{"gene":"UBR1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2021,"finding":"Cryo-EM structures of Ubr1 in complex with Ubc2, ubiquitin, and N-degron peptides revealed the structural basis for both initiation and elongation steps of ubiquitination. Key structural elements including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1 were identified and characterized by mutagenesis, explaining how Ubr1 mediates linkage-specific polyubiquitin chain formation through a single E2 enzyme.","method":"Cryo-EM structure determination with chemical mimics of reaction intermediates, plus mutagenesis of key structural elements","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures with chemical biology mimics of reaction intermediates, mutagenesis validation, multiple orthogonal methods in single rigorous study","pmids":["34789879"],"is_preprint":false},{"year":2008,"finding":"The type-1 and type-2 substrate-binding sites of UBR1 (recognizing basic and bulky hydrophobic N-terminal residues, respectively) reside within the first ~700 residues of the 1,950-residue protein. Selective inactivation of each site by distinct mutations was demonstrated, and binding affinities (Kd ~1 μM) were measured by fluorescence polarization and surface plasmon resonance. A third substrate-binding site targets the internal degron of CUP9, and CUP9 ubiquitylation dependent on dipeptide binding to type-1/2 sites was reconstituted in a fully defined in vitro system.","method":"Genetic screen for loss-of-function mutations, fluorescence polarization, surface plasmon resonance, in vitro reconstitution with purified components","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus biophysical binding measurements plus mutagenesis, multiple orthogonal methods","pmids":["18566452"],"is_preprint":false},{"year":2010,"finding":"The RING-type Ubr1 E3 and the HECT-type Ufd4 E3 physically interact and form a complex that is more processive in polyubiquitylation than Ubr1 alone, producing longer substrate-linked polyubiquitin chains. Ubr1 can also recognize the N-terminal ubiquitin moiety of UFD substrates, and can enhance polyubiquitylation of UFD substrates as part of the Ubr1-Ufd4 complex.","method":"Co-immunoprecipitation, in vitro ubiquitylation assays, genetic epistasis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal physical interaction plus in vitro functional reconstitution plus genetic epistasis in single study","pmids":["21076411"],"is_preprint":false},{"year":2009,"finding":"Ubr1 mediates chaperone-dependent ubiquitination of misfolded cytoplasmic proteins for proteasomal degradation in yeast, acting in parallel with nuclear San1. This cytoplasmic quality control function of Ubr1 is distinct from its N-end rule role and depends on chaperone assistance.","method":"Genetic epistasis (double mutant analysis), biochemical ubiquitination assays, phenotypic analysis of proteotoxic stress","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (biochemical + genetic), replicated across related studies","pmids":["20080635"],"is_preprint":false},{"year":2008,"finding":"Ubr1 is the E3 ligase responsible for targeting misfolded cytoplasmic proteins to proteasomal degradation in yeast, establishing its role as a cytoplasmic protein quality control ubiquitin ligase independent of N-end rule activity.","method":"Genetic deletion analysis, ubiquitination assay with misfolded reporter substrates","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with defined substrate phenotype, single lab","pmids":["19041308"],"is_preprint":false},{"year":2010,"finding":"Saccharomyces cerevisiae Ubr1 directly binds denatured (but not native) luciferase and catalyzes its ubiquitinylation in a purified system; Hsp70 stimulates polyubiquitinylation of the denatured substrate. Loss of Ubr1 and Ubr2 suppresses growth arrest caused by chaperone mutation, indicating these E3s compete with chaperones for misfolded substrates.","method":"In vitro ubiquitylation assay with purified components, genetic epistasis (suppressor analysis of chaperone mutants)","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified Ubr1 and denatured substrate plus genetic epistasis, single lab but multiple orthogonal methods","pmids":["20462952"],"is_preprint":false},{"year":2020,"finding":"Hsp70 ATP hydrolysis is directly required for Ubr1-mediated ubiquitination of misfolded substrates. Using a bead-based in vitro assay with covalently immobilized misfolded protein, only Hsp70 (with its ATPase cycle and cochaperones including nucleotide exchange factor Sse1 and J-proteins) was required for Ubr1-mediated ubiquitination.","method":"In vitro bead-based ubiquitination assay with immobilized misfolded substrate, ATPase cycle mutant analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with immobilized substrate to eliminate indirect effects, multiple cochaperone mutants tested, single lab","pmids":["32966159"],"is_preprint":false},{"year":2013,"finding":"The Type II Hsp40 Sis1 cooperates sequentially with Hsp70 and Ubr1 to target misfolded cytosolic proteins for proteasomal degradation. Sis1 is required for proteasomal degradation of a misfolded reporter (slGFP), acting upstream of Ubr1; in the absence of Ubr1, slGFP accumulates in relatively stable perinuclear and peripheral puncta.","method":"Genetic deletion/knockdown, fluorescence imaging of misfolded reporter localization, proteasomal degradation assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined substrate reporter and localization, single lab, multiple methods","pmids":["23341891"],"is_preprint":false},{"year":2013,"finding":"Cytosolic Ubr1 participates in ER-associated protein degradation (ERAD) in yeast. Two polytopic ERAD substrates (Ste6* and CFTR) undergo Ubr1-dependent degradation, with Ubr1 acting together with Hsp70 chaperone Ssa1 and the AAA-ATPase Cdc48 to direct retrotranslocated substrates to proteasomal degradation.","method":"Genetic epistasis (ubr1 deletion in combination with canonical ERAD ligase deletions), degradation assays, protein-protein interaction","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple ERAD substrates, interaction with Ssa1/Cdc48 demonstrated, single lab","pmids":["23988329"],"is_preprint":false},{"year":2006,"finding":"UBR1 interacts with c-Fos and ubiquitylates it in the cytoplasm, promoting its accelerated degradation. ERK5 inhibits this process by phosphorylating c-Fos at Ser32 (disrupting UBR1–c-Fos interaction) and Thr232 in the NES (blocking nuclear export). UBR1 expression is induced by STAT3 upon IL-6/gp130 stimulation.","method":"Co-immunoprecipitation, in vivo ubiquitylation assay, siRNA knockdown, phosphorylation site mutagenesis","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitylation assay, and mutagenesis in single study, single lab","pmids":["17018293"],"is_preprint":false},{"year":2004,"finding":"RECQL4 (mutated in Rothmund-Thomson syndrome) forms a stable complex with UBR1 and UBR2 in HeLa cells. Despite association with these E3 ligases, RECQL4 was not ubiquitylated in vivo and was a long-lived protein. The isolated RECQL4-UBR1/2 complex had DNA-stimulated ATPase activity.","method":"Co-immunoprecipitation with antibodies to RECQL4, in vivo ubiquitylation analysis, ATPase activity assay","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with functional characterization, single lab, multiple methods","pmids":["15317757"],"is_preprint":false},{"year":2008,"finding":"Ubr1 is phosphorylated in vivo at multiple sites including Ser300 and Tyr277. Casein kinases Yck1/Yck2 phosphorylate Ubr1 at Ser300, priming subsequent phosphorylations at Ser296, Ser292, Thr288, and Tyr277 by the GSK3-family kinase Mck1. Phosphorylation at Ser300 plays a major role in controlling peptide import regulation by the N-end rule pathway through modulation of Cup9 degradation.","method":"In vivo phosphorylation mapping, kinase deletion/mutation analysis, in vitro kinase assays, peptide import assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vivo phosphorylation site mapping combined with kinase identification and functional consequence (peptide import), multiple methods, single lab","pmids":["19033468"],"is_preprint":false},{"year":2010,"finding":"Phosphorylation of Ubc2/Rad6 at Ser120 by CDK1/2 negatively regulates N-end rule-dependent degradation by E3α/UBR1. The S120D phosphomimetic mutant shows 20-fold reduced activity with UBR1 (8-fold increased Km, 2.5-fold decreased Vmax for initial conjugation), while S120A shows reduced Vmax for polyubiquitin chain elongation.","method":"In vitro E3α/Ubr1-catalyzed ubiquitination kinetics with Ubc2 phosphorylation-site mutants, cell-based N-end rule degradation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous in vitro kinetic analysis but single lab","pmids":["21041297"],"is_preprint":false},{"year":2001,"finding":"Mouse UBR1, in the presence of a cognate mouse E2 enzyme, can rescue the N-end rule pathway in ubr1Δ S. cerevisiae, confirming its conserved E3 ubiquitin ligase activity. UBR1−/− mice lack the N-end rule pathway in skeletal muscle but not in fibroblasts (due to compensating UBR1-like proteins), and exhibit abnormal regulation of fatty acid synthase in skeletal muscle upon starvation.","method":"Functional complementation in yeast, knockout mouse construction, biochemical analysis of N-end rule activity in tissues","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — functional complementation assay plus tissue-specific pathway activity assay, replicated across yeast and mouse models","pmids":["11689692"],"is_preprint":false},{"year":2006,"finding":"UBR1−/−UBR2−/− double-knockout mouse embryos die at midgestation with defects in neurogenesis (reduced proliferation, precocious migration/differentiation of neural progenitor cells) and cardiovascular development. Altered expression of D-type cyclins and Notch1 was detected, placing UBR1 and UBR2 upstream of these developmental regulators.","method":"Double-knockout mouse construction, embryological analysis, molecular marker analysis (cyclin