation), and FBXO31-mediated Taxol chemoresistance in esophageal squamous cell carcinoma is at least partly dependent on cofilin-1, as cofilin-1 knockdown in FBXO31-overexpressing cells reversed FBXO31-induced suppression of apoptosis.","method":"Mass spectrometry, co-immunoprecipitation, RNAi knockdown epistasis, apoptosis assay (FACS/TUNEL), in vivo xenograft","journal":"Biochemical and Biophysical Research Communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and partial epistasis; mechanism of cofilin-1 ubiquitination by FBXO31 not demonstrated","pmids":["34839191"],"is_preprint":false}],"current_model":"FBXO31 is the substrate-recognition subunit of the SCF (SKP1-CUL1-RBX1-FBXO31) E3 ubiquitin ligase complex that targets multiple cell-cycle and signaling proteins — including cyclin D1, MDM2, Cdt1, FOXM1, MKK6, Snail1, DUSP6, GPX4, SIRT2, OGT, ABL2, c-Myc, and Par6c — for K48-linked polyubiquitination and proteasomal degradation; it is normally kept at low levels by APC/C (CDH1/CDC20, requiring AKT-mediated Ser33 phosphorylation) and by FBXO46, but following genotoxic stress, ATM phosphorylates FBXO31 at Ser278, disrupting these interactions and allowing FBXO31 to accumulate, degrade cyclin D1 and MDM2, stabilize p53, and enforce G1/S and G2/M checkpoints; additionally, FBXO31 recognizes C-terminal amide-bearing proteins via a conserved β-barrel binding pocket for broad surveillance of chemically damaged proteins, and functions in neuronal development by promoting proteasomal degradation of Par6c to control axon identity and migration in the developing cerebellum."},"narrative":{"mechanistic_narrative":"FBXO31 is the substrate-recognition subunit of an SCF (SKP1–CUL1–RBX1) E3 ubiquitin ligase that enforces cell-cycle checkpoints and genome stability by targeting cell-cycle and signaling regulators for K48-linked polyubiquitination and proteasomal degradation [PMID:16357137, PMID:19412162]. Its best-defined role is in the DNA-damage response: γ-irradiation triggers ATM-mediated phosphorylation of FBXO31, causing its accumulation and the degradation of cyclin D1 to impose G1 arrest [PMID:19412162], and FBXO31 likewise directs degradation of MDM2 to stabilize p53 and arrest growth after genotoxic stress [PMID:26124108]. FBXO31 also restrains cell-cycle progression at additional points by degrading the licensing factor Cdt1 in G2 to block re-replication [PMID:24828503], FOXM1 at the G2/M transition to preserve genomic stability [PMID:27568981], and by keeping cyclin A low during G1 to prevent premature replication and double-strand breaks [PMID:31413110]. A crystal structure of the SKP1–FBXO31 complex bound to a phosphorylated cyclin D1 C-terminal peptide showed that FBXO31 uses a unique two-β-barrel substrate-binding domain in which the free C-terminal carboxylate of cyclin D1 inserts into a cavity of the C-terminal β-barrel, and SCF-FBXO31 can ubiquitinate cyclin D1 independently of phosphorylation [PMID:29279382]; this β-barrel pocket was later shown to act as a reader of C-terminal amide-bearing proteins (CTAPs), recruiting chemically damaged amidated proteins to the SCF for degradation as a protein-quality-control surveillance mechanism, with a dominant neurodevelopmental disorder mutation reversing this substrate specificity [PMID:39880951]. FBXO31 levels are tightly controlled: APC/C (CDH1/CDC20) degrades FBXO31 via a D-box following AKT-mediated Ser33 phosphorylation, while DNA-damage-induced ATM phosphorylation at Ser278 disrupts this recognition to permit FBXO31 accumulation [PMID:29343641], and FBXO46 maintains basal FBXO31 levels through an RXXR-motif-dependent reciprocal feedback loop [PMID:30171069]. Beyond the checkpoint network, FBXO31 degrades a broad set of substrates—including MKK6 to dampen p38 MAPK and apoptosis [PMID:24936062], DUSP6 to tune ERK/PI3K-AKT signaling in prostate tumors [PMID:34686346], Snail1 to suppress EMT [PMID:29117943], c-Myc [PMID:34706096], GPX4 and ABL2 to sensitize cancer cells to ferroptosis [PMID:36269678, PMID:42170439], SIRT2 [PMID:38216561], and OGT to control O-GlcNAcylation homeostasis [PMID:39894887]—and it controls neuronal morphogenesis by degrading the polarity protein Par6c at the centrosome to specify axon identity and migration in the developing cerebellum [PMID:23469015]. Its C-terminal amide recognition pocket has been engineered to support targeted protein degradation using amidated chemical recruiters [PMID:41963263].","teleology":[{"year":2005,"claim":"Established FBXO31 as a genuine SCF E3 ligase subunit with cell-cycle inhibitory function, setting the framework for all subsequent substrate work.","evidence":"Co-IP showing association with Skp1/Roc-1/Cullin-1 plus ectopic expression causing G1 arrest in breast cancer cells","pmids":["16357137"],"confidence":"Medium","gaps":["No substrate identified at this stage","Mechanism of G1 arrest unknown"]},{"year":2009,"claim":"Defined the first FBXO31 substrate and linked it to the DNA-damage checkpoint, answering how FBXO31 enforces G1 