{"gene":"RFFL","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2004,"finding":"Rififylin (RFFL) contains an N-terminal FYVE-like domain that is critical for recruitment to recycling endocytic membranes and for inhibition of endocytic recycling from the endocytic recycling compartment (ERC) to the plasma membrane, in a manner independent of PtdIns(3)-kinase activity. The C-terminal RING finger domain is dispensable for this function. Overexpression induced condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region and delayed transferrin exit.","method":"Overexpression and deletion mutant analysis in HeLa cells; transferrin recycling assay; fluorescence microscopy","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion mutants with defined cellular phenotype, single lab, multiple orthogonal readouts","pmids":["15229288"],"is_preprint":false},{"year":2008,"finding":"RFFL (CARP-2) localizes to endocytic vesicles via its FYVE domain, where it interacts with the internalized TNF-receptor complex. RFFL ubiquitinates RIP (RIPK1), leading to its degradation and thereby acting as a negative regulator of TNF-induced NF-κB activation. Knockdown of CARP-2 stabilized TNFR1-associated polyubiquitinated RIP and enhanced NF-κB activation.","method":"siRNA knockdown; co-immunoprecipitation; ubiquitination assay; NF-κB reporter assay; endosomal localization by fluorescence microscopy","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, knockdown with defined signaling phenotype, subcellular localization linked to function, multiple orthogonal methods in single lab","pmids":["18450452"],"is_preprint":false},{"year":2018,"finding":"RFFL is an E3 ubiquitin ligase that directly and selectively recognizes unfolded ΔF508-CFTR through its disordered regions at the plasma membrane and mediates K63-linked poly-ubiquitination, leading to lysosomal degradation. This peripheral quality control mechanism is chaperone-independent. RFFL ablation enhanced functional cell-surface ΔF508-CFTR expression in the presence of folding correctors.","method":"Comprehensive siRNA screen; ubiquitination assay; linkage-specific ubiquitin analysis; siRNA knockdown with functional CFTR assay; direct binding assay","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide siRNA screen plus mechanistic validation with multiple orthogonal methods (ubiquitin linkage typing, binding assay, functional rescue), replicated in subsequent studies","pmids":["29503157"],"is_preprint":false},{"year":2018,"finding":"RFFL interacts with the core-glycosylated (ER-retained) form of hERG potassium channel. RFFL overexpression promotes polyubiquitination and proteasomal degradation of hERG in a RING domain-dependent manner, reducing IKr current. This degradation is partly mediated through the ERAD pathway, as RFFL interacts with VCP/p97 in vitro and a dominant-negative VCP partially abolishes RFFL-mediated hERG degradation.","method":"Co-immunoprecipitation; Western blotting; electrophysiology (patch clamp); RING domain mutant; dominant-negative VCP; adenoviral overexpression in cardiomyocytes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, RING domain mutagenesis, electrophysiology in native cardiomyocytes, in vitro VCP interaction, multiple orthogonal methods single lab","pmids":["30401747"],"is_preprint":false},{"year":2019,"finding":"RFFL regulates ubiquitylation of Rab11 effectors including EHD1, MICALL1, and class I Rab11-FIPs. A dominant-negative RFFL mutant induced clustering of ERCs and delayed endocytic cargo recycling without affecting lysosomal traffic. RFFL directly ubiquitylates these Rab11 effectors in vitro. Prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype. RFFL KO specifically reduced ubiquitylation of Rab11-FIP1 but had minimal effect on EHD1, MICALL1, and Rab11-FIP2, indicating redundancy with other E3 ligases.","method":"BioID interactome; dominant-negative mutant; RFFL knockout (KO); in vitro ubiquitylation assay; transferrin recycling assay; fluorescence microscopy","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro ubiquitylation reconstitution, BioID interactome, KO with defined phenotype, multiple orthogonal methods, single lab","pmids":["30659120"],"is_preprint":false},{"year":2011,"finding":"Overexpression of rififylin (Rffl) in rat cardiomyocytes delays endocytic recycling of transferrin, increases cardiomyocyte beat frequency, and is linked to shorter QT intervals and hypertension in a congenic rat model, consistent with RFFL's role as a regulator of endocytic recycling affecting cardiac function.","method":"Congenic rat model; transferrin recycling assay in isolated cardiomyocytes; ECG telemetry; mRNA/protein quantification","journal":"Hypertension (Dallas, Tex. : 1979)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional recycling assay in primary cardiomyocytes plus in vivo physiological readouts, single lab, no direct mechanistic dissection","pmids":["21357277"],"is_preprint":false},{"year":2012,"finding":"Increased Rffl expression in proximal tubules delays endosomal recycling of transferrin, increases intracellular polyubiquitinated proteins, and contributes to proteinuria, establishing a role for RFFL-mediated recycling regulation in renal proximal tubule function.","method":"Congenic rat model; transferrin recycling assay in isolated proximal tubules; transcriptome analysis; cell surface protein quantification; polyubiquitination assay","journal":"Frontiers in genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional recycling assay in primary tissue, multiple readouts, single lab, no direct mechanistic dissection","pmids":["22891072"],"is_preprint":false},{"year":2022,"finding":"RFFL associates with damaged mitochondria prior to PRKN/parkin recruitment and interacts with PRKN. RFFL KO substantially reduces stable PRKN recruitment to damaged mitochondria, indicating RFFL promotes PRKN-dependent mitophagy initiation from endosomes.","method":"RFFL knockout cells; co-immunoprecipitation; live confocal imaging; mitochondrial damage (CCCP) assay; PRKN recruitment quantification","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with defined phenotype, Co-IP, live imaging, single lab, mechanistic detail limited in