D, Notch1)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double-KO mouse with defined cellular phenotypes and molecular markers, single lab","pmids":["16606826"],"is_preprint":false},{"year":2005,"finding":"Loss-of-function mutations in UBR1 cause Johanson-Blizzard syndrome in humans. UBR1−/− mice exhibit exocrine pancreatic insufficiency with impaired stimulus-secretion coupling and increased susceptibility to pancreatic injury, indicating UBR1 is required for acinar cell function through degradation of N-end rule pathway substrates.","method":"Human genetics (mutation identification in 12 families), UBR1−/− mouse phenotypic analysis, pancreatic functional assays","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — human genetic evidence combined with mouse knockout functional characterization, replicated across multiple families and species","pmids":["16311597"],"is_preprint":false},{"year":2018,"finding":"Upon proteotoxic stress, the hydrophilin Roq1 is cleaved by the HtrA-type protease Ynm3, and cleaved Roq1 interacts with Ubr1, reprogramming its substrate specificity. This SHRED (stress-induced homeostatically regulated protein degradation) cascade accelerates proteasomal degradation of both misfolded and some native proteins at the ER membrane and in the cytosol.","method":"Genetic epistasis, protein interaction assays, substrate degradation assays under stress, protease cleavage assays","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus biochemical interaction demonstration, multiple methods, single lab","pmids":["29861160"],"is_preprint":false},{"year":2025,"finding":"Roq1 reprograms Ubr1 via two cooperating multifunctional motifs: an N-terminal arginine that engages the Ubr1 substrate-binding pocket and a short hydrophobic motif. The N-terminal arginine modulates ubiquitination of N-degron substrates and folded proteins, while the hydrophobic motif accelerates ubiquitination of misfolded proteins. This heterobivalent binding mechanism underlies Ubr1 reprogramming.","method":"Mutational analysis of Roq1 motifs, in vivo and in vitro ubiquitination assays, protein interaction assays","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of binding motifs plus in vitro functional assays, single lab","pmids":["39920309"],"is_preprint":false},{"year":2011,"finding":"UBR1 ubiquitin ligase activity, mediated by its RING-H2 domain, promotes degradation of the glucocorticoid receptor and androgen receptor (but not estrogen receptor α) upon Hsp90 inhibition in mammalian cells, demonstrating client-specific quality control activity beyond the N-end rule.","method":"UBR1 deletion mouse embryonic fibroblasts, geldanamycin treatment, Western blot for client degradation kinetics","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO cells with specific substrate readout and Hsp90 inhibitor treatment, single lab","pmids":["21983172"],"is_preprint":false},{"year":2013,"finding":"UBR1 deletion results in impaired degradation of the glucocorticoid receptor and androgen receptor, but not estrogen receptor α, upon Hsp90 inhibition, demonstrating specificity in UBR1's recognition of misfolded Hsp90 clients.","method":"UBR1 deletion cells, Hsp90 inhibitor treatment, receptor degradation assays","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic deletion with specific substrate panel, replicates and extends prior Caplan lab finding","pmids":["24251101"],"is_preprint":false},{"year":2010,"finding":"UBR1 and UBR2 bind leucine via their substrate-recognition domains, functioning as leucine-binding proteins identified from leucine-responsive cells using leucine-immobilized affinity beads. Overexpression of UBR1/UBR2 reduces mTOR-dependent S6K1 phosphorylation, while their knockdown increases S6K1 phosphorylation; leucine binding inhibits N-end rule substrate degradation in vitro, suggesting UBR1 is a negative regulator of leucine-mTOR signaling.","method":"Leucine-affinity bead pulldown, overexpression/knockdown with S6K1 phosphorylation readout, in vitro N-end rule substrate degradation assay","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity pulldown plus functional in vitro and in vivo assays, single lab, multiple methods","pmids":["20298436"],"is_preprint":false},{"year":2023,"finding":"Ubr1 targets the lipid droplet protein Plin2 for ubiquitin-proteasome degradation in an amino acid-dependent manner. Ubr1 uses its two canonical substrate-binding pockets (for basic and bulky hydrophobic amino acids) to bind amino acids, which allosterically activates Ubr1 by relieving auto-inhibition. This was directly demonstrated in cell-free reconstituted systems with purified proteins.","method":"In vitro cell-free reconstitution system with purified proteins, amino acid-binding assays, pulldown assays, in vitro ubiquitination assays, immunoprecipitation in mammalian cells","journal":"Journal of cachexia, sarcopenia and muscle","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified components plus binding assays plus cellular validation, multiple orthogonal methods, single lab","pmids":["37057345"],"is_preprint":false},{"year":2019,"finding":"Ubr1 preferentially targets mistranslocated secretory and mitochondrial proteins in the cytosol, recognizing cellular location signals encoded in the P2 (second amino acid) position. This establishes that the N-end rule pathway enforces protein compartmentalization by degrading proteins that fail to reach their intended subcellular locations.","method":"Systematic P2-position mutant analysis, bioinformatic proteome analysis, genetic deletion assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis combined with bioinformatics and genetic assays, single lab","pmids":["30940687"],"is_preprint":false},{"year":2022,"finding":"UBR1 functions as a protein quality control E3 ubiquitin ligase at endosomes, ubiquitinating mutant MLC1 via the ESCRT pathway. Under endosomal stress or cytosolic Ca2+ increase, UBR1 and SQSTM1/p62 cooperate to target arginylated and ubiquitinated cargo for selective endosomal autophagy (endophagy). Loss of UBR1 or arginylation causes endosomal compartment stress.","method":"Co-immunoprecipitation, ubiquitination assays, immunofluorescence of endosomal compartments, autophagy flux assays, siRNA knockdown","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods including co-IP, ubiquitination, and imaging, with loss-of-function phenotype, single lab","pmids":["35233680"],"is_preprint":false},{"year":2024,"finding":"UBR1 directly interacts with YAP and promotes its monoubiquitylation, which competitively suppresses YAP polyubiquitylation and extends YAP protein half-life, thereby stabilizing YAP and promoting anaplastic thyroid carcinoma cell proliferation and migration.","method":"siRNA screening, co-immunoprecipitation, ubiquitination assay (mono vs. poly), protein half-life assay, xenograft experiments","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay with mechanistic distinction of mono vs. polyubiquitylation, single lab","pmids":["39174635"],"is_preprint":false},{"year":2024,"finding":"UBR1 promotes sex-dependent ubiquitination and degradation of ACE2 in hypertension. UBR1 was identified by proteomics and validated by co-immunoprecipitation as a direct binding partner of ACE2. Testosterone (but not estradiol) upregulates UBR1, and in vivo UBR1 knockdown restores ACE2 levels and transiently reduces blood pressure in hypertensive male mice. UBR1 and Nedd4-2 appear to work synergistically to ubiquitinate ACE2.","method":"Proteomics, co-immunoprecipitation validation, siRNA knockdown in vivo and in vitro, blood pressure measurement, sex hormone treatment of cells","journal":"Hypertension (Dallas, Tex. : 1979)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics identification validated by Co-IP plus in vivo knockdown with functional readout, single lab","pmids":["39601126"],"is_preprint":false},{"year":2018,"finding":"In C. elegans, loss of UBR-1 leads to a specific motor deficit during reversal movements due to synchronized A-class motor neuron activation. This phenotype is rescued by removing GOT-1, a transaminase converting aspartate to glutamate. ubr-1 mutants show elevated glutamate levels and ubr-1 and got-1 are both required in premotor interneurons, placing UBR-1 in a pathway regulating glutamate metabolism for motor circuit function.","method":"C. elegans genetics (loss-of-function), epistasis with got-1, neuronal calcium imaging, metabolite measurement, cell-specific rescue experiments","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with cellular specificity and metabolite measurements, single lab","pmids":["29649217"],"is_preprint":false},{"year":2011,"finding":"In fission yeast, Ubr1 is concentrated in the nucleus and mediates degradation of activated (nuclear) Pap1 bZIP transcription factor, providing a mechanism to terminate the oxidative stress response. Loss of ubr1 causes increased Pap1 levels and hydrogen peroxide resistance; inactive mutations in the Pap1 bZIP domain stabilize the protein but rescue lethality in ubr1 mutants, showing that DNA-binding-competent Pap1 must be eliminated by Ubr1.","method":"Genetic deletion, epistasis with nuclear-export-deficient and DNA-binding mutants, Western blot, growth assays","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple alleles and nuclear localization component, single lab","pmids":["21410566"],"is_preprint":false},{"year":2011,"finding":"Missense mutations in the RING-H2 and UBR domains of human UBR1 (corresponding to JBS alleles) can be modeled in yeast Ubr1 and show graded activity defects: H160R (in UBR box) is completely inactive, Q1224E has weak activity, and V146L has reduced but significant activity, correlating with clinical severity of JBS in corresponding patients.","method":"Yeast-based functional complementation assay, N-end rule substrate degradation assay, site-directed mutagenesis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assay in heterologous system with multiple alleles correlated to clinical data, single lab","pmids":["21931868"],"is_preprint":false},{"year":2025,"finding":"In