arrest.","evidence":"Co-IP, F-box deletion, RNAi epistasis, proteasome assays, ATM dependency in a γ-irradiation model targeting cyclin D1","pmids":["19412162"],"confidence":"High","gaps":["Structural basis of cyclin D1 recognition not yet defined","ATM phosphorylation site on FBXO31 not mapped"]},{"year":2013,"claim":"Extended FBXO31 function to neuronal development, showing it acts at the centrosome to specify axon identity via degradation of a polarity factor.","evidence":"Centrosomal immunofluorescence, Co-IP of Par6c, RNAi and in vivo cerebellar migration assays","pmids":["23469015"],"confidence":"Medium","gaps":["Single lab","Whether Par6c degradation fully accounts for the axon phenotype unresolved"]},{"year":2014,"claim":"Broadened the cell-cycle role by showing FBXO31 degrades Cdt1 in G2 and MKK6 to control re-replication and stress signaling respectively.","evidence":"Co-IP, in-cell ubiquitylation assays, cell-cycle-synchronized degradation, Lys48 linkage specificity, re-replication and p38 readouts","pmids":["24828503","24936062"],"confidence":"High","gaps":["Degrons on these substrates only partially mapped","Coordination of multiple substrate targeting per cell-cycle phase unclear"]},{"year":2015,"claim":"Showed FBXO31 stabilizes p53 by degrading MDM2 after damage, connecting FBXO31 to the central tumor-suppressor axis.","evidence":"Reciprocal Co-IP, ATM epistasis, RNAi, proteasome inhibition with p53/MDM2 Western blots","pmids":["26124108"],"confidence":"High","gaps":["Relative contribution of cyclin D1 vs MDM2 degradation to arrest not quantified"]},{"year":2016,"claim":"Identified FOXM1 as a G2/M substrate and demonstrated FBXO31 has additional mitotic substrates beyond FOXM1.","evidence":"Co-IP, ubiquitination assay, double-knockdown epistasis, live-cell imaging of mitosis","pmids":["27568981"],"confidence":"High","gaps":["Identity of the additional mitotic substrate(s) unknown"]},{"year":2017,"claim":"Solved the structural basis of substrate recognition, revealing a unique two-β-barrel domain that reads the free C-terminal carboxylate and enables phosphorylation-independent ubiquitination.","evidence":"X-ray crystallography of Skp1–FBXO31 with phospho-cyclin D1 peptide, biophysical binding, in vitro ubiquitination, mutagenesis","pmids":["29279382"],"confidence":"High","gaps":["Generality of C-terminal-tail recognition to other substrates not yet tested at this stage"]},{"year":2017,"claim":"Showed FBXO31 degrades Snail1 to suppress EMT, expanding its tumor-suppressive substrate repertoire.","evidence":"Co-IP, ubiquitination assay, phospho/domain mutagenesis, EMT markers, xenograft","pmids":["29117943"],"confidence":"Medium","gaps":["Single lab","Snail1 phosphorylation kinase not identified"]},{"year":2018,"claim":"Defined the upstream control of FBXO31 abundance, showing APC/C and FBXO46 keep it low and that ATM-dependent phosphorylation switches that off after damage.","evidence":"Co-IP, D-box/Ser33/Ser278/RXXR mutagenesis, kinase inhibition, ubiquitination assays, demonstrated FBXO46–FBXO31 negative feedback","pmids":["29343641","30171069"],"confidence":"High","gaps":["How multiple competing degradation inputs are integrated quantitatively is unresolved"]},{"year":2019,"claim":"Connected FBXO31 loss directly to genomic instability through failure to restrain cyclin A in G1, and linked it to TGF-β/fibrogenesis via Smad7.","evidence":"DNA combing, γH2AX foci, flow cytometry for cyclin A; Co-IP, co-localization and ubiquitination for Smad7","pmids":["31413110","31680332"],"confidence":"Medium","gaps":["Whether cyclin A is a direct FBXO31 substrate not established","Single labs"]},{"year":2022,"claim":"Established FBXO31 as a regulator of ERK/AKT signaling and ferroptosis, and revealed reciprocal transcriptional repression by its own substrate c-Myc.","evidence":"Co-IP, ubiquitylation assays, in vivo tumor models, pharmacological rescue (DUSP6 inhibitor), GPX4 rescue, ChIP for c-Myc at the FBXO31 promoter","pmids":["34686346","36269678","34706096"],"confidence":"High","gaps":["Context-dependence of tumor-suppressive vs other roles across tissues unresolved","Some substrate findings from single labs"]},{"year":2025,"claim":"Reframed the β-barrel pocket as a quality-control reader of C-terminal amide-bearing damaged proteins, generalizing the structural recognition principle and explaining a neurodevelopmental disorder mutation.","evidence":"CRISPR screen, semi-synthetic chemical biology, structure-informed pocket analysis, in vitro ubiquitylation, mutagenesis, disease-mutation characterization; also OGT identified as a substrate via CRISPR screen","pmids":["39880951","39894887"],"confidence":"High","gaps":["Endogenous physiological CTAP substrate landscape not enumerated","Mechanism by which the disorder mutation rewires specificity in vivo incompletely defined"]},{"year":2026,"claim":"Demonstrated FBXO31 can be co-opted for targeted protein degradation and identified ABL2 and SIRT2 as further substrates, broadening both its biology and its therapeutic