abstract","pmids":["35373701"],"is_preprint":false},{"year":2023,"finding":"α-Tocopherol succinate (αTOS) directly binds to RFFL's substrate-binding region (confirmed by NMR) without affecting E3 enzymatic activity, thereby inhibiting RFFL-substrate interaction and preventing ΔF508-CFTR ubiquitination and elimination from the plasma membrane. The proapoptotic effect of αTOS is also dependent on RFFL expression.","method":"Chemical array screening; NMR binding assay; ubiquitination assay; cell surface CFTR functional assay; RFFL knockdown/expression controls","journal":"Biochemical pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural binding plus functional assay with mechanistic mutagenesis-equivalent (substrate-binding region identified), multiple orthogonal methods, single lab","pmids":["37543348"],"is_preprint":false},{"year":2023,"finding":"RFFL ubiquitinates DHX9, targeting it for proteasomal degradation. LINC01016 lncRNA competitively binds DHX9 to prevent RFFL from accessing DHX9, thereby stabilizing DHX9 protein levels and activating PI3K/AKT signaling in breast cancer cells.","method":"RNA pull-down; mass spectrometry; co-immunoprecipitation; ubiquitination assay; proteasome inhibitor assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, RNA pull-down, single lab, mechanistic detail about RFFL-DHX9 substrate relationship","pmids":["37550275"],"is_preprint":false},{"year":2024,"finding":"RFFL overexpression reduces Kv4.3 channel expression and Ito,f in a RING domain-dependent manner, while simultaneously increasing Kv1.4 expression and Ito,s, also in a RING domain-dependent manner. These opposing effects on cardiac potassium channel subunits affect action potential morphology and repolarization reserve.","method":"Adenoviral overexpression in adult rabbit ventricular cardiomyocytes; patch clamp electrophysiology; RING domain mutant; HEK293A transfection; Western blotting; computational cardiac model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RING domain mutant with electrophysiology and expression readouts, single lab, mechanistic link via ubiquitination implied but not directly shown for these channels","pmids":["38367666"],"is_preprint":false},{"year":2025,"finding":"RFFL is an E3 ubiquitin ligase for mitofusin 2 (MFN2). RFFL interacts endogenously with MFN2 and contributes to its ubiquitylation upon mitochondrial damage. Recombinant RFFL directly ubiquitylates MFN2 in vitro. RFFL KO cells exhibit enlarged (hyperfused) mitochondrial morphology. RFFL overexpression in a ligase-dependent manner reduces exogenous MFN1 and MFN2, but not DRP1, and perturbs lipid homeostasis. Co-expression of RFFL rescues hyperfused mitochondrial morphology caused by pathogenic MFN2 mutants (T206I and R364W) associated with Charcot-Marie-Tooth disease type 2A.","method":"In vitro ubiquitylation assay (reconstitution); RFFL KO (CRISPR); co-immunoprecipitation; electron microscopy; confocal imaging; RING domain mutant; exogenous protein expression","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of ubiquitylation, KO phenotype with EM, RING domain dependency, disease-mutant rescue, multiple orthogonal methods, single lab","pmids":["40444323"],"is_preprint":false},{"year":2025,"finding":"RFFL ubiquitinates and degrades JMJD6 and DNAJB11 via the proteasomal pathway, identified as endogenous substrates by label-free quantitative mass spectrometry proteomics comparing RFFL KO, RFFL rescue, and wild-type cells. RFFL also has a role in lipid metabolism.","method":"CRISPR/Cas9 KO; quantitative mass spectrometry proteomics (label-free); in vivo ubiquitination assay; proteasome inhibitor experiments; three cell line comparison","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO plus quantitative proteomics plus in vivo ubiquitination validation, single lab, orthogonal methods","pmids":["40568870"],"is_preprint":false},{"year":2026,"finding":"RFFL plays a critical role in peripheral quality control of full-length CFTR proteins restored by translational readthrough-inducing drugs (TRIDs) at nonsense mutation sites. RFFL knockdown markedly reduces CFTR ubiquitination, stabilizes mature CFTR at the plasma membrane, and significantly enhances functional rescue when TRIDs are combined with CFTR modulators.","method":"siRNA knockdown; ubiquitination assay; cell surface CFTR functional assay (Ussing chamber/patch clamp); Western blotting in epithelial cells","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined ubiquitination and functional readouts, single lab, extends prior RFFL-CFTR mechanism to new context","pmids":["41723327"],"is_preprint":false}],"current_model":"RFFL is a RING-finger and FYVE-like domain-containing E3 ubiquitin ligase that localizes to endosomes and, via its FYVE-like domain, is recruited to endocytic recycling compartments where it: (1) mediates K63-linked polyubiquitination of misfolded plasma membrane proteins (e.g., ΔF508-CFTR) for chaperone-independent lysosomal degradation; (2) ubiquitinates Rab11 effectors (EHD1, MICALL1, Rab11-FIPs) to regulate endocytic recycling compartment function; (3) ubiquitinates RIP/RIPK1 on endosomes to negatively regulate TNF-induced NF-κB signaling; (4) ubiquitinates hERG, Kv4.3 (reducing expression), and Kv1.4 (increasing expression) through ERAD-linked and direct mechanisms to modulate cardiac repolarization; (5) associates with damaged mitochondria to facilitate PRKN recruitment for mitophagy initiation; (6) ubiquitinates MFN2 to regulate mitochondrial morphology; and (7) degrades substrates JMJD6 and DNAJB11 via the proteasome."