C. elegans, UBR-1 physically associates with glutamate metabolic enzymes (GLN-3, GOT-2.2, GFAT-1, GDH-1) identified by CRISPR-based proteomics. Genetic interaction studies show UBR-1, GLN-3, and GOT-2.2 form a signaling axis regulating glutamate homeostasis required for locomotion and developmental viability, with UBR-1 functioning via both ubiquitin ligase and scaffolding mechanisms.","method":"CRISPR-based proteomics, genetic interaction analysis, super-resolution imaging, pharmacological rescue","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR proteomics plus multi-gene interaction studies plus pharmacology, preprint, single lab","pmids":["40766417"],"is_preprint":true}],"current_model":"UBR1 is a RING-type E3 ubiquitin ligase with three substrate-binding sites: a type-1 site (UBR box) for basic N-terminal residues, a type-2 site (N domain) for bulky hydrophobic N-terminal residues, and an internal-degron site; structural studies reveal it mediates both ubiquitin chain initiation and K48-linked elongation through a single E2 (Ubc2/Rad6), whose activity is modulated by CDK-dependent phosphorylation, while the Ubr1–Ufd4 complex enhances processivity; beyond canonical N-degron substrates, UBR1 performs chaperone-dependent cytoplasmic protein quality control of misfolded proteins requiring Hsp70 ATPase activity, participates in ERAD, endosomal autophagy, and ER stress responses, ubiquitinates substrates including c-Fos, Plin2, RECQL4, ACE2, and YAP, is allosterically activated by amino acid binding, and is reprogrammed under stress by the cleaved intrinsically disordered protein Roq1 through a heterobivalent binding mechanism that shifts substrate specificity toward misfolded proteins."},"narrative":{"mechanistic_narrative":"UBR1 is a RING-type E3 ubiquitin ligase that serves as a central recognition component of the N-end rule (N-degron) pathway and as a broad protein quality-control ligase [PMID:18566452, PMID:11689692]. Substrate recognition is partitioned across distinct sites within its N-terminal region: a type-1 site for basic N-terminal residues and a type-2 site for bulky hydrophobic N-terminal residues, both with micromolar affinity, plus a separate internal-degron site that drives CUP9 ubiquitylation [PMID:18566452]. Cryo-EM structures show that UBR1 mediates both ubiquitin chain initiation and linkage-specific polyubiquitin elongation through a single E2 enzyme, Ubc2/Rad6, using a defined E2-binding region and an acceptor-ubiquitin-binding loop [PMID:34789879]; processivity is further enhanced by physical partnership with the HECT ligase Ufd4 [PMID:21076411], and the system is tuned by CDK1/2 phosphorylation of Ubc2 at Ser120 [PMID:21041297] and by casein-kinase/Mck1 phosphorylation of UBR1 itself that controls peptide-import regulation via CUP9 [PMID:19033468]. Beyond canonical N-degron substrates, UBR1 performs chaperone-dependent cytoplasmic quality control of misfolded proteins, directly binding denatured substrate and requiring Hsp70 ATPase activity together with the Hsp40 Sis1 and nucleotide-exchange cofactors [PMID:20080635, PMID:20462952, PMID:32966159, PMID:23341891]; it extends this surveillance to ERAD substrates in concert with Hsp70 (Ssa1) and Cdc48 [PMID:23988329], to mistranslocated secretory/mitochondrial proteins recognized via the P2 residue [PMID:30940687], and to endosomal quality control and selective endophagy with SQSTM1/p62 [PMID:35233680]. UBR1 activity is allosterically gated by amino-acid binding at its canonical pockets, which relieves auto-inhibition to drive degradation of substrates such as the lipid-droplet protein Plin2 and links UBR1 to leucine–mTOR signaling [PMID:20298436, PMID:37057345], and under proteotoxic stress its specificity is reprogrammed by the protease-cleaved hydrophilin Roq1 through a heterobivalent mechanism using an N-terminal arginine and a hydrophobic motif [PMID:29861160, PMID:39920309]. Loss-of-function mutations in UBR1 cause Johanson-Blizzard syndrome, and graded activity defects of disease alleles in the RING-H2 and UBR domains correlate with clinical severity [PMID:16311597, PMID:21931868]. Additional reported substrates include c-Fos, the glucocorticoid and androgen receptors as Hsp90 clients, ACE2, and YAP [PMID:17018293, PMID:21983172, PMID:39174635, PMID:39601126].","teleology":[{"year":2001,"claim":"Established that mammalian UBR1 is a functionally conserved N-end rule E3 ligase, not merely a yeast factor, and that it operates redundantly in some tissues.","evidence":"Functional complementation of yeast ubr1Δ by mouse UBR1 with a cognate E2, plus tissue-specific N-end rule activity analysis in UBR1-knockout mice","pmids":["11689692"],"confidence":"High","gaps":["Identity of compensating UBR1-like proteins in fibroblasts not resolved","Physiological substrate in skeletal muscle beyond fatty acid synthase regulation not defined"]},{"year":2004,"claim":"Showed UBR1 can form stable complexes with substrates without degrading them, indicating association does not always equal ubiquitylation.","evidence":"Reciprocal Co-IP of RECQL4 with UBR1/UBR2 in HeLa cells, in vivo ubiquitylation analysis, and ATPase assay of the isolated complex","pmids":["15317757"],"confidence":"Medium","gaps":["Functional consequence of the RECQL4–UBR1/2 complex unknown","Why a bound substrate escapes ubiquitylation not explained"]},{"year":2005,"claim":"Linked UBR1 loss to a human Mendelian disease, defining its physiological importance in exocrine pancreatic function.","evidence":"Human mutation identification in 12 Johanson-Blizzard families plus pancreatic functional analysis of UBR1-knockout mice","pmids":["16311597"],"confidence":"High","gaps":["Specific acinar-cell N-end rule substrates whose stabilization causes pathology not identified"]},{"year":2006,"claim":"Extended UBR1 to regulated turnover of a signaling transcription factor, showing its activity is gated by phosphorylation and induced transcriptionally.","evidence":"Co-IP, in vivo ubiquitylation, siRNA, and phospho-site mutagenesis linking UBR1, c-Fos, ERK5, and STAT3/IL-6","pmids":["17018293"],"confidence":"Medium","gaps":["Whether c-Fos is an N-degron or internal-degron substrate not defined","Generality beyond the cell systems tested unknown"]},{"year":2006,"claim":"Defined an essential developmental role by removing redundancy, placing UBR1/UBR2 upstream of neurogenic and cardiovascular regulators.","evidence":"UBR1/UBR2 double-knockout mouse embryology with cyclin D and Notch1 marker analysis","pmids":["16606826"],"confidence":"Medium","gaps":["Direct substrates connecting UBR1 to cyclin D/Notch1 not identified","Relative contributions of UBR1 vs UBR2 not separated"]},{"year":2008,"claim":"Mapped UBR1's substrate recognition architecture, defining separable type-1, type-2, and internal-degron sites and their affinities.","evidence":"Loss-of-function genetic screen, fluorescence polarization and SPR binding measurements, and fully reconstituted CUP9 ubiquitylation in vitro","pmids":["18566452"],"confidence":"High","gaps":["Structural basis of site cooperativity not resolved at this stage","Internal-degron recognition determinants only defined for CUP9"]},{"year":2008,"claim":"Established that UBR1 itself is phosphoregulated, connecting kinase signaling to N-end rule control of peptide import.","evidence":"In vivo phospho-site mapping, Yck1/2 and Mck1 kinase analysis, in vitro kinase assays, and peptide-import readouts","pmids":["19033468"],"confidence":"High","gaps":["Structural effect of Ser300 phosphorylation on UBR1 conformation unknown","Whether phosphoregulation extends beyond CUP9-dependent import not tested"]},{"year":2008,"claim":"Identified UBR1 as the cytoplasmic quality-control ligase for misfolded proteins, a role distinct from N-end rule recognition.","evidence":"Genetic deletion and ubiquitination assays with misfolded reporter substrates in yeast","pmids":["19041308"],"confidence":"Medium","gaps":["Single lab; chaperone dependence not yet dissected here","Degron recognized on misfolded substrates undefined"]},{"year":2009,"claim":"Showed cytoplasmic misfolded-protein quality control by UBR1 is chaperone-dependent and operates in parallel with nuclear San1.","evidence":"Genetic epistasis (double-mutant analysis), biochemical ubiquitination assays, and proteotoxic-stress phenotyping","pmids":["20080635"],"confidence":"High","gaps":["Mechanistic basis of cytoplasmic/nuclear division of labor not fully mapped"]},{"year":2010,"claim":"Demonstrated direct UBR1 recognition of denatured substrate and reconstituted chaperone-stimulated ubiquitination, with E3s competing with chaperones.","evidence":"In vitro ubiquitylation with purified Ubr1 and denatured luciferase plus genetic suppressor analysis of chaperone mutants","pmids":["20462952"],"confidence":"High","gaps":["Molecular feature of denatured substrate recognized by Ubr1 not defined"]},{"year":2010,"claim":"Explained how a single E2 supports both initiation and elongation by showing CDK phosphorylation of Ubc2 tunes each step differently.","evidence":"In vitro Ubr1-catalyzed ubiquitination kinetics with Ubc2 Ser120 phosphomimetic/null mutants plus cell-based N-end rule assay","pmids":["21041297"],"confidence":"Medium","gaps":["Single lab; in vivo CDK regulation of N-end rule degradation not directly shown","Cell-cycle timing of this control not established"]},{"year":2010,"claim":"Increased polyubiquitin processivity by partnering UBR1 with a HECT ligase and recognition of the UFD ubiquitin moiety.","evidence":"Co-IP, in vitro ubiquitylation with Ufd4, and genetic epistasis on UFD substrates","pmids":["21076411"],"confidence":"High","gaps":["Stoichiometry and architecture of the Ubr1–Ufd4 complex not resolved","Generality of cooperativity across substrate classes not tested"]},{"year":2010,"claim":"Connected UBR1 to nutrient signaling by identifying it as a leucine-binding negative regulator of mTOR-S6K signaling.","evidence":"Leucine-affinity pulldown, overexpression/knockdown with S6K1 phospho-readout, and in vitro N-end rule degradation