utility.","evidence":"Ternary complex and TPD assays with amidated recruiters, F-box mutagenesis, Co-IP, ubiquitination and ferroptosis assays, tumor models; m6A/YTHDF1 control of FBXO31 translation for SIRT2","pmids":["41963263","42170439","38216561"],"confidence":"Medium","gaps":["TPD work is biochemical/cellular, not validated in vivo","Single labs"]},{"year":null,"claim":"The full endogenous substrate spectrum of FBXO31 and how its expanding roles (checkpoint enforcement, protein-quality surveillance, ferroptosis, signaling) are coordinated within a given cell remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unbiased proteome-wide substrate map reported","Determinants of tissue-specific substrate choice unknown","Whether cyclin A and cofilin-1 are direct substrates not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,3,4,5,6,13,17]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,5,9]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,3,6,12]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,13]}],"complexes":["SCF (SKP1-CUL1-RBX1-FBXO31) E3 ubiquitin ligase"],"partners":["SKP1","CUL1","RBX1","CCND1","MDM2","CDT1","FOXM1","FBXO46"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5XUX0","full_name":"F-box only protein 31","aliases":[],"length_aa":539,"mass_kda":60.7,"function":"Substrate-recognition component of the SCF(FBXO31) protein ligase complex, which specifically mediates the ubiquitination of proteins amidated at their C-terminus in response to oxidative stress, leading to their degradation by the proteasome (PubMed:39880951). FBXO31 specifically recognizes and binds C-terminal peptides bearing an amide: C-terminal amidation in response to oxidative stress takes place following protein fragmentation (PubMed:39880951). The SCF(FBXO31) also plays a role in G1 arrest following DNA damage by mediating ubiquitination of phosphorylated cyclin-D1 (CCND1), promoting its degradation by the proteasome, resulting in G1 arrest (PubMed:19412162, PubMed:29279382). The SCF(FBXO31) complex is however not a major regulator of CCND1 stability during the G1/S transition (By similarity). In response to genotoxic stress, the SCF(FBXO31) complex directs ubiquitination and degradation of phosphorylated MDM2, thereby promoting p53/TP53-mediated DNA damage response (PubMed:26124108). SCF(FBXO31) complex is required for genomic integrity by catalyzing ubiquitination and degradation of cyclin-A (CCNA1 and/or CCNA2) during the G1 phase (PubMed:31413110). In response to genotoxic stress, the SCF(FBXO31) complex directs ubiquitination and degradation of phosphorylated FBXO46 and MAP2K6 (PubMed:24936062, PubMed:30171069). SCF(FBXO31) complex promotes ubiquitination and degradation of CDT1 during the G2 phase to prevent re-replication (PubMed:24828503). The SCF(FBXO31) complex also mediates ubiquitination and degradation of DUSP6, OGT and PARD6A (PubMed:23469015, PubMed:34686346, PubMed:39894887)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/Q5XUX0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FBXO31","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FBXO31","total_profiled":1310},"omim":[{"mim_id":"615979","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 45; MRT45","url":"https://www.omim.org/entry/615979"},{"mim_id":"609102","title":"F-BOX ONLY PROTEIN 31; FBXO31","url":"https://www.omim.org/entry/609102"},{"mim_id":"168461","title":"CYCLIN D1; CCND1","url":"https://www.omim.org/entry/168461"},{"mim_id":"164757","title":"B-RAF PROTOONCOGENE, SERINE/THREONINE KINASE; BRAF","url":"https://www.omim.org/entry/164757"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal 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DNA damage (gamma-irradiation) induces FBXO31 accumulation through ATM-mediated phosphorylation of FBXO31, and FBXO31 knockdown prevents efficient G1 arrest after DNA damage.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, ectopic expression, proteasome inhibitor assays, domain mutagenesis (F-box deletion), ATM kinase epistasis, gamma-irradiation DNA damage model\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, mutagenesis, RNAi epistasis, in vivo kinase dependency), replicated in subsequent studies\",\n      \"pmids\": [\"19412162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"FBXO31-SCF localizes to the centrosome and regulates neuronal morphogenesis, axonal identity, dendrite growth, and neuronal migration in developing cerebellar cortex. FBXO31 interacts with and targets the polarity protein Par6c for proteasomal degradation to control axon (but not dendrite) growth.\",\n      \"method\": \"Centrosomal localization (immunofluorescence), Co-immunoprecipitation, RNAi knockdown in cerebellar neurons, in vivo migration assays in cerebellar cortex\",\n      \"journal\": \"PLoS One\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization tied to functional consequence, Co-IP of substrate, in vivo neuronal migration assay, single lab\",\n      \"pmids\": [\"23469015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FBXO31 interacts with Cdt1 (DNA replication licensing factor) and mediates its ubiquitylation and proteasomal degradation specifically during G2 phase, independently of previously described Cdt1 proteolysis pathways (CRL4-Cdt2 and SCF-Skp2), preventing re-replication. Targeting is mediated through the N-terminus of Cdt1.