},"narrative":{"mechanistic_narrative":"RFFL (Rififylin/CARP-2) is a RING-finger E3 ubiquitin ligase that organizes endosomal protein quality control and membrane trafficking through an N-terminal FYVE-like domain that targets it to recycling endocytic membranes and a C-terminal RING domain that confers ligase activity [PMID:15229288, PMID:18450452]. At the endocytic recycling compartment it restrains cargo recycling to the plasma membrane: it directly ubiquitylates Rab11 effectors including EHD1, MICALL1, and Rab11-FIPs, and its prolonged engagement of these effectors clusters recycling tubulovesicular membranes and delays transferrin exit [PMID:15229288, PMID:30659120]. RFFL provides a chaperone-independent peripheral quality-control pathway by directly recognizing the disordered regions of unfolded ΔF508-CFTR at the plasma membrane and appending K63-linked polyubiquitin to route it for lysosomal degradation; loss of RFFL stabilizes surface CFTR and potentiates pharmacological rescue by folding correctors and translational readthrough drugs [PMID:29503157, PMID:41723327]. Through RING-dependent ubiquitination RFFL also degrades the internalized TNF receptor effector RIPK1 to dampen TNF-induced NF-κB signaling [PMID:18450452], promotes ERAD-coupled proteasomal turnover of the hERG channel in association with VCP/p97 [PMID:30401747], and exerts opposing RING-dependent control over cardiac Kv4.3 and Kv1.4 channel levels, shaping repolarization [PMID:38367666]. At damaged mitochondria RFFL acts upstream of mitophagy, interacting with PRKN/parkin to promote its stable recruitment and directly ubiquitylating mitofusin 2 to control mitochondrial morphology, with overexpression rescuing the hyperfused phenotype of Charcot-Marie-Tooth type 2A MFN2 mutants [PMID:35373701, PMID:40444323]. Additional proteasomal substrates identified by proteomics and biochemistry include DHX9, JMJD6, and DNAJB11, and RFFL influences lipid homeostasis [PMID:37550275, PMID:40444323, PMID:40568870].","teleology":[{"year":2004,"claim":"Established that RFFL is a recycling-compartment protein whose FYVE-like domain, not its RING domain, governs its membrane localization and its inhibitory effect on endocytic recycling.","evidence":"Overexpression and deletion-mutant analysis with transferrin recycling assays in HeLa cells","pmids":["15229288"],"confidence":"Medium","gaps":["Did not identify ubiquitination substrates at the ERC","RING-domain catalytic role left undefined","Mechanism of recycling inhibition not resolved to specific effectors"]},{"year":2008,"claim":"Defined a signaling function for endosomal RFFL by showing it ubiquitinates and degrades RIPK1 within the internalized TNF-receptor complex to negatively regulate NF-κB.","evidence":"siRNA knockdown, reciprocal Co-IP, ubiquitination and NF-κB reporter assays with endosomal imaging","pmids":["18450452"],"confidence":"High","gaps":["Ubiquitin linkage type on RIPK1 not specified","How receptor internalization recruits RFFL not detailed"]},{"year":2011,"claim":"Connected RFFL-driven recycling delay to organ physiology, linking cardiomyocyte Rffl overexpression to altered beat frequency, QT interval, and hypertension.","evidence":"Congenic rat model with transferrin recycling assays and ECG telemetry","pmids":["21357277"],"confidence":"Medium","gaps":["No direct ubiquitination substrate identified for the cardiac phenotype","Correlative physiology without molecular dissection"]},{"year":2012,"claim":"Extended RFFL recycling control to renal proximal tubules, where increased expression delays endosomal recycling and contributes to proteinuria.","evidence":"Congenic rat model with primary proximal tubule recycling and polyubiquitination assays","pmids":["22891072"],"confidence":"Medium","gaps":["Specific surface cargo affected not pinned down","Direct substrate-ligase relationship not established"]},{"year":2018,"claim":"Identified RFFL as the E3 ligase mediating chaperone-independent peripheral quality control of ΔF508-CFTR via direct recognition of disordered regions and K63-linked polyubiquitination.","evidence":"Genome-wide siRNA screen, linkage-specific ubiquitin analysis, direct binding and functional CFTR assays","pmids":["29503157"],"confidence":"High","gaps":["Structure of the RFFL substrate-binding region not solved here","Whether the mechanism generalizes to other misfolded surface proteins untested"]},{"year":2018,"claim":"Showed RFFL couples to ERAD by degrading the ER-retained hERG channel in a RING- and VCP/p97-dependent manner, implicating it in cardiac repolarization.","evidence":"Reciprocal Co-IP, RING-domain mutant, dominant-negative VCP, and patch-clamp electrophysiology in cardiomyocytes","pmids":["30401747"],"confidence":"High","gaps":["Direct ubiquitin transfer to hERG in vitro not reconstituted","ERAD versus peripheral pathway contributions not fully separated"]},{"year":2019,"claim":"Resolved the molecular basis of RFFL's recycling phenotype by showing it directly ubiquitylates Rab11 effectors (EHD1, MICALL1, Rab11-FIPs), with redundancy among E3 ligases revealed by KO.","evidence":"BioID interactome, in vitro ubiquitylation reconstitution, RFFL KO, and dominant-negative mutant with recycling assays","pmids":["30659120"],"confidence":"High","gaps":["Ubiquitin linkage and fate of effectors not fully defined","Identity of redundant E3 ligases unknown"]},{"year":2022,"claim":"Placed RFFL upstream of mitophagy by showing it associates with damaged mitochondria and interacts with PRKN to promote stable parkin recruitment.","evidence":"RFFL KO cells, Co-IP, live confocal imaging, and CCCP mitochondrial damage assays","pmids":["35373701"],"confidence":"Medium","gaps":["Substrate ubiquitinated to recruit PRKN not identified","Whether endosomal localization is required for mitochondrial function unclear"]},{"year":2023,"claim":"Demonstrated that RFFL's substrate-binding region is pharmacologically targetable, as α-tocopherol succinate binds it and blocks ΔF508-CFTR ubiquitination without affecting catalysis.","evidence":"Chemical array screening with NMR binding, ubiquitination assays, and functional CFTR rescue","pmids":["37543348"],"confidence":"High","gaps":["High-resolution structure of the binding pocket not determined","Selectivity across RFFL substrates not mapped"]},{"year":2023,"claim":"Expanded the RFFL substrate repertoire to DHX9, whose RFFL-mediated proteasomal degradation is antagonized by LINC01016 to control PI3K/AKT signaling in breast cancer.","evidence":"RNA pull-down, mass spectrometry, Co-IP, and ubiquitination/proteasome inhibitor assays","pmids":["37550275"],"confidence":"Medium","gaps":["Direct in vitro ubiquitylation of DHX9 not shown","Ubiquitin linkage type unspecified"]},{"year":2024,"claim":"Showed RFFL exerts divergent RING-dependent control over cardiac potassium channels, lowering Kv4.3 while raising Kv1.4, thereby tuning repolarization reserve.","evidence":"Adenoviral overexpression in rabbit cardiomyocytes, patch clamp, RING-domain