assay","pmids":["20298436"],"confidence":"Medium","gaps":["Mechanistic link between leucine binding and mTOR activity not fully defined","Direct mTOR-pathway substrate of UBR1 not identified"]},{"year":2011,"claim":"Showed UBR1 acts as a quality-control ligase for specific Hsp90 clients, distinguishing client recognition by receptor identity.","evidence":"UBR1-deletion MEFs treated with geldanamycin and degradation kinetics of glucocorticoid, androgen, and estrogen receptors","pmids":["21983172"],"confidence":"Medium","gaps":["Structural basis for client selectivity (GR/AR vs ERα) unknown"]},{"year":2011,"claim":"Provided nuclear, organism-specific evidence that UBR1 terminates a stress transcriptional response by degrading DNA-binding-competent Pap1.","evidence":"Fission-yeast genetic deletion, epistasis with export- and DNA-binding-mutant Pap1, and Western/growth assays","pmids":["21410566"],"confidence":"Medium","gaps":["Degron on activated Pap1 not defined","Generalizability of nuclear UBR1 localization to other species unclear"]},{"year":2011,"claim":"Linked specific Johanson-Blizzard alleles to graded enzymatic defects, establishing genotype–activity–severity correlation.","evidence":"Yeast functional complementation and N-end rule degradation assays with H160R, Q1224E, and V146L UBR domain/RING mutants","pmids":["21931868"],"confidence":"Medium","gaps":["Modeled in yeast rather than native human context","Effects on chaperone-dependent quality-control activity not assessed"]},{"year":2013,"claim":"Ordered the chaperone-handoff to UBR1 by placing the Hsp40 Sis1 upstream of Ubr1 in misfolded-substrate triage.","evidence":"Genetic deletion/knockdown, imaging of misfolded reporter localization, and proteasomal degradation assays","pmids":["23341891"],"confidence":"Medium","gaps":["Physical handoff between Sis1/Hsp70 and Ubr1 not biochemically resolved"]},{"year":2013,"claim":"Expanded cytosolic UBR1 quality control to ERAD of polytopic membrane substrates acting with Hsp70 and Cdc48.","evidence":"Genetic epistasis with canonical ERAD ligase deletions, degradation assays of Ste6*/CFTR, and interaction with Ssa1/Cdc48","pmids":["23988329"],"confidence":"Medium","gaps":["How retrotranslocated substrates are routed to Ubr1 vs canonical ERAD ligases unclear"]},{"year":2018,"claim":"Revealed stress-induced reprogramming of UBR1 specificity by a cleaved hydrophilin, defining the SHRED pathway.","evidence":"Genetic epistasis, Roq1–Ubr1 interaction assays, Ynm3 cleavage assays, and stress-dependent substrate degradation","pmids":["29861160"],"confidence":"Medium","gaps":["Molecular mechanism of the specificity shift not yet defined at this stage","Conservation of SHRED beyond yeast not established"]},{"year":2018,"claim":"Uncovered a neuronal role for UBR-1 in glutamate metabolism controlling motor circuit output.","evidence":"C. elegans loss-of-function genetics, got-1 epistasis, neuronal calcium imaging, metabolite measurement, and cell-specific rescue","pmids":["29649217"],"confidence":"Medium","gaps":["Whether the motor phenotype reflects ligase activity or a non-catalytic role unresolved at this stage"]},{"year":2019,"claim":"Showed the N-end rule pathway enforces protein compartmentalization by Ubr1 degrading mistranslocated proteins via P2-position signals.","evidence":"Systematic P2-position mutant analysis, proteome bioinformatics, and genetic deletion assays","pmids":["30940687"],"confidence":"Medium","gaps":["Direct biochemical demonstration of P2 recognition not provided","Breadth of endogenous mislocalized substrate pool not quantified"]},{"year":2021,"claim":"Provided the structural mechanism for how UBR1 uses a single E2 to perform linkage-specific chain initiation and elongation.","evidence":"Cryo-EM of Ubr1–Ubc2–ubiquitin–N-degron complexes with reaction-intermediate mimics and mutagenesis validation","pmids":["34789879"],"confidence":"High","gaps":["Structures of chaperone-dependent quality-control engagement not captured","Conformational coupling to allosteric amino-acid regulation not visualized"]},{"year":2022,"claim":"Defined UBR1 as an endosomal quality-control ligase driving selective endophagy of arginylated/ubiquitinated cargo.","evidence":"Co-IP, ubiquitination assays, endosomal imaging, autophagy flux, and siRNA knockdown of mutant MLC1 turnover","pmids":["35233680"],"confidence":"Medium","gaps":["How UBR1 is recruited to endosomes mechanistically unknown","Direct ESCRT-component interactions not mapped"]},{"year":2023,"claim":"Demonstrated that amino-acid binding allosterically activates UBR1 by relieving auto-inhibition, driving Plin2 degradation.","evidence":"Cell-free reconstitution with purified proteins, amino-acid binding and pulldown assays, in vitro ubiquitination, and cellular IP","pmids":["37057345"],"confidence":"High","gaps":["Structural conformation of the auto-inhibited vs activated states not resolved","Physiological amino-acid concentrations governing this switch in vivo not defined"]},{"year":2024,"claim":"Showed UBR1 can stabilize rather than degrade a substrate via monoubiquitylation, with pro-tumorigenic consequences.","evidence":"siRNA screening, Co-IP, mono- vs poly-ubiquitination assays, half-life measurement, and xenografts for YAP in anaplastic thyroid carcinoma","pmids":["39174635"],"confidence":"Medium","gaps":["Mechanism preventing chain elongation on YAP unknown","Whether N-degron or internal-degron recognition is used not defined"]},{"year":2024,"claim":"Connected UBR1 to sex-dependent blood-pressure regulation through hormone-driven ACE2 degradation.","evidence":"Proteomics, Co-IP validation, in vivo/in vitro siRNA knockdown, blood-pressure measurement, and sex-hormone treatment","pmids":["39601126"],"confidence":"Medium","gaps":["Direct ubiquitination site on ACE2 not mapped","Relative roles of UBR1 vs Nedd4-2 not separated mechanistically"]},{"year":2025,"claim":"Dissected the heterobivalent logic of Roq1-mediated reprogramming, separating N-degron-substrate modulation from misfolded-protein targeting.","evidence":"Mutational analysis of Roq1 N-terminal arginine and hydrophobic motifs with in vivo and in vitro ubiquitination and interaction assays","pmids":["39920309"],"confidence":"Medium","gaps":["Structure of the reprogrammed Ubr1–Roq1 complex not determined","Mammalian functional homolog of SHRED reprogramming not established"]},{"year":2025,"claim":"Proposed that UBR-1 acts through both catalytic and scaffolding modes within a glutamate-homeostasis enzyme axis.","evidence":"CRISPR-based proteomics identifying enzyme partners, multi-gene genetic interactions, super-resolution imaging, and pharmacological rescue in C. elegans (preprint)","pmids":["40766417"],"confidence":"Medium","gaps":["Preprint; not peer-reviewed","Scaffolding (non-catalytic) function not biochemically separated from ligase activity","Conservation of the axis in mammals untested"]},{"year":null,"claim":"How UBR1 integrates its multiple substrate-binding sites, allosteric amino-acid sensing, chaperone partnerships, and stress-induced reprogramming into a unified conformational regulatory logic remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure captures the chaperone-dependent or allosterically activated states","Determinants choosing degradation vs stabilization (e.g., mono- vs polyubiquitylation) not defined","Mammalian counterpart of SHRED reprogramming not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2,13]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,3,9,21,24]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,12,20,21]},{"term_id":"GO:0140313","term_label":"molecular sequestering 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(HSP70/SSA1)","SIS1","CDC48","SQSTM1","ROQ1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IWV7","full_name":"E3 ubiquitin-protein ligase UBR1","aliases":["N-recognin-1","Ubiquitin-protein ligase E3-alpha-1","Ubiquitin-protein ligase E3-alpha-I"],"length_aa":1749,"mass_kda":200.2,"function":"E3 ubiquitin-protein ligase which is a component of the N-end rule pathway (PubMed:15548684, PubMed:16311597, PubMed:18162545, PubMed:20835242, PubMed:28392261). Recognizes and binds proteins bearing specific N-terminal residues that are destabilizing according to the N-end rule, leading to their ubiquitination and subsequent degradation (PubMed:18162545, PubMed:20835242, PubMed:28392261). Recognizes both type-1 and type-2 N-degrons, containing positively charged amino acids (Arg, Lys and His) and bulky and hydrophobic amino acids, respectively (PubMed:18162545). Does not ubiquitinate proteins that are acetylated at the N-terminus (PubMed:20835242). In contrast, it strongly binds methylated N-degrons (PubMed:28392261). Binds leucine and is a negative regulator of the leucine-mTOR signaling pathway, thereby controlling cell growth (PubMed:20298436)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q8IWV7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBR1","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/UBR1","total_profiled":1310},"omim":[{"mim_id":"620846","title":"N-TERMINAL GLUTAMINE AMIDASE 1; NTAQ1","url":"https://www.omim.org/entry/620846"},{"mim_id":"620743","title":"SDE2 TELOMERE MAINTENANCE HOMOLOG; SDE2","url":"https://www.omim.org/entry/620743"},{"mim_id":"615367","title":"N-TERMINAL ASPARAGINE AMIDASE; NTAN1","url":"https://www.omim.org/entry/615367"},{"mim_id":"613831","title":"UBIQUITIN PROTEIN LIGASE E3 COMPONENT N-RECOGNIN 3; UBR3","url":"https://www.omim.org/entry/613831"},{"mim_id":"609134","title":"UBIQUITIN-PROTEIN LIGASE E3 COMPONENT N-RECOGNIN 2; UBR2","url":"https://www.omim.org/entry/609134"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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Key structural elements including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1 were identified and characterized by mutagenesis, explaining how Ubr1 mediates linkage-specific polyubiquitin chain formation through a single E2 enzyme.