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, cell cycle-synchronized degradation assay, RNAi knockdown with re-replication readout (flow cytometry)\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — Co-IP, in-cell ubiquitylation assay, domain mapping, functional re-replication phenotype with KD, multiple orthogonal methods\",\n      \"pmids\": [\"24828503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FBXO31 (as part of SCF complex) binds MKK6 and mediates its Lys48-linked polyubiquitination and proteasomal degradation, thereby negatively regulating p38 MAPK signaling and protecting cells from stress-induced apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin linkage-specific assay (Lys48), RNAi knockdown, ectopic expression with p38 activation readout\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, Lys48 linkage specificity assay, and functional p38 signaling readout, single lab\",\n      \"pmids\": [\"24936062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FBXO31 directs MDM2 degradation following genotoxic stress: ATM phosphorylates both FBXO31 (increasing its levels) and MDM2 (enabling recognition); FBXO31 then interacts with MDM2 and promotes its proteasomal degradation, resulting in elevated p53 levels and growth arrest. FBXO31 depletion prevents MDM2 degradation and p53 accumulation after DNA damage.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, ATM kinase inhibition/epistasis, proteasome inhibitor assays, Western blot for p53/MDM2 levels\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ATM epistasis, RNAi phenotype with multiple molecular readouts, multiple orthogonal methods\",\n      \"pmids\": [\"26124108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FBXO31 interacts with and ubiquitinates FOXM1 specifically during the G2/M transition, promoting its degradation. Loss of FBXO31 leads to increased FOXM1 levels, spindle checkpoint activation, lagging chromosomes, and anaphase bridges; co-depletion of FOXM1 rescues genomic instability but not the mitotic delay, indicating FBXO31 has additional mitotic substrates.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RNAi double knockdown epistasis, flow cytometry, live-cell imaging for mitotic progression\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitination assay, genetic epistasis by double KD, multiple functional readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27568981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structures of Skp1-FBXO31 complex alone and bound to phosphorylated cyclin D1 C-terminal peptide revealed that FBXO31 possesses a unique substrate-binding domain with two β-barrel motifs, and cyclin D1 binds by inserting its free C-terminal carboxylate tail into a cavity of the C-terminal β-barrel. Biophysical and functional studies showed SCFFBXO31 can ubiquitinate cyclin D1 in a phosphorylation-independent manner.\",\n      \"method\": \"X-ray crystallography, biophysical binding assays, in vitro ubiquitination assay, mutagenesis\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by in vitro ubiquitination and mutagenesis, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"29279382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FBXO31 interacts with Snail1 (SNAI1) and mediates its ubiquitin- and proteasome-dependent degradation in gastric cancer cells, suppressing EMT. The F-box domain of FBXO31 and phosphorylation of Snail1 are required for the interaction.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown with EMT markers, domain/phosphorylation mutagenesis, mouse xenograft model\",\n      \"journal\": \"Molecular Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, mutagenesis of interaction determinants, in vivo confirmation, single lab\",\n      \"pmids\": [\"29117943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"APC/C (with coactivators CDH1 and CDC20) degrades FBXO31 via a D-box motif in a cell-cycle-regulated manner, maintaining low basal FBXO31 levels. AKT phosphorylates FBXO31 at Ser33 to enable APC/C-mediated degradation, while ATM phosphorylation of FBXO31 at Ser278 following DNA damage disrupts interaction with CDH1/CDC20, preventing FBXO31 degradation and allowing its levels to rise.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (Ser33, Ser278, D-box), kinase inhibition, RNAi knockdown, cell-cycle synchronization, ubiquitination assay\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP, mutagenesis of multiple phosphorylation sites and D-box, kinase inhibitor epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"29343641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The SCF-E3 ligase FBXO46 recognizes an RXXR motif at the C-terminus of FBXO31 and directs its polyubiquitination and proteasomal degradation to maintain basal FBXO31 levels in unstressed cells, preventing premature senescence. Following DNA damage, ATM phosphorylates FBXO46 at Ser21/Ser67, leading to FBXO46 degradation via FBXO31 (negative feedback loop).