mutant, and computational modeling","pmids":["38367666"],"confidence":"Medium","gaps":["Direct ubiquitination of Kv4.3/Kv1.4 not demonstrated","Mechanism producing opposite effects on the two channels unexplained"]},{"year":2025,"claim":"Established RFFL as a direct E3 ligase for MFN2 controlling mitochondrial morphology, with disease relevance shown by rescue of CMT2A MFN2 mutant hyperfusion.","evidence":"In vitro ubiquitylation reconstitution, CRISPR KO with EM, Co-IP, RING-domain mutant, and disease-mutant rescue","pmids":["40444323"],"confidence":"High","gaps":["Whether RFFL acts on MFN2 at endosome-mitochondria contacts unresolved","Link between MFN turnover and observed lipid homeostasis defects not mechanistically detailed"]},{"year":2025,"claim":"Used unbiased proteomics to identify JMJD6 and DNAJB11 as endogenous proteasomal RFFL substrates and reinforced a role in lipid metabolism.","evidence":"CRISPR KO with label-free quantitative proteomics, in vivo ubiquitination, and proteasome inhibitor experiments across three cell lines","pmids":["40568870"],"confidence":"Medium","gaps":["Direct binding/ubiquitylation not reconstituted for each substrate","Biological consequences of JMJD6/DNAJB11 degradation not defined"]},{"year":2026,"claim":"Showed RFFL controls peripheral quality control of full-length CFTR restored by readthrough drugs, with knockdown enhancing functional rescue.","evidence":"siRNA knockdown with ubiquitination and Ussing chamber/patch clamp CFTR assays in epithelial cells","pmids":["41723327"],"confidence":"Medium","gaps":["Whether RFFL inhibition is safe/selective in vivo untested","Extent of overlap with the ΔF508 mechanism not fully delineated"]},{"year":null,"claim":"How RFFL's single FYVE-like/RING architecture coordinates its diverse roles across recycling endosomes, the plasma membrane, and damaged mitochondria, and what determines its substrate selectivity and ubiquitin-linkage choice, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate recognition","Spatial regulation of RFFL recruitment to distinct organelles unexplained","Linkage-type determinants across substrates undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,3,4,11,12]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[2,4,11]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,13]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[7,11]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,12,13]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[7]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,4]}],"complexes":[],"partners":["RIPK1","EHD1","MICALL1","VCP","PRKN","MFN2","DHX9","JMJD6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8WZ73","full_name":"E3 ubiquitin-protein ligase rififylin","aliases":["Caspase regulator CARP2","Caspases-8 and -10-associated RING finger protein 2","CARP-2","FYVE-RING finger protein Sakura","Fring","RING finger and FYVE-like domain-containing protein 1","RING finger protein 189","RING finger protein 34-like","RING-type E3 ubiquitin transferase rififylin"],"length_aa":363,"mass_kda":40.5,"function":"E3 ubiquitin-protein ligase that regulates several biological processes through the ubiquitin-mediated proteasomal degradation of various target proteins. Mediates 'Lys-48'-linked polyubiquitination of PRR5L and its subsequent proteasomal degradation thereby indirectly regulating cell migration through the mTORC2 complex. Ubiquitinates the caspases CASP8 and CASP10, promoting their proteasomal degradation, to negatively regulate cell death downstream of death domain receptors in the extrinsic pathway of apoptosis. Negatively regulates the tumor necrosis factor-mediated signaling pathway through targeting of RIPK1 to ubiquitin-mediated proteasomal degradation. Negatively regulates p53/TP53 through its direct ubiquitination and targeting to proteasomal degradation. Indirectly, may also negatively regulate p53/TP53 through ubiquitination and degradation of SFN. May also play a role in endocytic recycling","subcellular_location":"Cytoplasm, cytosol; Cell membrane; Recycling endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q8WZ73/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RFFL","classification":"Not Classified","n_dependent_lines":108,"n_total_lines":1208,"dependency_fraction":0.08940397350993377},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RFFL","total_profiled":1310},"omim":[{"mim_id":"609735","title":"RING FINGER AND FYVE-LIKE DOMAIN CONTAINING 1; RFFL","url":"https://www.omim.org/entry/609735"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RFFL"},"hgnc":{"alias_symbol":["rififylin","fring","RNF189","RNF34L","CARP2","CARP-2"],"prev_symbol":[]},"alphafold":{"accession":"Q8WZ73","domains":[{"cath_id":"1.10.720.140","chopping":"56-135","consensus_level":"high","plddt":94.5765,"start":56,"end":135},{"cath_id":"3.30.40.10","chopping":"311-358","consensus_level":"high","plddt":92.4281,"start":311,"end":358},{"cath_id":"1.10.720","chopping":"240-296","consensus_level":"high","plddt":92.5388,"start":240,"end":296}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WZ73","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WZ73-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WZ73-F1-predicted_aligned_error_v6.png","plddt_mean":71.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RFFL","jax_strain_url":"https://www.jax.org/strain/search?query=RFFL"},"sequence":{"accession":"Q8WZ73","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WZ73.