\",\n      \"method\": \"Cryo-EM structure determination with chemical mimics of reaction intermediates, plus mutagenesis of key structural elements\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures with chemical biology mimics of reaction intermediates, mutagenesis validation, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"34789879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The type-1 and type-2 substrate-binding sites of UBR1 (recognizing basic and bulky hydrophobic N-terminal residues, respectively) reside within the first ~700 residues of the 1,950-residue protein. Selective inactivation of each site by distinct mutations was demonstrated, and binding affinities (Kd ~1 μM) were measured by fluorescence polarization and surface plasmon resonance. A third substrate-binding site targets the internal degron of CUP9, and CUP9 ubiquitylation dependent on dipeptide binding to type-1/2 sites was reconstituted in a fully defined in vitro system.\",\n      \"method\": \"Genetic screen for loss-of-function mutations, fluorescence polarization, surface plasmon resonance, in vitro reconstitution with purified components\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus biophysical binding measurements plus mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"18566452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The RING-type Ubr1 E3 and the HECT-type Ufd4 E3 physically interact and form a complex that is more processive in polyubiquitylation than Ubr1 alone, producing longer substrate-linked polyubiquitin chains. Ubr1 can also recognize the N-terminal ubiquitin moiety of UFD substrates, and can enhance polyubiquitylation of UFD substrates as part of the Ubr1-Ufd4 complex.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitylation assays, genetic epistasis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal physical interaction plus in vitro functional reconstitution plus genetic epistasis in single study\",\n      \"pmids\": [\"21076411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Ubr1 mediates chaperone-dependent ubiquitination of misfolded cytoplasmic proteins for proteasomal degradation in yeast, acting in parallel with nuclear San1. This cytoplasmic quality control function of Ubr1 is distinct from its N-end rule role and depends on chaperone assistance.\",\n      \"method\": \"Genetic epistasis (double mutant analysis), biochemical ubiquitination assays, phenotypic analysis of proteotoxic stress\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (biochemical + genetic), replicated across related studies\",\n      \"pmids\": [\"20080635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ubr1 is the E3 ligase responsible for targeting misfolded cytoplasmic proteins to proteasomal degradation in yeast, establishing its role as a cytoplasmic protein quality control ubiquitin ligase independent of N-end rule activity.\",\n      \"method\": \"Genetic deletion analysis, ubiquitination assay with misfolded reporter substrates\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with defined substrate phenotype, single lab\",\n      \"pmids\": [\"19041308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Saccharomyces cerevisiae Ubr1 directly binds denatured (but not native) luciferase and catalyzes its ubiquitinylation in a purified system; Hsp70 stimulates polyubiquitinylation of the denatured substrate. Loss of Ubr1 and Ubr2 suppresses growth arrest caused by chaperone mutation, indicating these E3s compete with chaperones for misfolded substrates.\",\n      \"method\": \"In vitro ubiquitylation assay with purified components, genetic epistasis (suppressor analysis of chaperone mutants)\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified Ubr1 and denatured substrate plus genetic epistasis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"20462952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Hsp70 ATP hydrolysis is directly required for Ubr1-mediated ubiquitination of misfolded substrates. Using a bead-based in vitro assay with covalently immobilized misfolded protein, only Hsp70 (with its ATPase cycle and cochaperones including nucleotide exchange factor Sse1 and J-proteins) was required for Ubr1-mediated ubiquitination.\",\n      \"method\": \"In vitro bead-based ubiquitination assay with immobilized misfolded substrate, ATPase cycle mutant analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with immobilized substrate to eliminate indirect effects, multiple cochaperone mutants tested, single lab\",\n      \"pmids\": [\"32966159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The Type II Hsp40 Sis1 cooperates sequentially with Hsp70 and Ubr1 to target misfolded cytosolic proteins for proteasomal degradation. Sis1 is required for proteasomal degradation of a misfolded reporter (slGFP), acting upstream of Ubr1; in the absence of Ubr1, slGFP accumulates in relatively stable perinuclear and peripheral puncta.\",\n      \"method\": \"Genetic deletion/knockdown, fluorescence imaging of misfolded reporter localization, proteasomal degradation assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined substrate reporter and localization, single lab, multiple methods\",\n      \"pmids\": [\"23341891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cytosolic Ubr1 participates in ER-associated protein degradation (ERAD) in yeast. Two polytopic ERAD substrates (Ste6* and CFTR) undergo Ubr1-dependent degradation, with Ubr1 acting together with Hsp70 chaperone Ssa1 and the AAA-ATPase Cdc48 to direct retrotranslocated substrates to proteasomal degradation.\",\n      \"method\": \"Genetic epistasis (ubr1 deletion in combination with canonical ERAD ligase deletions), degradation assays, protein-protein interaction\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple ERAD substrates, interaction with Ssa1/Cdc48 demonstrated, single lab\",\n      \"pmids\": [\"23988329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"UBR1 interacts with c-Fos and ubiquitylates it in the cytoplasm, promoting its accelerated degradation. ERK5 inhibits this process by phosphorylating c-Fos at Ser32 (disrupting UBR1–c-Fos interaction) and Thr232 in the NES (blocking nuclear export). UBR1 expression is induced by STAT3 upon IL-6/gp130 stimulation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitylation assay, siRNA knockdown, phosphorylation site mutagenesis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitylation assay, and mutagenesis in single study, single lab\",\n      \"pmids\": [\"17018293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RECQL4 (mutated in Rothmund-Thomson syndrome) forms a stable complex with UBR1 and UBR2 in HeLa cells. Despite association with these E3 ligases, RECQL4 was not ubiquitylated in vivo and was a long-lived protein. The isolated RECQL4-UBR1/2 complex had DNA-stimulated ATPase activity.\",\n      \"method\": \"Co-immunoprecipitation with antibodies to RECQL4, in vivo ubiquitylation analysis, ATPase activity assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with functional characterization, single lab, multiple methods\",\n      \"pmids\": [\"15317757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ubr1 is phosphorylated in vivo at multiple sites including Ser300 and Tyr277. Casein kinases Yck1/Yck2 phosphorylate Ubr1 at Ser300, priming subsequent phosphorylations at Ser296, Ser292, Thr288, and Tyr277 by the GSK3-family kinase Mck1. Phosphorylation at Ser300 plays a major role in controlling peptide import regulation by the N-end rule pathway through modulation of Cup9 degradation.\",\n      \"method\": \"In vivo phosphorylation mapping, kinase deletion/mutation analysis, in vitro kinase assays, peptide import assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vivo phosphorylation site mapping combined with kinase identification and functional consequence (peptide import), multiple methods, single lab\",\n      \"pmids\": [\"19033468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Phosphorylation of Ubc2/Rad6 at Ser120 by CDK1/2 negatively regulates N-end rule-dependent degradation by E3α/UBR1. The S120D phosphomimetic mutant shows 20-fold reduced activity with UBR1 (8-fold increased Km, 2.5-fold decreased Vmax for initial conjugation), while S120A shows reduced Vmax for polyubiquitin chain elongation.\",\n      \"method\": \"In vitro E3α/Ubr1-catalyzed ubiquitination kinetics with Ubc2 phosphorylation-site mutants, cell-based N-end rule degradation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous in vitro kinetic analysis but single lab\",\n      \"pmids\": [\"21041297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mouse UBR1, in the presence of a cognate mouse E2 enzyme, can rescue the N-end rule pathway in ubr1Δ S. cerevisiae, confirming its conserved E3 ubiquitin ligase activity. UBR1−/− mice lack the N-end rule pathway in skeletal muscle but not in fibroblasts (due to compensating UBR1-like proteins), and exhibit abnormal regulation of fatty acid synthase in skeletal muscle upon starvation.\",\n      \"method\": \"Functional complementation in yeast, knockout mouse construction, biochemical analysis of N-end rule activity in tissues\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — functional complementation assay plus tissue-specific pathway activity assay, replicated across yeast and mouse models\",\n      \"pmids\": [\"11689692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"UBR1−/−UBR2−/− double-knockout mouse embryos die at midgestation with defects in neurogenesis (reduced proliferation, precocious migration/differentiation of neural progenitor cells) and cardiovascular development. Altered expression of D-type cyclins and Notch1 was detected, placing UBR1 and UBR2 upstream of these developmental regulators.\",\n      \"method\": \"Double-knockout mouse construction, embryological analysis, molecular marker analysis (cyclin D, Notch1)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double-KO mouse with defined cellular phenotypes and molecular markers, single lab\",\n      \"pmids\": [\"16606826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Loss-of-function mutations in UBR1 cause Johanson-Blizzard syndrome in humans. UBR1−/− mice exhibit exocrine pancreatic insufficiency with impaired stimulus-secretion coupling and increased susceptibility to pancreatic injury, indicating UBR1 is required for acinar cell function through degradation of N-end rule pathway substrates.