\",\n      \"method\": \"Co-immunoprecipitation, molecular docking, RXXR motif mutagenesis, RNAi knockdown, ubiquitination assay, ATM kinase inhibitor\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — Co-IP, mutagenesis of recognition motif, ubiquitination assay, ATM epistasis, negative feedback experimentally demonstrated\",\n      \"pmids\": [\"30171069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FBXO31 interacts with Smad7 (negative regulator of TGF-β/Smad signaling) and enhances its ubiquitination, promoting fibrogenesis in hepatic stellate cells. FBXO31 and Smad7 co-localize in HSC-T6 cells and in mouse liver fibrosis tissues.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, ubiquitination assay, ectopic expression/knockdown with α-SMA and Col-1 readouts\",\n      \"journal\": \"Journal of Cellular Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, co-localization, functional phenotype, single lab\",\n      \"pmids\": [\"31680332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FBXO31 is essential for maintaining low cyclin A levels during G1 phase. Stable FBXO31 knockdown causes atypical cyclin A accumulation in G1, leading to premature DNA replication, compromised MCM loading, replication from fewer origins, and DNA double-strand breaks, resulting in genomic instability.\",\n      \"method\": \"Flow cytometry, Western blot, immunofluorescence, RNAi stable knockdown, DNA combing for replication origin analysis, γH2AX foci for DSBs\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (flow cytometry, DNA combing, immunofluorescence), single lab\",\n      \"pmids\": [\"31413110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CRL1FBXO31 promotes ubiquitylation-mediated degradation of DUSP6, a dual-specificity phosphatase that inactivates ERK1/2. FBXO31 depletion stabilizes DUSP6, suppresses ERK signaling, and activates PI3K-AKT signaling, promoting prostate tumor development. Pharmacological inhibition of DUSP6 rescues the tumor-promoting effects of FBXO31 loss.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, RNAi knockdown, mouse orthotopic tumor model, DUSP6 inhibitor (BCI) treatment\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitylation assay, in vivo tumor model, pharmacological rescue, multiple orthogonal methods\",\n      \"pmids\": [\"34686346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBXO31 interacts with and promotes ubiquitination and proteasomal degradation of GPX4, sensitizing cholangiocarcinoma cancer stem cell-like cells to cisplatin-induced ferroptosis. GPX4 overexpression reverses FBXO31-promoted ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown, ferroptosis assay, GPX4 rescue experiment, in vivo tumor model\",\n      \"journal\": \"Liver International\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, rescue experiment, functional ferroptosis readout, single lab\",\n      \"pmids\": [\"36269678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"c-Myc suppresses FBXO31 transcription by binding its promoter (shown by ChIP), while FBXO31 reciprocally interacts with c-Myc and directs its polyubiquitination via the SCF complex and proteasomal degradation in a phosphorylation-independent manner, inhibiting ovarian cancer growth.\",\n      \"method\": \"Chromatin immunoprecipitation (c-Myc at FBXO31 promoter), Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"International Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for transcriptional regulation, Co-IP and ubiquitination for degradation mechanism, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"34706096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FBXO31 promotes proteasome-dependent degradation of SIRT2 by interacting with its sirtuin-type domain, and this is regulated upstream by METTL3-mediated m6A modification of FBXO31 mRNA, enhancing FBXO31 translation in a YTHDF1-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, protein half-life assay, domain mapping (sirtuin-type domain of SIRT2), m6A RNA modification analysis, YTHDF1 knockdown\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, domain mapping, m6A mechanism with YTHDF1 epistasis, single lab multiple orthogonal methods\",\n      \"pmids\": [\"38216561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO31 is a reader of C-terminal amide-bearing proteins (CTAPs), recognizing their C-terminal amide modification via a conserved binding pocket in its β-barrel domain. FBXO31 recruits CTAPs to the SCF ubiquitin ligase for ubiquitylation and proteasomal degradation, enabling surveillance of chemically damaged proteins. A dominant neurodevelopmental disorder mutation reverses substrate specificity such that non-amidated neo-substrates are now degraded.