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WZ73/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WZ73"}},"corpus_meta":[{"pmid":"29503157","id":"PMC_29503157","title":"Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase.","date":"2018","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/29503157","citation_count":64,"is_preprint":false},{"pmid":"18450452","id":"PMC_18450452","title":"CARP-2 is an endosome-associated ubiquitin ligase for RIP and regulates TNF-induced NF-kappaB activation.","date":"2008","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/18450452","citation_count":57,"is_preprint":false},{"pmid":"21357277","id":"PMC_21357277","title":"Augmented rififylin is a risk factor linked to aberrant cardiomyocyte function, short-QT interval and hypertension.","date":"2011","source":"Hypertension (Dallas, Tex. : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/21357277","citation_count":32,"is_preprint":false},{"pmid":"15229288","id":"PMC_15229288","title":"Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17981469","citation_count":8,"is_preprint":false},{"pmid":"39047427","id":"PMC_39047427","title":"Enhanced CFTR modulator efficacy in ΔF508 CFTR mouse organoids by ablation of RFFL ubiquitin ligase.","date":"2024","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/39047427","citation_count":6,"is_preprint":false},{"pmid":"40444323","id":"PMC_40444323","title":"Endosomal RFFL ubiquitin ligase regulates mitochondrial morphology by targeting mitofusin 2.","date":"2025","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/40444323","citation_count":3,"is_preprint":false},{"pmid":"40578289","id":"PMC_40578289","title":"RFFL inhibition increases cell surface CFTR and reduces IL-8 production in airway epithelial cells upon COPD-associated environmental pathogen exposure.","date":"2025","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/40578289","citation_count":2,"is_preprint":false},{"pmid":"38367666","id":"PMC_38367666","title":"E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38367666","citation_count":2,"is_preprint":false},{"pmid":"40568870","id":"PMC_40568870","title":"Quantitative Proteomic Analysis Reveals JMJD6 and DNAJB11 as Endogenous Substrates of E3 Ligase RFFL.","date":"2025","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/40568870","citation_count":1,"is_preprint":false},{"pmid":"41323797","id":"PMC_41323797","title":"Antisense oligonucleotide targeting the E3 ligase RFFL potentiates CFTR modulator efficacy in CF primary bronchial epithelial cells.","date":"2025","source":"Molecular therapy. Nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/41323797","citation_count":1,"is_preprint":false},{"pmid":"41723327","id":"PMC_41723327","title":"RFFL-mediated protein quality control limits functional rescue of TRID-CFTR modulator combination therapy for cystic fibrosis nonsense mutations.","date":"2026","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/41723327","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11966,"output_tokens":4064,"usd":0.048429,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11914,"output_tokens":4677,"usd":0.088248,"stage2_stop_reason":"end_turn"},"total_usd":0.136677,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"Rififylin (RFFL) contains an N-terminal FYVE-like domain that is critical for recruitment to recycling endocytic membranes and for inhibition of endocytic recycling from the endocytic recycling compartment (ERC) to the plasma membrane, in a manner independent of PtdIns(3)-kinase activity. The C-terminal RING finger domain is dispensable for this function. Overexpression induced condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region and delayed transferrin exit.\",\n      \"method\": \"Overexpression and deletion mutant analysis in HeLa cells; transferrin recycling assay; fluorescence microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mutants with defined cellular phenotype, single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"15229288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RFFL (CARP-2) localizes to endocytic vesicles via its FYVE domain, where it interacts with the internalized TNF-receptor complex. RFFL ubiquitinates RIP (RIPK1), leading to its degradation and thereby acting as a negative regulator of TNF-induced NF-κB activation. Knockdown of CARP-2 stabilized TNFR1-associated polyubiquitinated RIP and enhanced NF-κB activation.\",\n      \"method\": \"siRNA knockdown; co-immunoprecipitation; ubiquitination assay; NF-κB reporter assay; endosomal localization by fluorescence microscopy\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, knockdown with defined signaling phenotype, subcellular localization linked to function, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"18450452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RFFL is an E3 ubiquitin ligase that directly and selectively recognizes unfolded ΔF508-CFTR through its disordered regions at the plasma membrane and mediates K63-linked poly-ubiquitination, leading to lysosomal degradation. This peripheral quality control mechanism is chaperone-independent. RFFL ablation enhanced functional cell-surface ΔF508-CFTR expression in the presence of folding correctors.\",\n      \"method\": \"Comprehensive siRNA screen; ubiquitination assay; linkage-specific ubiquitin analysis; siRNA knockdown with functional CFTR assay; direct binding assay\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide siRNA screen plus mechanistic validation with multiple orthogonal methods (ubiquitin linkage typing, binding assay, functional rescue), replicated in subsequent studies\",\n      \"pmids\": [\"29503157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RFFL interacts with the core-glycosylated (ER-retained) form of hERG potassium channel. RFFL overexpression promotes polyubiquitination and proteasomal degradation of hERG in a RING domain-dependent manner, reducing IKr current. This degradation is partly mediated through the ERAD pathway, as RFFL interacts with VCP/p97 in vitro and a dominant-negative VCP partially abolishes RFFL-mediated hERG degradation.\",\n      \"method\": \"Co-immunoprecipitation; Western blotting; electrophysiology (patch clamp); RING domain mutant; dominant-negative VCP; adenoviral overexpression in cardiomyocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, RING domain mutagenesis, electrophysiology in native cardiomyocytes, in vitro VCP interaction, multiple orthogonal methods single lab\",\n      \"pmids\": [\"30401747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RFFL regulates ubiquitylation of Rab11 effectors including EHD1, MICALL1, and class I Rab11-FIPs. A dominant-negative RFFL mutant induced clustering of ERCs and delayed endocytic cargo recycling without affecting lysosomal traffic. RFFL directly ubiquitylates these Rab11 effectors in vitro. Prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype. RFFL KO specifically reduced ubiquitylation of Rab11-FIP1 but had minimal effect on EHD1, MICALL1, and Rab11-FIP2, indicating redundancy with other E3 ligases.