\",\n      \"method\": \"Human genetics (mutation identification in 12 families), UBR1−/− mouse phenotypic analysis, pancreatic functional assays\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human genetic evidence combined with mouse knockout functional characterization, replicated across multiple families and species\",\n      \"pmids\": [\"16311597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Upon proteotoxic stress, the hydrophilin Roq1 is cleaved by the HtrA-type protease Ynm3, and cleaved Roq1 interacts with Ubr1, reprogramming its substrate specificity. This SHRED (stress-induced homeostatically regulated protein degradation) cascade accelerates proteasomal degradation of both misfolded and some native proteins at the ER membrane and in the cytosol.\",\n      \"method\": \"Genetic epistasis, protein interaction assays, substrate degradation assays under stress, protease cleavage assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus biochemical interaction demonstration, multiple methods, single lab\",\n      \"pmids\": [\"29861160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Roq1 reprograms Ubr1 via two cooperating multifunctional motifs: an N-terminal arginine that engages the Ubr1 substrate-binding pocket and a short hydrophobic motif. The N-terminal arginine modulates ubiquitination of N-degron substrates and folded proteins, while the hydrophobic motif accelerates ubiquitination of misfolded proteins. This heterobivalent binding mechanism underlies Ubr1 reprogramming.\",\n      \"method\": \"Mutational analysis of Roq1 motifs, in vivo and in vitro ubiquitination assays, protein interaction assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of binding motifs plus in vitro functional assays, single lab\",\n      \"pmids\": [\"39920309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"UBR1 ubiquitin ligase activity, mediated by its RING-H2 domain, promotes degradation of the glucocorticoid receptor and androgen receptor (but not estrogen receptor α) upon Hsp90 inhibition in mammalian cells, demonstrating client-specific quality control activity beyond the N-end rule.\",\n      \"method\": \"UBR1 deletion mouse embryonic fibroblasts, geldanamycin treatment, Western blot for client degradation kinetics\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO cells with specific substrate readout and Hsp90 inhibitor treatment, single lab\",\n      \"pmids\": [\"21983172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"UBR1 deletion results in impaired degradation of the glucocorticoid receptor and androgen receptor, but not estrogen receptor α, upon Hsp90 inhibition, demonstrating specificity in UBR1's recognition of misfolded Hsp90 clients.\",\n      \"method\": \"UBR1 deletion cells, Hsp90 inhibitor treatment, receptor degradation assays\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic deletion with specific substrate panel, replicates and extends prior Caplan lab finding\",\n      \"pmids\": [\"24251101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"UBR1 and UBR2 bind leucine via their substrate-recognition domains, functioning as leucine-binding proteins identified from leucine-responsive cells using leucine-immobilized affinity beads. Overexpression of UBR1/UBR2 reduces mTOR-dependent S6K1 phosphorylation, while their knockdown increases S6K1 phosphorylation; leucine binding inhibits N-end rule substrate degradation in vitro, suggesting UBR1 is a negative regulator of leucine-mTOR signaling.\",\n      \"method\": \"Leucine-affinity bead pulldown, overexpression/knockdown with S6K1 phosphorylation readout, in vitro N-end rule substrate degradation assay\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity pulldown plus functional in vitro and in vivo assays, single lab, multiple methods\",\n      \"pmids\": [\"20298436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Ubr1 targets the lipid droplet protein Plin2 for ubiquitin-proteasome degradation in an amino acid-dependent manner. Ubr1 uses its two canonical substrate-binding pockets (for basic and bulky hydrophobic amino acids) to bind amino acids, which allosterically activates Ubr1 by relieving auto-inhibition. This was directly demonstrated in cell-free reconstituted systems with purified proteins.\",\n      \"method\": \"In vitro cell-free reconstitution system with purified proteins, amino acid-binding assays, pulldown assays, in vitro ubiquitination assays, immunoprecipitation in mammalian cells\",\n      \"journal\": \"Journal of cachexia, sarcopenia and muscle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified components plus binding assays plus cellular validation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"37057345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Ubr1 preferentially targets mistranslocated secretory and mitochondrial proteins in the cytosol, recognizing cellular location signals encoded in the P2 (second amino acid) position. This establishes that the N-end rule pathway enforces protein compartmentalization by degrading proteins that fail to reach their intended subcellular locations.\",\n      \"method\": \"Systematic P2-position mutant analysis, bioinformatic proteome analysis, genetic deletion assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis combined with bioinformatics and genetic assays, single lab\",\n      \"pmids\": [\"30940687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBR1 functions as a protein quality control E3 ubiquitin ligase at endosomes, ubiquitinating mutant MLC1 via the ESCRT pathway. Under endosomal stress or cytosolic Ca2+ increase, UBR1 and SQSTM1/p62 cooperate to target arginylated and ubiquitinated cargo for selective endosomal autophagy (endophagy). Loss of UBR1 or arginylation causes endosomal compartment stress.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, immunofluorescence of endosomal compartments, autophagy flux assays, siRNA knockdown\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods including co-IP, ubiquitination, and imaging, with loss-of-function phenotype, single lab\",\n      \"pmids\": [\"35233680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBR1 directly interacts with YAP and promotes its monoubiquitylation, which competitively suppresses YAP polyubiquitylation and extends YAP protein half-life, thereby stabilizing YAP and promoting anaplastic thyroid carcinoma cell proliferation and migration.\",\n      \"method\": \"siRNA screening, co-immunoprecipitation, ubiquitination assay (mono vs. poly), protein half-life assay, xenograft experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay with mechanistic distinction of mono vs. polyubiquitylation, single lab\",\n      \"pmids\": [\"39174635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBR1 promotes sex-dependent ubiquitination and degradation of ACE2 in hypertension. UBR1 was identified by proteomics and validated by co-immunoprecipitation as a direct binding partner of ACE2. Testosterone (but not estradiol) upregulates UBR1, and in vivo UBR1 knockdown restores ACE2 levels and transiently reduces blood pressure in hypertensive male mice. UBR1 and Nedd4-2 appear to work synergistically to ubiquitinate ACE2.\",\n      \"method\": \"Proteomics, co-immunoprecipitation validation, siRNA knockdown in vivo and in vitro, blood pressure measurement, sex hormone treatment of cells\",\n      \"journal\": \"Hypertension (Dallas, Tex. : 1979)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics identification validated by Co-IP plus in vivo knockdown with functional readout, single lab\",\n      \"pmids\": [\"39601126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In C. elegans, loss of UBR-1 leads to a specific motor deficit during reversal movements due to synchronized A-class motor neuron activation. This phenotype is rescued by removing GOT-1, a transaminase converting aspartate to glutamate. ubr-1 mutants show elevated glutamate levels and ubr-1 and got-1 are both required in premotor interneurons, placing UBR-1 in a pathway regulating glutamate metabolism for motor circuit function.\",\n      \"method\": \"C. elegans genetics (loss-of-function), epistasis with got-1, neuronal calcium imaging, metabolite measurement, cell-specific rescue experiments\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with cellular specificity and metabolite measurements, single lab\",\n      \"pmids\": [\"29649217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In fission yeast, Ubr1 is concentrated in the nucleus and mediates degradation of activated (nuclear) Pap1 bZIP transcription factor, providing a mechanism to terminate the oxidative stress response. Loss of ubr1 causes increased Pap1 levels and hydrogen peroxide resistance; inactive mutations in the Pap1 bZIP domain stabilize the protein but rescue lethality in ubr1 mutants, showing that DNA-binding-competent Pap1 must be eliminated by Ubr1.\",\n      \"method\": \"Genetic deletion, epistasis with nuclear-export-deficient and DNA-binding mutants, Western blot, growth assays\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple alleles and nuclear localization component, single lab\",\n      \"pmids\": [\"21410566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Missense mutations in the RING-H2 and UBR domains of human UBR1 (corresponding to JBS alleles) can be modeled in yeast Ubr1 and show graded activity defects: H160R (in UBR box) is completely inactive, Q1224E has weak activity, and V146L has reduced but significant activity, correlating with clinical severity of JBS in corresponding patients.