\",\n      \"method\": \"CRISPR screen to identify FBXO31, semi-synthetic chemical biology approach, crystal structure-informed binding pocket analysis, in vitro ubiquitylation, mutagenesis of binding pocket residues, cellular degradation assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — CRISPR screen, structural basis of recognition, in vitro ubiquitylation, mutagenesis, disease mutation functional characterization — multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"39880951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO31 mediates ubiquitination and proteasomal degradation of OGT (O-GlcNAc transferase), thereby regulating O-GlcNAcylation homeostasis in endometrial cancer cells.\",\n      \"method\": \"CRISPR screen, Co-immunoprecipitation, ubiquitination assay, ectopic expression/knockdown, endometrial organoid models, mouse tumor model\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen identification, Co-IP, ubiquitination assay, in vivo model, single lab\",\n      \"pmids\": [\"39894887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO31 phosphorylation at Thr37 and Ser523 contributes to FBXO31 protein stabilization, as shown by identification of five phosphorylation sites (Thr28, Thr37, Ser33, Ser400, Ser523) by LC-MS/MS in HEK293T cells, with mutagenesis demonstrating differential effects on protein turnover.\",\n      \"method\": \"LC-MS/MS phosphoproteomics, site-directed mutagenesis, cycloheximide chase assay, Western blot, flow cytometry\",\n      \"journal\": \"Advanced Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mass spectrometry identification plus mutagenesis and cycloheximide chase, single lab\",\n      \"pmids\": [\"40847744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FBXO31 can be exploited as a TPD-competent E3 ligase using C-terminal amide-bearing degrons (amidated Ala-Phe motif as chemical recruiter), forming ternary complexes with neo-substrates (FKBP12, multiple kinases, BRD2/3/4) for targeted degradation. Key residues in FBXO31 required for recruiter engagement were identified.\",\n      \"method\": \"Ternary complex formation assay, targeted protein degradation assays, mutagenesis of FBXO31 residues, biochemical degradation assays for multiple substrates\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ternary complex confirmation, mutagenesis, degradation of multiple neo-substrates, single lab\",\n      \"pmids\": [\"41963263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FBXO31 interacts with ABL2 via its F-box motif, promotes ABL2 ubiquitination and proteasomal degradation, and thereby induces xCT-mediated ferroptosis and inhibits progression in triple-negative breast cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, F-box domain mutagenesis, ectopic expression/knockdown, ferroptosis assay, rescue experiments, mouse tumor model\",\n      \"journal\": \"American Journal of Translational Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, F-box mutagenesis, rescue experiment with functional ferroptosis readout, single lab\",\n      \"pmids\": [\"42170439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FBXO31 binds cofilin-1 (shown by mass spectrometry and co-immunoprecipitation), and FBXO31-mediated Taxol chemoresistance in esophageal squamous cell carcinoma is at least partly dependent on cofilin-1, as cofilin-1 knockdown in FBXO31-overexpressing cells reversed FBXO31-induced suppression of apoptosis.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, RNAi knockdown epistasis, apoptosis assay (FACS/TUNEL), in vivo xenograft\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and partial epistasis; mechanism of cofilin-1 ubiquitination by FBXO31 not demonstrated\",\n      \"pmids\": [\"34839191\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FBXO31 is the substrate-recognition subunit of the SCF (SKP1-CUL1-RBX1-FBXO31) E3 ubiquitin ligase complex that targets multiple cell-cycle and signaling proteins — including cyclin D1, MDM2, Cdt1, FOXM1, MKK6, Snail1, DUSP6, GPX4, SIRT2, OGT, ABL2, c-Myc, and Par6c — for K48-linked polyubiquitination and proteasomal degradation; it is normally kept at low levels by APC/C (CDH1/CDC20, requiring AKT-mediated Ser33 phosphorylation) and by FBXO46, but following genotoxic stress, ATM phosphorylates FBXO31 at Ser278, disrupting these interactions and allowing FBXO31 to accumulate, degrade cyclin D1 and MDM2, stabilize p53, and enforce G1/S and G2/M checkpoints; additionally, FBXO31 recognizes C-terminal amide-bearing proteins via a conserved β-barrel binding pocket for broad surveillance of chemically damaged proteins, and functions in neuronal development by promoting proteasomal degradation of Par6c to control axon identity and migration in the developing cerebellum.