\",\n      \"method\": \"BioID interactome; dominant-negative mutant; RFFL knockout (KO); in vitro ubiquitylation assay; transferrin recycling assay; fluorescence microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro ubiquitylation reconstitution, BioID interactome, KO with defined phenotype, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"30659120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Overexpression of rififylin (Rffl) in rat cardiomyocytes delays endocytic recycling of transferrin, increases cardiomyocyte beat frequency, and is linked to shorter QT intervals and hypertension in a congenic rat model, consistent with RFFL's role as a regulator of endocytic recycling affecting cardiac function.\",\n      \"method\": \"Congenic rat model; transferrin recycling assay in isolated cardiomyocytes; ECG telemetry; mRNA/protein quantification\",\n      \"journal\": \"Hypertension (Dallas, Tex. : 1979)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional recycling assay in primary cardiomyocytes plus in vivo physiological readouts, single lab, no direct mechanistic dissection\",\n      \"pmids\": [\"21357277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Increased Rffl expression in proximal tubules delays endosomal recycling of transferrin, increases intracellular polyubiquitinated proteins, and contributes to proteinuria, establishing a role for RFFL-mediated recycling regulation in renal proximal tubule function.\",\n      \"method\": \"Congenic rat model; transferrin recycling assay in isolated proximal tubules; transcriptome analysis; cell surface protein quantification; polyubiquitination assay\",\n      \"journal\": \"Frontiers in genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional recycling assay in primary tissue, multiple readouts, single lab, no direct mechanistic dissection\",\n      \"pmids\": [\"22891072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RFFL associates with damaged mitochondria prior to PRKN/parkin recruitment and interacts with PRKN. RFFL KO substantially reduces stable PRKN recruitment to damaged mitochondria, indicating RFFL promotes PRKN-dependent mitophagy initiation from endosomes.\",\n      \"method\": \"RFFL knockout cells; co-immunoprecipitation; live confocal imaging; mitochondrial damage (CCCP) assay; PRKN recruitment quantification\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with defined phenotype, Co-IP, live imaging, single lab, mechanistic detail limited in abstract\",\n      \"pmids\": [\"35373701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"α-Tocopherol succinate (αTOS) directly binds to RFFL's substrate-binding region (confirmed by NMR) without affecting E3 enzymatic activity, thereby inhibiting RFFL-substrate interaction and preventing ΔF508-CFTR ubiquitination and elimination from the plasma membrane. The proapoptotic effect of αTOS is also dependent on RFFL expression.\",\n      \"method\": \"Chemical array screening; NMR binding assay; ubiquitination assay; cell surface CFTR functional assay; RFFL knockdown/expression controls\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural binding plus functional assay with mechanistic mutagenesis-equivalent (substrate-binding region identified), multiple orthogonal methods, single lab\",\n      \"pmids\": [\"37543348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RFFL ubiquitinates DHX9, targeting it for proteasomal degradation. LINC01016 lncRNA competitively binds DHX9 to prevent RFFL from accessing DHX9, thereby stabilizing DHX9 protein levels and activating PI3K/AKT signaling in breast cancer cells.\",\n      \"method\": \"RNA pull-down; mass spectrometry; co-immunoprecipitation; ubiquitination assay; proteasome inhibitor assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, RNA pull-down, single lab, mechanistic detail about RFFL-DHX9 substrate relationship\",\n      \"pmids\": [\"37550275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RFFL overexpression reduces Kv4.3 channel expression and Ito,f in a RING domain-dependent manner, while simultaneously increasing Kv1.4 expression and Ito,s, also in a RING domain-dependent manner. These opposing effects on cardiac potassium channel subunits affect action potential morphology and repolarization reserve.\",\n      \"method\": \"Adenoviral overexpression in adult rabbit ventricular cardiomyocytes; patch clamp electrophysiology; RING domain mutant; HEK293A transfection; Western blotting; computational cardiac model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RING domain mutant with electrophysiology and expression readouts, single lab, mechanistic link via ubiquitination implied but not directly shown for these channels\",\n      \"pmids\": [\"38367666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFFL is an E3 ubiquitin ligase for mitofusin 2 (MFN2). RFFL interacts endogenously with MFN2 and contributes to its ubiquitylation upon mitochondrial damage. Recombinant RFFL directly ubiquitylates MFN2 in vitro. RFFL KO cells exhibit enlarged (hyperfused) mitochondrial morphology. RFFL overexpression in a ligase-dependent manner reduces exogenous MFN1 and MFN2, but not DRP1, and perturbs lipid homeostasis. Co-expression of RFFL rescues hyperfused mitochondrial morphology caused by pathogenic MFN2 mutants (T206I and R364W) associated with Charcot-Marie-Tooth disease type 2A.\",\n      \"method\": \"In vitro ubiquitylation assay (reconstitution); RFFL KO (CRISPR); co-immunoprecipitation; electron microscopy; confocal imaging; RING domain mutant; exogenous protein expression\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of ubiquitylation, KO phenotype with EM, RING domain dependency, disease-mutant rescue, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"40444323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFFL ubiquitinates and degrades JMJD6 and DNAJB11 via the proteasomal pathway, identified as endogenous substrates by label-free quantitative mass spectrometry proteomics comparing RFFL KO, RFFL rescue, and wild-type cells. RFFL also has a role in lipid metabolism.