\",\n      \"method\": \"Yeast-based functional complementation assay, N-end rule substrate degradation assay, site-directed mutagenesis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assay in heterologous system with multiple alleles correlated to clinical data, single lab\",\n      \"pmids\": [\"21931868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In C. elegans, UBR-1 physically associates with glutamate metabolic enzymes (GLN-3, GOT-2.2, GFAT-1, GDH-1) identified by CRISPR-based proteomics. Genetic interaction studies show UBR-1, GLN-3, and GOT-2.2 form a signaling axis regulating glutamate homeostasis required for locomotion and developmental viability, with UBR-1 functioning via both ubiquitin ligase and scaffolding mechanisms.\",\n      \"method\": \"CRISPR-based proteomics, genetic interaction analysis, super-resolution imaging, pharmacological rescue\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR proteomics plus multi-gene interaction studies plus pharmacology, preprint, single lab\",\n      \"pmids\": [\"40766417\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"UBR1 is a RING-type E3 ubiquitin ligase with three substrate-binding sites: a type-1 site (UBR box) for basic N-terminal residues, a type-2 site (N domain) for bulky hydrophobic N-terminal residues, and an internal-degron site; structural studies reveal it mediates both ubiquitin chain initiation and K48-linked elongation through a single E2 (Ubc2/Rad6), whose activity is modulated by CDK-dependent phosphorylation, while the Ubr1–Ufd4 complex enhances processivity; beyond canonical N-degron substrates, UBR1 performs chaperone-dependent cytoplasmic protein quality control of misfolded proteins requiring Hsp70 ATPase activity, participates in ERAD, endosomal autophagy, and ER stress responses, ubiquitinates substrates including c-Fos, Plin2, RECQL4, ACE2, and YAP, is allosterically activated by amino acid binding, and is reprogrammed under stress by the cleaved intrinsically disordered protein Roq1 through a heterobivalent binding mechanism that shifts substrate specificity toward misfolded proteins.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBR1 is a RING-type E3 ubiquitin ligase that serves as a central recognition component of the N-end rule (N-degron) pathway and as a broad protein quality-control ligase [#1, #13]. Substrate recognition is partitioned across distinct sites within its N-terminal region: a type-1 site for basic N-terminal residues and a type-2 site for bulky hydrophobic N-terminal residues, both with micromolar affinity, plus a separate internal-degron site that drives CUP9 ubiquitylation [#1]. Cryo-EM structures show that UBR1 mediates both ubiquitin chain initiation and linkage-specific polyubiquitin elongation through a single E2 enzyme, Ubc2/Rad6, using a defined E2-binding region and an acceptor-ubiquitin-binding loop [#0]; processivity is further enhanced by physical partnership with the HECT ligase Ufd4 [#2], and the system is tuned by CDK1/2 phosphorylation of Ubc2 at Ser120 [#12] and by casein-kinase/Mck1 phosphorylation of UBR1 itself that controls peptide-import regulation via CUP9 [#11]. Beyond canonical N-degron substrates, UBR1 performs chaperone-dependent cytoplasmic quality control of misfolded proteins, directly binding denatured substrate and requiring Hsp70 ATPase activity together with the Hsp40 Sis1 and nucleotide-exchange cofactors [#3, #5, #6, #7]; it extends this surveillance to ERAD substrates in concert with Hsp70 (Ssa1) and Cdc48 [#8], to mistranslocated secretory/mitochondrial proteins recognized via the P2 residue [#22], and to endosomal quality control and selective endophagy with SQSTM1/p62 [#23]. UBR1 activity is allosterically gated by amino-acid binding at its canonical pockets, which relieves auto-inhibition to drive degradation of substrates such as the lipid-droplet protein Plin2 and links UBR1 to leucine–mTOR signaling [#20, #21], and under proteotoxic stress its specificity is reprogrammed by the protease-cleaved hydrophilin Roq1 through a heterobivalent mechanism using an N-terminal arginine and a hydrophobic motif [#16, #17]. Loss-of-function mutations in UBR1 cause Johanson-Blizzard syndrome, and graded activity defects of disease alleles in the RING-H2 and UBR domains correlate with clinical severity [#15, #28]. Additional reported substrates include c-Fos, the glucocorticoid and androgen receptors as Hsp90 clients, ACE2, and YAP [#9, #18, #24, #25].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established that mammalian UBR1 is a functionally conserved N-end rule E3 ligase, not merely a yeast factor, and that it operates redundantly in some tissues.\",\n      \"evidence\": \"Functional complementation of yeast ubr1Δ by mouse UBR1 with a cognate E2, plus tissue-specific N-end rule activity analysis in UBR1-knockout mice\",\n      \"pmids\": [\"11689692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of compensating UBR1-like proteins in fibroblasts not resolved\", \"Physiological substrate in skeletal muscle beyond fatty acid synthase regulation not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed UBR1 can form stable complexes with substrates without degrading them, indicating association does not always equal ubiquitylation.\",\n      \"evidence\": \"Reciprocal Co-IP of RECQL4 with UBR1/UBR2 in HeLa cells, in vivo ubiquitylation analysis, and ATPase assay of the isolated complex\",\n      \"pmids\": [\"15317757\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the RECQL4–UBR1/2 complex unknown\", \"Why a bound substrate escapes ubiquitylation not explained\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked UBR1 loss to a human Mendelian disease, defining its physiological importance in exocrine pancreatic function.\",\n      \"evidence\": \"Human mutation identification in 12 Johanson-Blizzard families plus pancreatic functional analysis of UBR1-knockout mice\",\n      \"pmids\": [\"16311597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific acinar-cell N-end rule substrates whose stabilization causes pathology not identified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended UBR1 to regulated turnover of a signaling transcription factor, showing its activity is gated by phosphorylation and induced transcriptionally.\",\n      \"evidence\": \"Co-IP, in vivo ubiquitylation, siRNA, and phospho-site mutagenesis linking UBR1, c-Fos, ERK5, and STAT3/IL-6\",\n      \"pmids\": [\"17018293\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether c-Fos is an N-degron or internal-degron substrate not defined\", \"Generality beyond the cell systems tested unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined an essential developmental role by removing redundancy, placing UBR1/UBR2 upstream of neurogenic and cardiovascular regulators.\",\n      \"evidence\": \"UBR1/UBR2 double-knockout mouse embryology with cyclin D and Notch1 marker analysis\",\n      \"pmids\": [\"16606826\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct substrates connecting UBR1 to cyclin D/Notch1 not identified\", \"Relative contributions of UBR1 vs UBR2 not separated\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapped UBR1's substrate recognition architecture, defining separable type-1, type-2, and internal-degron sites and their affinities.\",\n      \"evidence\": \"Loss-of-function genetic screen, fluorescence polarization and SPR binding measurements, and fully reconstituted CUP9 ubiquitylation in vitro\",\n      \"pmids\": [\"18566452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of site cooperativity not resolved at this stage\", \"Internal-degron recognition determinants only defined for CUP9\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Established that UBR1 itself is phosphoregulated, connecting kinase signaling to N-end rule control of peptide import.\",\n      \"evidence\": \"In vivo phospho-site mapping, Yck1/2 and Mck1 kinase analysis, in vitro kinase assays, and peptide-import readouts\",\n      \"pmids\": [\"19033468\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural effect of Ser300 phosphorylation on UBR1 conformation unknown\", \"Whether phosphoregulation extends beyond CUP9-dependent import not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified UBR1 as the cytoplasmic quality-control ligase for misfolded proteins, a role distinct from N-end rule recognition.\",\n      \"evidence\": \"Genetic deletion and ubiquitination assays with misfolded reporter substrates in yeast\",\n      \"pmids\": [\"19041308\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; chaperone dependence not yet dissected here\", \"Degron recognized on misfolded substrates undefined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed cytoplasmic misfolded-protein quality control by UBR1 is chaperone-dependent and operates in parallel with nuclear San1.\",\n      \"evidence\": \"Genetic epistasis (double-mutant analysis), biochemical ubiquitination assays, and proteotoxic-stress phenotyping\",\n      \"pmids\": [\"20080635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic basis of cytoplasmic/nuclear division of labor not fully mapped\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated direct UBR1 recognition of denatured substrate and reconstituted chaperone-stimulated ubiquitination, with E3s competing with chaperones.\",\n      \"evidence\": \"In vitro ubiquitylation with purified Ubr1 and denatured luciferase plus genetic suppressor analysis of chaperone mutants\",\n      \"pmids\": [\"20462952\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular feature of denatured substrate recognized by Ubr1 not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Explained how a single E2 supports both initiation and elongation by showing CDK phosphorylation of Ubc2 tunes each step differently.\",\n      \"evidence\": \"In vitro Ubr1-catalyzed ubiquitination kinetics with Ubc2 Ser120 phosphomimetic/null mutants plus cell-based N-end rule assay\",\n      \"pmids\": [\"21041297\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; in vivo CDK regulation of N-end rule degradation not directly shown\", \"Cell-cycle timing of this control not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Increased polyubiquitin processivity by partnering UBR1 with a HECT ligase and recognition of the UFD ubiquitin moiety.