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FBXO31 is the substrate-recognition subunit of an SCF (SKP1–CUL1–RBX1) E3 ubiquitin ligase that enforces cell-cycle checkpoints and genome stability by targeting cell-cycle and signaling regulators for K48-linked polyubiquitination and proteasomal degradation [#0, #1]. Its best-defined role is in the DNA-damage response: γ-irradiation triggers ATM-mediated phosphorylation of FBXO31, causing its accumulation and the degradation of cyclin D1 to impose G1 arrest [#1], and FBXO31 likewise directs degradation of MDM2 to stabilize p53 and arrest growth after genotoxic stress [#5]. FBXO31 also restrains cell-cycle progression at additional points by degrading the licensing factor Cdt1 in G2 to block re-replication [#3], FOXM1 at the G2/M transition to preserve genomic stability [#6], and by keeping cyclin A low during G1 to prevent premature replication and double-strand breaks [#12]. A crystal structure of the SKP1–FBXO31 complex bound to a phosphorylated cyclin D1 C-terminal peptide showed that FBXO31 uses a unique two-β-barrel substrate-binding domain in which the free C-terminal carboxylate of cyclin D1 inserts into a cavity of the C-terminal β-barrel, and SCF-FBXO31 can ubiquitinate cyclin D1 independently of phosphorylation [#7]; this β-barrel pocket was later shown to act as a reader of C-terminal amide-bearing proteins (CTAPs), recruiting chemically damaged amidated proteins to the SCF for degradation as a protein-quality-control surveillance mechanism, with a dominant neurodevelopmental disorder mutation reversing this substrate specificity [#17]. FBXO31 levels are tightly controlled: APC/C (CDH1/CDC20) degrades FBXO31 via a D-box following AKT-mediated Ser33 phosphorylation, while DNA-damage-induced ATM phosphorylation at Ser278 disrupts this recognition to permit FBXO31 accumulation [#9], and FBXO46 maintains basal FBXO31 levels through an RXXR-motif-dependent reciprocal feedback loop [#10]. Beyond the checkpoint network, FBXO31 degrades a broad set of substrates—including MKK6 to dampen p38 MAPK and apoptosis [#4], DUSP6 to tune ERK/PI3K-AKT signaling in prostate tumors [#13], Snail1 to suppress EMT [#8], c-Myc [#15], GPX4 and ABL2 to sensitize cancer cells to ferroptosis [#14, #21], SIRT2 [#16], and OGT to control O-GlcNAcylation homeostasis [#18]—and it controls neuronal morphogenesis by degrading the polarity protein Par6c at the centrosome to specify axon identity and migration in the developing cerebellum [#2]. Its C-terminal amide recognition pocket has been engineered to support targeted protein degradation using amidated chemical recruiters [#20].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established FBXO31 as a genuine SCF E3 ligase subunit with cell-cycle inhibitory function, setting the framework for all subsequent substrate work.\",\n      \"evidence\": \"Co-IP showing association with Skp1/Roc-1/Cullin-1 plus ectopic expression causing G1 arrest in breast cancer cells\",\n      \"pmids\": [\"16357137\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrate identified at this stage\", \"Mechanism of G1 arrest unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the first FBXO31 substrate and linked it to the DNA-damage checkpoint, answering how FBXO31 enforces G1 arrest.\",\n      \"evidence\": \"Co-IP, F-box deletion, RNAi epistasis, proteasome assays, ATM dependency in a γ-irradiation model targeting cyclin D1\",\n      \"pmids\": [\"19412162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of cyclin D1 recognition not yet defined\", \"ATM phosphorylation site on FBXO31 not mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended FBXO31 function to neuronal development, showing it acts at the centrosome to specify axon identity via degradation of a polarity factor.\",\n      \"evidence\": \"Centrosomal immunofluorescence, Co-IP of Par6c, RNAi and in vivo cerebellar migration assays\",\n      \"pmids\": [\"23469015\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Whether Par6c degradation fully accounts for the axon phenotype unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Broadened the cell-cycle role by showing FBXO31 degrades Cdt1 in G2 and MKK6 to control re-replication and stress signaling respectively.\",\n      \"evidence\": \"Co-IP, in-cell ubiquitylation assays, cell-cycle-synchronized degradation, Lys48 linkage specificity, re-replication and p38 readouts\",\n      \"pmids\": [\"24828503\", \"24936062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degrons on these substrates only partially mapped\", \"Coordination of multiple substrate targeting per cell-cycle phase unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed FBXO31 stabilizes p53 by degrading MDM2 after damage, connecting FBXO31 to the central tumor-suppressor axis.\",\n      \"evidence\": \"Reciprocal Co-IP, ATM epistasis, RNAi, proteasome inhibition with p53/MDM2 Western blots\",\n      \"pmids\": [\"26124108\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of cyclin D1 vs MDM2 degradation to arrest not quantified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified FOXM1 as a G2/M substrate and demonstrated FBXO31 has additional mitotic substrates beyond FOXM1.