\",\n      \"method\": \"CRISPR/Cas9 KO; quantitative mass spectrometry proteomics (label-free); in vivo ubiquitination assay; proteasome inhibitor experiments; three cell line comparison\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO plus quantitative proteomics plus in vivo ubiquitination validation, single lab, orthogonal methods\",\n      \"pmids\": [\"40568870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RFFL plays a critical role in peripheral quality control of full-length CFTR proteins restored by translational readthrough-inducing drugs (TRIDs) at nonsense mutation sites. RFFL knockdown markedly reduces CFTR ubiquitination, stabilizes mature CFTR at the plasma membrane, and significantly enhances functional rescue when TRIDs are combined with CFTR modulators.\",\n      \"method\": \"siRNA knockdown; ubiquitination assay; cell surface CFTR functional assay (Ussing chamber/patch clamp); Western blotting in epithelial cells\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined ubiquitination and functional readouts, single lab, extends prior RFFL-CFTR mechanism to new context\",\n      \"pmids\": [\"41723327\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RFFL is a RING-finger and FYVE-like domain-containing E3 ubiquitin ligase that localizes to endosomes and, via its FYVE-like domain, is recruited to endocytic recycling compartments where it: (1) mediates K63-linked polyubiquitination of misfolded plasma membrane proteins (e.g., ΔF508-CFTR) for chaperone-independent lysosomal degradation; (2) ubiquitinates Rab11 effectors (EHD1, MICALL1, Rab11-FIPs) to regulate endocytic recycling compartment function; (3) ubiquitinates RIP/RIPK1 on endosomes to negatively regulate TNF-induced NF-κB signaling; (4) ubiquitinates hERG, Kv4.3 (reducing expression), and Kv1.4 (increasing expression) through ERAD-linked and direct mechanisms to modulate cardiac repolarization; (5) associates with damaged mitochondria to facilitate PRKN recruitment for mitophagy initiation; (6) ubiquitinates MFN2 to regulate mitochondrial morphology; and (7) degrades substrates JMJD6 and DNAJB11 via the proteasome.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RFFL (Rififylin/CARP-2) is a RING-finger E3 ubiquitin ligase that organizes endosomal protein quality control and membrane trafficking through an N-terminal FYVE-like domain that targets it to recycling endocytic membranes and a C-terminal RING domain that confers ligase activity [#0, #1]. At the endocytic recycling compartment it restrains cargo recycling to the plasma membrane: it directly ubiquitylates Rab11 effectors including EHD1, MICALL1, and Rab11-FIPs, and its prolonged engagement of these effectors clusters recycling tubulovesicular membranes and delays transferrin exit [#0, #4]. RFFL provides a chaperone-independent peripheral quality-control pathway by directly recognizing the disordered regions of unfolded \\u0394F508-CFTR at the plasma membrane and appending K63-linked polyubiquitin to route it for lysosomal degradation; loss of RFFL stabilizes surface CFTR and potentiates pharmacological rescue by folding correctors and translational readthrough drugs [#2, #13]. Through RING-dependent ubiquitination RFFL also degrades the internalized TNF receptor effector RIPK1 to dampen TNF-induced NF-\\u03baB signaling [#1], promotes ERAD-coupled proteasomal turnover of the hERG channel in association with VCP/p97 [#3], and exerts opposing RING-dependent control over cardiac Kv4.3 and Kv1.4 channel levels, shaping repolarization [#10]. At damaged mitochondria RFFL acts upstream of mitophagy, interacting with PRKN/parkin to promote its stable recruitment and directly ubiquitylating mitofusin 2 to control mitochondrial morphology, with overexpression rescuing the hyperfused phenotype of Charcot-Marie-Tooth type 2A MFN2 mutants [#7, #11]. Additional proteasomal substrates identified by proteomics and biochemistry include DHX9, JMJD6, and DNAJB11, and RFFL influences lipid homeostasis [#9, #11, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that RFFL is a recycling-compartment protein whose FYVE-like domain, not its RING domain, governs its membrane localization and its inhibitory effect on endocytic recycling.\",\n      \"evidence\": \"Overexpression and deletion-mutant analysis with transferrin recycling assays in HeLa cells\",\n      \"pmids\": [\"15229288\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify ubiquitination substrates at the ERC\", \"RING-domain catalytic role left undefined\", \"Mechanism of recycling inhibition not resolved to specific effectors\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined a signaling function for endosomal RFFL by showing it ubiquitinates and degrades RIPK1 within the internalized TNF-receptor complex to negatively regulate NF-\\u03baB.\",\n      \"evidence\": \"siRNA knockdown, reciprocal Co-IP, ubiquitination and NF-\\u03baB reporter assays with endosomal imaging\",\n      \"pmids\": [\"18450452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin linkage type on RIPK1 not specified\", \"How receptor internalization recruits RFFL not detailed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected RFFL-driven recycling delay to organ physiology, linking cardiomyocyte Rffl overexpression to altered beat frequency, QT interval, and hypertension.\",\n      \"evidence\": \"Congenic rat model with transferrin recycling assays and ECG telemetry\",\n      \"pmids\": [\"21357277\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct ubiquitination substrate identified for the cardiac phenotype\", \"Correlative physiology without molecular dissection\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended RFFL recycling control to renal proximal tubules, where increased expression delays endosomal recycling and contributes to proteinuria.\",\n      \"evidence\": \"Congenic rat model with primary proximal tubule recycling and polyubiquitination assays\",\n      \"pmids\": [\"22891072\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific surface cargo affected not pinned down\", \"Direct substrate-ligase relationship not established\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified RFFL as the E3 ligase mediating chaperone-independent peripheral quality control of \\u0394F508-CFTR via direct recognition of disordered regions and K63-linked polyubiquitination.