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitylation with Ufd4, and genetic epistasis on UFD substrates\",\n      \"pmids\": [\"21076411\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the Ubr1–Ufd4 complex not resolved\", \"Generality of cooperativity across substrate classes not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connected UBR1 to nutrient signaling by identifying it as a leucine-binding negative regulator of mTOR-S6K signaling.\",\n      \"evidence\": \"Leucine-affinity pulldown, overexpression/knockdown with S6K1 phospho-readout, and in vitro N-end rule degradation assay\",\n      \"pmids\": [\"20298436\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between leucine binding and mTOR activity not fully defined\", \"Direct mTOR-pathway substrate of UBR1 not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed UBR1 acts as a quality-control ligase for specific Hsp90 clients, distinguishing client recognition by receptor identity.\",\n      \"evidence\": \"UBR1-deletion MEFs treated with geldanamycin and degradation kinetics of glucocorticoid, androgen, and estrogen receptors\",\n      \"pmids\": [\"21983172\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for client selectivity (GR/AR vs ERα) unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided nuclear, organism-specific evidence that UBR1 terminates a stress transcriptional response by degrading DNA-binding-competent Pap1.\",\n      \"evidence\": \"Fission-yeast genetic deletion, epistasis with export- and DNA-binding-mutant Pap1, and Western/growth assays\",\n      \"pmids\": [\"21410566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degron on activated Pap1 not defined\", \"Generalizability of nuclear UBR1 localization to other species unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linked specific Johanson-Blizzard alleles to graded enzymatic defects, establishing genotype–activity–severity correlation.\",\n      \"evidence\": \"Yeast functional complementation and N-end rule degradation assays with H160R, Q1224E, and V146L UBR domain/RING mutants\",\n      \"pmids\": [\"21931868\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Modeled in yeast rather than native human context\", \"Effects on chaperone-dependent quality-control activity not assessed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Ordered the chaperone-handoff to UBR1 by placing the Hsp40 Sis1 upstream of Ubr1 in misfolded-substrate triage.\",\n      \"evidence\": \"Genetic deletion/knockdown, imaging of misfolded reporter localization, and proteasomal degradation assays\",\n      \"pmids\": [\"23341891\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physical handoff between Sis1/Hsp70 and Ubr1 not biochemically resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Expanded cytosolic UBR1 quality control to ERAD of polytopic membrane substrates acting with Hsp70 and Cdc48.\",\n      \"evidence\": \"Genetic epistasis with canonical ERAD ligase deletions, degradation assays of Ste6*/CFTR, and interaction with Ssa1/Cdc48\",\n      \"pmids\": [\"23988329\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How retrotranslocated substrates are routed to Ubr1 vs canonical ERAD ligases unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed stress-induced reprogramming of UBR1 specificity by a cleaved hydrophilin, defining the SHRED pathway.\",\n      \"evidence\": \"Genetic epistasis, Roq1–Ubr1 interaction assays, Ynm3 cleavage assays, and stress-dependent substrate degradation\",\n      \"pmids\": [\"29861160\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of the specificity shift not yet defined at this stage\", \"Conservation of SHRED beyond yeast not established\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Uncovered a neuronal role for UBR-1 in glutamate metabolism controlling motor circuit output.\",\n      \"evidence\": \"C. elegans loss-of-function genetics, got-1 epistasis, neuronal calcium imaging, metabolite measurement, and cell-specific rescue\",\n      \"pmids\": [\"29649217\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the motor phenotype reflects ligase activity or a non-catalytic role unresolved at this stage\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed the N-end rule pathway enforces protein compartmentalization by Ubr1 degrading mistranslocated proteins via P2-position signals.\",\n      \"evidence\": \"Systematic P2-position mutant analysis, proteome bioinformatics, and genetic deletion assays\",\n      \"pmids\": [\"30940687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical demonstration of P2 recognition not provided\", \"Breadth of endogenous mislocalized substrate pool not quantified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the structural mechanism for how UBR1 uses a single E2 to perform linkage-specific chain initiation and elongation.\",\n      \"evidence\": \"Cryo-EM of Ubr1–Ubc2–ubiquitin–N-degron complexes with reaction-intermediate mimics and mutagenesis validation\",\n      \"pmids\": [\"34789879\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures of chaperone-dependent quality-control engagement not captured\", \"Conformational coupling to allosteric amino-acid regulation not visualized\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined UBR1 as an endosomal quality-control ligase driving selective endophagy of arginylated/ubiquitinated cargo.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, endosomal imaging, autophagy flux, and siRNA knockdown of mutant MLC1 turnover\",\n      \"pmids\": [\"35233680\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How UBR1 is recruited to endosomes mechanistically unknown\", \"Direct ESCRT-component interactions not mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that amino-acid binding allosterically activates UBR1 by relieving auto-inhibition, driving Plin2 degradation.\",\n      \"evidence\": \"Cell-free reconstitution with purified proteins, amino-acid binding and pulldown assays, in vitro ubiquitination, and cellular IP\",\n      \"pmids\": [\"37057345\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural conformation of the auto-inhibited vs activated states not resolved\", \"Physiological amino-acid concentrations governing this switch in vivo not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed UBR1 can stabilize rather than degrade a substrate via monoubiquitylation, with pro-tumorigenic consequences.\",\n      \"evidence\": \"siRNA screening, Co-IP, mono- vs poly-ubiquitination assays, half-life measurement, and xenografts for YAP in anaplastic thyroid carcinoma\",\n      \"pmids\": [\"39174635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism preventing chain elongation on YAP unknown\", \"Whether N-degron or internal-degron recognition is used not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected UBR1 to sex-dependent blood-pressure regulation through hormone-driven ACE2 degradation.\",\n      \"evidence\": \"Proteomics, Co-IP validation, in vivo/in vitro siRNA knockdown, blood-pressure measurement, and sex-hormone treatment\",\n      \"pmids\": [\"39601126\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination site on ACE2 not mapped\", \"Relative roles of UBR1 vs Nedd4-2 not separated mechanistically\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Dissected the heterobivalent logic of Roq1-mediated reprogramming, separating N-degron-substrate modulation from misfolded-protein targeting.\",\n      \"evidence\": \"Mutational analysis of Roq1 N-terminal arginine and hydrophobic motifs with in vivo and in vitro ubiquitination and interaction assays\",\n      \"pmids\": [\"39920309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure of the reprogrammed Ubr1–Roq1 complex not determined\", \"Mammalian functional homolog of SHRED reprogramming not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proposed that UBR-1 acts through both catalytic and scaffolding modes within a glutamate-homeostasis enzyme axis.\",\n      \"evidence\": \"CRISPR-based proteomics identifying enzyme partners, multi-gene genetic interactions, super-resolution imaging, and pharmacological rescue in C. elegans (preprint)\",\n      \"pmids\": [\"40766417\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint; not peer-reviewed\", \"Scaffolding (non-catalytic) function not biochemically separated from ligase activity\", \"Conservation of the axis in mammals untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UBR1 integrates its multiple substrate-binding sites, allosteric amino-acid sensing, chaperone partnerships, and stress-induced reprogramming into a unified conformational regulatory logic remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure captures the chaperone-dependent or allosterically activated states\", \"Determinants choosing degradation vs stabilization (e.g., mono- vs polyubiquitylation) not defined\", \"Mammalian counterpart of SHRED reprogramming not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2, 13]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 3, 9, 21, 24]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 12, 20, 21]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [10, 20]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 8, 9, 22]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [27]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 3, 8]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [16, 27]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [23]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [14, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [15, 28, 24, 25]}\n    ],\n    \"complexes\": [\n      \"Ubr1–Ufd4 complex\",\n      \"RECQL4–UBR1/UBR2 complex\"\n    ],\n    \"partners\": [\n      \"RAD6/UBC2\",\n      \"UFD4\",\n      \"RECQL4\",\n      \"HSPA (Hsp70/Ssa1)\",\n      \"SIS1\",\n      \"CDC48\",\n      \"SQSTM1\",\n      \"ROQ1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}