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, double-knockdown epistasis, live-cell imaging of mitosis\",\n      \"pmids\": [\"27568981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the additional mitotic substrate(s) unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Solved the structural basis of substrate recognition, revealing a unique two-β-barrel domain that reads the free C-terminal carboxylate and enables phosphorylation-independent ubiquitination.\",\n      \"evidence\": \"X-ray crystallography of Skp1–FBXO31 with phospho-cyclin D1 peptide, biophysical binding, in vitro ubiquitination, mutagenesis\",\n      \"pmids\": [\"29279382\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of C-terminal-tail recognition to other substrates not yet tested at this stage\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed FBXO31 degrades Snail1 to suppress EMT, expanding its tumor-suppressive substrate repertoire.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, phospho/domain mutagenesis, EMT markers, xenograft\",\n      \"pmids\": [\"29117943\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Snail1 phosphorylation kinase not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the upstream control of FBXO31 abundance, showing APC/C and FBXO46 keep it low and that ATM-dependent phosphorylation switches that off after damage.\",\n      \"evidence\": \"Co-IP, D-box/Ser33/Ser278/RXXR mutagenesis, kinase inhibition, ubiquitination assays, demonstrated FBXO46–FBXO31 negative feedback\",\n      \"pmids\": [\"29343641\", \"30171069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How multiple competing degradation inputs are integrated quantitatively is unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected FBXO31 loss directly to genomic instability through failure to restrain cyclin A in G1, and linked it to TGF-β/fibrogenesis via Smad7.\",\n      \"evidence\": \"DNA combing, γH2AX foci, flow cytometry for cyclin A; Co-IP, co-localization and ubiquitination for Smad7\",\n      \"pmids\": [\"31413110\", \"31680332\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether cyclin A is a direct FBXO31 substrate not established\", \"Single labs\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established FBXO31 as a regulator of ERK/AKT signaling and ferroptosis, and revealed reciprocal transcriptional repression by its own substrate c-Myc.\",\n      \"evidence\": \"Co-IP, ubiquitylation assays, in vivo tumor models, pharmacological rescue (DUSP6 inhibitor), GPX4 rescue, ChIP for c-Myc at the FBXO31 promoter\",\n      \"pmids\": [\"34686346\", \"36269678\", \"34706096\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Context-dependence of tumor-suppressive vs other roles across tissues unresolved\", \"Some substrate findings from single labs\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reframed the β-barrel pocket as a quality-control reader of C-terminal amide-bearing damaged proteins, generalizing the structural recognition principle and explaining a neurodevelopmental disorder mutation.\",\n      \"evidence\": \"CRISPR screen, semi-synthetic chemical biology, structure-informed pocket analysis, in vitro ubiquitylation, mutagenesis, disease-mutation characterization; also OGT identified as a substrate via CRISPR screen\",\n      \"pmids\": [\"39880951\", \"39894887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous physiological CTAP substrate landscape not enumerated\", \"Mechanism by which the disorder mutation rewires specificity in vivo incompletely defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrated FBXO31 can be co-opted for targeted protein degradation and identified ABL2 and SIRT2 as further substrates, broadening both its biology and its therapeutic utility.\",\n      \"evidence\": \"Ternary complex and TPD assays with amidated recruiters, F-box mutagenesis, Co-IP, ubiquitination and ferroptosis assays, tumor models; m6A/YTHDF1 control of FBXO31 translation for SIRT2\",\n      \"pmids\": [\"41963263\", \"42170439\", \"38216561\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TPD work is biochemical/cellular, not validated in vivo\", \"Single labs\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full endogenous substrate spectrum of FBXO31 and how its expanding roles (checkpoint enforcement, protein-quality surveillance, ferroptosis, signaling) are coordinated within a given cell remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unbiased proteome-wide substrate map reported\", \"Determinants of tissue-specific substrate choice unknown\", \"Whether cyclin A and cofilin-1 are direct substrates not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 3, 4, 5, 6, 13, 17]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 5, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 3, 6, 12]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 13]}\n    ],\n    \"complexes\": [\"SCF (SKP1-CUL1-RBX1-FBXO31) E3 ubiquitin ligase\"],\n    \"partners\": [\"SKP1\", \"CUL1\", \"RBX1\", \"CCND1\", \"MDM2\", \"CDT1\", \"FOXM1\", \"FBXO46\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}