\",\n      \"evidence\": \"Genome-wide siRNA screen, linkage-specific ubiquitin analysis, direct binding and functional CFTR assays\",\n      \"pmids\": [\"29503157\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the RFFL substrate-binding region not solved here\", \"Whether the mechanism generalizes to other misfolded surface proteins untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed RFFL couples to ERAD by degrading the ER-retained hERG channel in a RING- and VCP/p97-dependent manner, implicating it in cardiac repolarization.\",\n      \"evidence\": \"Reciprocal Co-IP, RING-domain mutant, dominant-negative VCP, and patch-clamp electrophysiology in cardiomyocytes\",\n      \"pmids\": [\"30401747\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitin transfer to hERG in vitro not reconstituted\", \"ERAD versus peripheral pathway contributions not fully separated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved the molecular basis of RFFL's recycling phenotype by showing it directly ubiquitylates Rab11 effectors (EHD1, MICALL1, Rab11-FIPs), with redundancy among E3 ligases revealed by KO.\",\n      \"evidence\": \"BioID interactome, in vitro ubiquitylation reconstitution, RFFL KO, and dominant-negative mutant with recycling assays\",\n      \"pmids\": [\"30659120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin linkage and fate of effectors not fully defined\", \"Identity of redundant E3 ligases unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed RFFL upstream of mitophagy by showing it associates with damaged mitochondria and interacts with PRKN to promote stable parkin recruitment.\",\n      \"evidence\": \"RFFL KO cells, Co-IP, live confocal imaging, and CCCP mitochondrial damage assays\",\n      \"pmids\": [\"35373701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate ubiquitinated to recruit PRKN not identified\", \"Whether endosomal localization is required for mitochondrial function unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that RFFL's substrate-binding region is pharmacologically targetable, as \\u03b1-tocopherol succinate binds it and blocks \\u0394F508-CFTR ubiquitination without affecting catalysis.\",\n      \"evidence\": \"Chemical array screening with NMR binding, ubiquitination assays, and functional CFTR rescue\",\n      \"pmids\": [\"37543348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the binding pocket not determined\", \"Selectivity across RFFL substrates not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Expanded the RFFL substrate repertoire to DHX9, whose RFFL-mediated proteasomal degradation is antagonized by LINC01016 to control PI3K/AKT signaling in breast cancer.\",\n      \"evidence\": \"RNA pull-down, mass spectrometry, Co-IP, and ubiquitination/proteasome inhibitor assays\",\n      \"pmids\": [\"37550275\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro ubiquitylation of DHX9 not shown\", \"Ubiquitin linkage type unspecified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed RFFL exerts divergent RING-dependent control over cardiac potassium channels, lowering Kv4.3 while raising Kv1.4, thereby tuning repolarization reserve.\",\n      \"evidence\": \"Adenoviral overexpression in rabbit cardiomyocytes, patch clamp, RING-domain mutant, and computational modeling\",\n      \"pmids\": [\"38367666\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of Kv4.3/Kv1.4 not demonstrated\", \"Mechanism producing opposite effects on the two channels unexplained\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established RFFL as a direct E3 ligase for MFN2 controlling mitochondrial morphology, with disease relevance shown by rescue of CMT2A MFN2 mutant hyperfusion.\",\n      \"evidence\": \"In vitro ubiquitylation reconstitution, CRISPR KO with EM, Co-IP, RING-domain mutant, and disease-mutant rescue\",\n      \"pmids\": [\"40444323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RFFL acts on MFN2 at endosome-mitochondria contacts unresolved\", \"Link between MFN turnover and observed lipid homeostasis defects not mechanistically detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Used unbiased proteomics to identify JMJD6 and DNAJB11 as endogenous proteasomal RFFL substrates and reinforced a role in lipid metabolism.\",\n      \"evidence\": \"CRISPR KO with label-free quantitative proteomics, in vivo ubiquitination, and proteasome inhibitor experiments across three cell lines\",\n      \"pmids\": [\"40568870\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding/ubiquitylation not reconstituted for each substrate\", \"Biological consequences of JMJD6/DNAJB11 degradation not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed RFFL controls peripheral quality control of full-length CFTR restored by readthrough drugs, with knockdown enhancing functional rescue.\",\n      \"evidence\": \"siRNA knockdown with ubiquitination and Ussing chamber/patch clamp CFTR assays in epithelial cells\",\n      \"pmids\": [\"41723327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RFFL inhibition is safe/selective in vivo untested\", \"Extent of overlap with the \\u0394F508 mechanism not fully delineated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RFFL's single FYVE-like/RING architecture coordinates its diverse roles across recycling endosomes, the plasma membrane, and damaged mitochondria, and what determines its substrate selectivity and ubiquitin-linkage choice, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate recognition\", \"Spatial regulation of RFFL recruitment to distinct organelles unexplained\", \"Linkage-type determinants across substrates undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0061630\", \"supporting_discovery_ids\": [1, 2, 3, 4, 11]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 3, 4, 11, 12]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [2, 4, 11]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 13]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [7, 11]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 12, 13]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RIPK1\", \"EHD1\", \"MICALL1\", \"VCP\", \"PRKN\", \"MFN2\", \"DHX9\", \"JMJD6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}