id: Q7L1T6 gene_symbol: CYB5R4 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: >- CYB5R4 (NADH-cytochrome b5 reductase 4; also known as NCB5OR or b5+b5R) is a multidomain, soluble flavohemoprotein and NAD(P)H-dependent oxidoreductase. It contains an N-terminal CS/Hsp20 (p23-like) domain, a cytochrome b5-like heme-binding domain (with axial His-89 and His-112 ligating a six-coordinate low-spin heme) joined by a long hinge to a C-terminal FAD-dependent cytochrome-b5-reductase (FNR-type FAD- and NAD-binding) module. It binds stoichiometric heme and FAD and catalyzes EC 1.6.2.2 (NADH:cytochrome b5 oxidoreductase), reducing electron acceptors including cytochrome b5, cytochrome c, methemoglobin and ferricyanide; unlike the classical single-domain cytochrome b5 reductase it lacks a membrane anchor. The protein localizes to the endoplasmic reticulum and perinuclear cytoplasm. It functions in the response to oxidative and endoplasmic reticulum stress, protecting cells (notably pancreatic beta cells) from excess reactive oxygen species; loss of the orthologous gene in mice causes diabetes and lipoatrophy. Although early work proposed it as an NAD(P)H oxidase / candidate oxygen sensor, subsequent enzymology showed it preferentially reduces substrates rather than transferring electrons to molecular oxygen and is not an efficient superoxide-generating oxidase. references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000024 title: Manual transfer of experimentally-verified manual GO annotation data to orthologs by curator judgment of sequence similarity findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000107 title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara findings: [] - id: GO_REF:0000117 title: Electronic Gene Ontology annotations created by ARBA machine learning models findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:10611283 title: Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells. findings: - statement: Human b5+b5R (NCB5OR) is a flavohemoprotein with cytochrome b5 and b5 reductase domains that binds heme, FAD and NAD(P)H, is reduced by NAD(P)H, reduces cytochrome c in vitro, and localizes to the cytosolic perinuclear space; proposed as a candidate oxygen sensor. reference_section_type: ABSTRACT - id: PMID:15131110 title: NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum. findings: - statement: Recombinant NCB5OR is soluble with stoichiometric heme and FAD, colocalizes with calreticulin (ER marker), actively reduces cytochrome c, and produces superoxide only with very slow turnover, indicating it preferentially reduces substrates rather than acting as an NAD(P)H oxidase. reference_section_type: ABSTRACT - id: Reactome:R-HSA-1237044 title: Erythrocytes take up carbon dioxide and release oxygen findings: [] - id: Reactome:R-HSA-6806831 title: CYB5Rs reduce MetHb to HbA findings: [] - id: file:human/CYB5R4/CYB5R4-uniprot.txt title: UniProt entry Q7L1T6 (NB5R4_HUMAN), Cytochrome b5 reductase 4 findings: [] existing_annotations: - term: id: GO:0004128 label: cytochrome-b5 reductase activity, acting on NAD(P)H evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: Phylogenetic (IBA) assignment of the core cytochrome-b5 reductase activity. This matches the experimentally demonstrated EC 1.6.2.2 activity and is the core molecular function. action: ACCEPT reason: The cytochrome-b5 reductase activity acting on NAD(P)H is directly supported by experimental characterization of NCB5OR, which contains a cytochrome-b5-reductase domain and reduces cytochrome b5-type acceptors using NAD(P)H. supported_by: - reference_id: PMID:10611283 supporting_text: We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. - term: id: GO:0020037 label: heme binding evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: Phylogenetic assignment of heme binding, supported by experimental evidence that NCB5OR binds stoichiometric heme via its cytochrome b5-like domain. action: ACCEPT reason: NCB5OR contains a cytochrome b5-like heme-binding domain with axial His ligands and binds stoichiometric heme experimentally. supported_by: - reference_id: PMID:15131110 supporting_text: Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. - term: id: GO:0005783 label: endoplasmic reticulum evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in review: summary: Phylogenetic assignment of ER localization, consistent with experimental colocalization of NCB5OR with the ER marker calreticulin. action: ACCEPT reason: NCB5OR colocalizes with the ER marker calreticulin and is annotated by UniProt to the endoplasmic reticulum. supported_by: - reference_id: PMID:15131110 supporting_text: we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. - term: id: GO:0006801 label: superoxide metabolic process evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in review: summary: Phylogenetic assignment of superoxide metabolic process. NCB5OR can produce superoxide in vitro, but only with very slow turnover, and later enzymology concluded it preferentially reduces substrates rather than transferring electrons to oxygen, so this process is not a core in vivo function. action: KEEP_AS_NON_CORE reason: Superoxide production by NCB5OR occurs only at very low turnover rates in vitro; the protein is concluded to be a substrate reductase rather than an oxidase, so superoxide metabolism is at most a minor/non-core activity. supported_by: - reference_id: PMID:15131110 supporting_text: both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates - term: id: GO:0005783 label: endoplasmic reticulum evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: located_in review: summary: Electronic (IEA) ER localization, redundant with and supported by the experimental IDA ER annotation. action: ACCEPT reason: ER localization is experimentally established by colocalization with calreticulin. supported_by: - reference_id: PMID:15131110 supporting_text: we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. - term: id: GO:0016491 label: oxidoreductase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: Very general InterPro-based oxidoreductase activity. Correct but uninformative given the more specific cytochrome-b5 reductase activity annotations. action: MARK_AS_OVER_ANNOTATED reason: This is a high-level parent of the specific cytochrome-b5 reductase activity that is already annotated; it is correct but too general to be informative. supported_by: - reference_id: PMID:10611283 supporting_text: Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase - term: id: GO:0016653 label: oxidoreductase activity, acting on NAD(P)H, heme protein as acceptor evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: enables review: summary: ARBA machine-learning annotation of oxidoreductase activity acting on NAD(P)H with a heme protein as acceptor, redundant with the experimental IDA annotation of the same term. action: ACCEPT reason: This activity is directly supported by experimental demonstration that NCB5OR uses NAD(P)H to reduce heme-protein acceptors such as cytochrome b5/cytochrome c and methemoglobin. supported_by: - reference_id: PMID:15131110 supporting_text: reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro - term: id: GO:0020037 label: heme binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: InterPro-based heme binding annotation, redundant with and supported by the IBA heme binding annotation and experimental evidence of stoichiometric heme. action: ACCEPT reason: Heme binding is experimentally established and the protein has a cytochrome b5-like heme-binding domain with axial His ligands. supported_by: - reference_id: PMID:15131110 supporting_text: Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. - term: id: GO:0090524 label: cytochrome-b5 reductase activity, acting on NADH evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: EC/Rhea-based annotation of NADH-specific cytochrome-b5 reductase activity (EC 1.6.2.2). Supported by experimental catalytic activity; closely related to the NAD(P)H form. action: ACCEPT reason: UniProt records the catalytic reaction 2 Fe(III)-[cytochrome b5] + NADH = 2 Fe(II)-[cytochrome b5] + NAD+ + H+ (EC 1.6.2.2), demonstrated experimentally. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: 'EC=1.6.2.2 {ECO:0000305|PubMed:10611283}; Reaction=2 Fe(III)-[cytochrome b5] + NADH = 2 Fe(II)-[cytochrome b5] + NAD(+) + H(+)' - term: id: GO:0004128 label: cytochrome-b5 reductase activity, acting on NAD(P)H evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: enables review: summary: Ensembl Compara ortholog transfer of the core cytochrome-b5 reductase activity, redundant with the experimental IDA annotation of the same term. action: ACCEPT reason: The core cytochrome-b5 reductase activity acting on NAD(P)H is directly demonstrated experimentally. supported_by: - reference_id: PMID:10611283 supporting_text: The recombinant b5+b5R protein can be reduced by NAD(P)H ... and by cytochrome c reduction in vitro. - term: id: GO:0015701 label: bicarbonate transport evidence_type: TAS original_reference_id: Reactome:R-HSA-1237044 qualifier: involved_in review: summary: Reactome pathway-context annotation ("Erythrocytes take up carbon dioxide and release oxygen"). CYB5R4 is a redox enzyme, not a bicarbonate transporter; this is a pathway-membership artifact rather than a true molecular role. action: MARK_AS_OVER_ANNOTATED reason: CYB5R4 has no bicarbonate transport activity; the term derives from inclusion in a broad erythrocyte gas-exchange Reactome pathway and does not reflect the protein's function. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: NADH-cytochrome b5 reductase involved in endoplasmic reticulum stress response pathway. - term: id: GO:0004128 label: cytochrome-b5 reductase activity, acting on NAD(P)H evidence_type: TAS original_reference_id: Reactome:R-HSA-6806831 qualifier: enables review: summary: Reactome TAS annotation of the core cytochrome-b5 reductase activity (in the context of methemoglobin reduction), consistent with experimental data. action: ACCEPT reason: The cytochrome-b5 reductase activity is experimentally demonstrated and NCB5OR reduces methemoglobin in vitro. supported_by: - reference_id: PMID:15131110 supporting_text: reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro - term: id: GO:0072593 label: reactive oxygen species metabolic process evidence_type: IDA original_reference_id: PMID:15131110 qualifier: involved_in review: summary: ROS metabolic process supported by NCB5OR's redox chemistry and role in protecting cells against oxidant stress; consistent with the slow superoxide-producing activity and substrate-reducing behavior. action: KEEP_AS_NON_CORE reason: NCB5OR participates in cellular ROS handling/oxidant-stress protection, but this is a downstream physiological consequence of its redox activity rather than the core catalytic function. supported_by: - reference_id: PMID:15131110 supporting_text: both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates - term: id: GO:0005789 label: endoplasmic reticulum membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-6806831 qualifier: located_in review: summary: Reactome annotation to ER membrane. NCB5OR is explicitly a soluble protein lacking a membrane anchor, so an ER membrane localization is not accurate; it is associated with the ER but as a soluble protein. action: MARK_AS_OVER_ANNOTATED reason: Unlike classical single-domain cytochrome b5 reductase, NCB5OR has no membrane anchor and is soluble; the appropriate localization is the ER compartment, not the ER membrane. supported_by: - reference_id: PMID:10611283 supporting_text: b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. - term: id: GO:0030073 label: insulin secretion evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: involved_in review: summary: ISS transfer from the mouse ortholog (Q3TDX8). The Ncb5or-null mouse has a diabetes/beta-cell phenotype, so a role in insulin secretion is plausible but indirect and phenotype-driven, not a core molecular function. action: KEEP_AS_NON_CORE reason: A role in insulin secretion reflects the physiological consequence of beta-cell dysfunction in Ncb5or-deficient mice rather than a direct biochemical function of the enzyme. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: Plays a critical role in protecting pancreatic beta-cells against oxidant stress - term: id: GO:0042593 label: glucose homeostasis evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: involved_in review: summary: ISS transfer from the mouse ortholog. Consistent with the diabetic phenotype of Ncb5or-null mice, but a downstream physiological role rather than a core molecular function. action: KEEP_AS_NON_CORE reason: Glucose homeostasis is a systemic phenotype linked to beta-cell protection in mouse models, not a direct enzymatic function of CYB5R4. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: Plays a critical role in protecting pancreatic beta-cells against oxidant stress - term: id: GO:0046677 label: response to antibiotic evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: involved_in review: summary: ISS transfer from the mouse ortholog. There is no biological evidence linking CYB5R4 to an antibiotic response; this appears to be a spurious/non-specific transferred annotation. action: REMOVE reason: No experimental or mechanistic support connects CYB5R4 to a response to antibiotic; the term is implausible for a soluble ER redox enzyme and is not supported by the literature. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: NADH-cytochrome b5 reductase involved in endoplasmic reticulum stress response pathway. - term: id: GO:0048468 label: cell development evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: involved_in review: summary: Very general ISS transfer ("cell development"). Too broad to be informative and not directly supported by mechanistic data on CYB5R4. action: MARK_AS_OVER_ANNOTATED reason: Cell development is a high-level, non-specific process term that does not capture the redox/stress-protection function of CYB5R4 and is only indirectly inferred from mouse phenotypes. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: Plays a critical role in protecting pancreatic beta-cells against oxidant stress - term: id: GO:0003032 label: detection of oxygen evidence_type: NAS original_reference_id: PMID:10611283 qualifier: involved_in review: summary: NAS annotation based on the early hypothesis that b5+b5R is a candidate oxygen sensor. Later enzymology showed it is a substrate reductase, not an efficient oxygen-utilizing oxidase, undermining the oxygen-sensing proposal. action: REMOVE reason: The oxygen-sensor/oxygen-detection role was a speculative proposal in the 1999 paper that was superseded by 2004 enzymology showing NCB5OR preferentially reduces substrates rather than reacting with molecular oxygen; there is no direct evidence for oxygen detection. supported_by: - reference_id: PMID:15131110 supporting_text: endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen - term: id: GO:0004128 label: cytochrome-b5 reductase activity, acting on NAD(P)H evidence_type: IDA original_reference_id: PMID:10611283 qualifier: enables review: summary: Direct experimental demonstration of the core NAD(P)H-dependent cytochrome-b5 reductase activity. This is the central molecular function of CYB5R4. action: ACCEPT reason: The recombinant protein is reduced by NAD(P)H and reduces cytochrome b5-type/cytochrome c acceptors, directly demonstrating cytochrome-b5 reductase activity (EC 1.6.2.2). supported_by: - reference_id: PMID:10611283 supporting_text: The recombinant b5+b5R protein can be reduced by NAD(P)H ... and by cytochrome c reduction in vitro. - term: id: GO:0005783 label: endoplasmic reticulum evidence_type: IDA original_reference_id: PMID:15131110 qualifier: located_in review: summary: Direct experimental ER localization via colocalization with the ER marker calreticulin. This is the well-supported subcellular localization. action: ACCEPT reason: Subcellular fractionation and confocal microscopy show colocalization with calreticulin, establishing ER localization. supported_by: - reference_id: PMID:15131110 supporting_text: we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. - term: id: GO:0006801 label: superoxide metabolic process evidence_type: IDA original_reference_id: PMID:10611283 qualifier: involved_in review: summary: Based on in vitro superoxide production observed with air and excess NAD(P)H. Later work showed this occurs only at very slow turnover and that NCB5OR is not a bona fide oxidase, making superoxide metabolism a non-core activity. action: KEEP_AS_NON_CORE reason: Superoxide production is a slow, minor in vitro activity; NCB5OR is concluded to preferentially reduce substrates rather than generate superoxide, so this is not a core function. supported_by: - reference_id: PMID:10611283 supporting_text: superoxide production in the presence of air and excess NAD(P)H - term: id: GO:0016174 label: NAD(P)H oxidase H2O2-forming activity evidence_type: IDA original_reference_id: PMID:15131110 qualifier: enables negated: true review: summary: Correctly negated annotation. The 2004 enzymology concluded that NCB5OR is not an NAD(P)H oxidase for superoxide/H2O2 production, refuting the earlier oxidase interpretation. action: ACCEPT reason: The NOT annotation accurately reflects the experimental conclusion that NCB5OR is a substrate reductase, not an NAD(P)H oxidase. supported_by: - reference_id: PMID:15131110 supporting_text: therefore not an NAD(P)H oxidase for superoxide production - term: id: GO:0016174 label: NAD(P)H oxidase H2O2-forming activity evidence_type: IDA original_reference_id: PMID:10611283 qualifier: enables review: summary: Positive (non-negated) NAD(P)H oxidase annotation from the original 1999 report. This interpretation was explicitly refuted by the 2004 study, which established the NOT annotation for the same activity. action: REMOVE reason: The proposed NAD(P)H oxidase activity was superseded and refuted; NCB5OR produces superoxide only at negligible rates and is concluded not to be an oxidase, so this positive annotation is incorrect. supported_by: - reference_id: PMID:15131110 supporting_text: endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production - term: id: GO:0016653 label: oxidoreductase activity, acting on NAD(P)H, heme protein as acceptor evidence_type: IDA original_reference_id: PMID:15131110 qualifier: enables review: summary: Direct experimental demonstration that NCB5OR uses NAD(P)H to reduce heme-protein acceptors (cytochrome c, methemoglobin). This is a core, well-supported molecular function. action: ACCEPT reason: NCB5OR actively reduces cytochrome c and methemoglobin using NAD(P)H, directly demonstrating oxidoreductase activity acting on NAD(P)H with a heme protein as acceptor. supported_by: - reference_id: PMID:15131110 supporting_text: reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro - term: id: GO:0048471 label: perinuclear region of cytoplasm evidence_type: IDA original_reference_id: PMID:10611283 qualifier: located_in review: summary: Direct observation of cytosolic perinuclear localization in COS-7 cells. Consistent with the soluble nature of the protein and its ER/perinuclear association. action: ACCEPT reason: Confocal microscopy of myc-tagged b5+b5R showed cytosolic localization in the perinuclear space, supporting this localization term. supported_by: - reference_id: PMID:10611283 supporting_text: confocal microscopy revealed a cytosolic localization at the perinuclear space. - term: id: GO:0050660 label: flavin adenine dinucleotide binding evidence_type: IC original_reference_id: PMID:15131110 qualifier: enables review: summary: Proposed annotation not present in the current GOA for CYB5R4. action: NEW reason: NCB5OR contains a defined FAD-binding domain and was shown to contain stoichiometric FAD, yet GO:0050660 is absent from existing annotations. supported_by: - reference_id: PMID:15131110 supporting_text: Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. - term: id: GO:0034976 label: response to endoplasmic reticulum stress evidence_type: IC original_reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt qualifier: involved_in review: summary: Proposed annotation not present in the current GOA for CYB5R4. action: NEW reason: The protein is annotated by UniProt as functioning in the ER stress response pathway and is ER-localized, supporting a response to ER stress process term. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: NADH-cytochrome b5 reductase involved in endoplasmic reticulum stress response pathway. core_functions: - description: NAD(P)H-dependent cytochrome-b5 reductase that transfers electrons from NAD(P)H, via bound FAD and a cytochrome b5-like heme center, to heme-protein acceptors (cytochrome b5, cytochrome c, methemoglobin), catalyzing EC 1.6.2.2. supported_by: - reference_id: PMID:10611283 supporting_text: The recombinant b5+b5R protein can be reduced by NAD(P)H ... and by cytochrome c reduction in vitro. - reference_id: PMID:15131110 supporting_text: reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro molecular_function: id: GO:0004128 label: cytochrome-b5 reductase activity, acting on NAD(P)H - description: Soluble ER/perinuclear flavohemoprotein binding stoichiometric heme (via a cytochrome b5-like domain) and FAD, supporting electron transfer. supported_by: - reference_id: PMID:15131110 supporting_text: Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. molecular_function: id: GO:0016653 label: oxidoreductase activity, acting on NAD(P)H, heme protein as acceptor - description: Contributes to protection of cells (notably pancreatic beta cells) from oxidative and endoplasmic reticulum stress, helping limit excess reactive oxygen species. supported_by: - reference_id: file:human/CYB5R4/CYB5R4-uniprot.txt supporting_text: Plays a critical role in protecting pancreatic beta-cells against oxidant stress, possibly by protecting the cell from excess buildup of reactive oxygen species (ROS). proposed_new_terms: [] suggested_questions: - question: What are the physiological electron acceptor(s) of CYB5R4 in vivo, given that it lacks a membrane anchor and is soluble within the ER/perinuclear compartment? - question: How does the N-terminal CS/Hsp20 (p23-like) domain contribute to function, folding, or partner interactions of CYB5R4? - question: By what mechanism does CYB5R4 protect pancreatic beta cells from oxidative and ER stress, and is this a direct antioxidant role or an indirect consequence of its reductase activity? suggested_experiments: - description: Identify the endogenous electron acceptor(s) and interacting partners of CYB5R4 in beta cells using proximity labeling (BioID/APEX) and reconstituted electron-transfer assays with candidate cytochrome b5 / desaturase systems. - description: Generate beta-cell-specific CYB5R4 knockout or catalytic-dead (heme/FAD-binding mutant) cell lines and measure ER stress markers, ROS levels, and insulin secretion under oxidative challenge to define the mechanism of cytoprotection. - description: Perform steady-state and stopped-flow enzymology comparing reduction of physiological heme-protein acceptors versus molecular oxygen to quantitatively confirm that substrate reduction, not oxidase activity, dominates in vivo.