Affinage

CYB5A

Cytochrome b5 · UniProt P00167

Length
134 aa
Mass
15.3 kDa
Annotated
2026-06-09
26 papers in source corpus 10 papers cited in narrative 10 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/6 claims corpus-supported (83%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CYB5A encodes microsomal cytochrome b5, an electron-transfer hemoprotein that functions principally as an obligate cofactor for CYP17A1 17,20-lyase activity in steroidogenesis (PMID:25890314, PMID:32051920). CYB5A physically interacts with CYP17A1 through defined surface residues (including N62S, N21K, L28V, R52M), and this interaction selectively stimulates the 17,20-lyase and delta-16-synthase reactions to drive production of DHEA and 16-androstene from pregnenolone (PMID:31509771). Consistent with this cofactor role, CYB5A is required for adrenal androstenedione synthesis at the zona fasciculata/reticularis border where it is co-expressed with HSD3B2 (PMID:21185375), and its expression and activity quantitatively gate lyase output and androstenone (16-androstene) biosynthesis (PMID:25890314, PMID:16104384, PMID:25516134). Homozygous loss-of-function in CYB5A causes isolated 17,20-lyase deficiency together with methemoglobinemia in humans, establishing the in vivo requirement, while steroid metabolomics shows CYB5A dependence is developmentally staged and that an independent lyase activity handles cortisol side-chain cleavage (PMID:32051920, PMID:33626548). Beyond steroidogenesis, CYB5A acts redundantly with mitochondrial CYB5B to support the sterol-C4 oxidation/demethylation step of cholesterol biosynthesis, with either paralog sufficient to sustain this reaction (PMID:39489939). CYB5A additionally promotes autophagy via the AKT/mTOR/ULK1 axis in osteogenic differentiation (PMID:40246926).

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 2005 Medium

    Established that CYB5A enzymatic activity itself, not merely its abundance, causally drives 16-androstene biosynthesis, linking a regulatory variant to a steroid phenotype.

    Evidence Functional CYB5A activity assay across porcine 5'UTR genotypes with androstenone measurement in testis samples

    PMID:16104384

    Open questions at the time
    • Did not resolve the molecular interaction with CYP17A1
    • Associative link between activity and androstenone in tissue, not reconstituted
  2. 2010 Medium

    Defined the cellular basis for adrenal androgen synthesis by showing CYB5A is required for androstenedione production and is co-localized with HSD3B2 at the ZF/ZR border.

    Evidence siRNA knockdown in H295R adrenal cells with androstenedione readout plus co-immunolocalization in human adrenal tissue

    PMID:21185375

    Open questions at the time
    • Did not establish direct physical interaction with CYP17A1
    • Mechanism of CYB5A action on the lyase reaction not addressed
  3. 2014 Medium

    Identified promoter SNPs that alter transcription factor binding and CYB5A expression, explaining how regulatory variation translates into differential steroid output.

    Evidence Luciferase reporter assays, EMSA, and qPCR of liver tissue with in silico TF prediction in pigs

    PMID:25516134

    Open questions at the time
    • Predicted MAZ binding site not confirmed by direct factor identification
    • Connection to steroidogenic tissue regulation inferred from liver expression
  4. 2014 Low

    First implicated CYB5A in a non-steroidogenic role, suppressing oncogenic phenotypes through autophagy induction in pancreatic cancer cells.

    Evidence Loss-of-function in vitro and in vivo studies with autophagy readouts and TRAF6 pathway analysis

    PMID:24448000

    Open questions at the time
    • Limited methodological detail; no orthogonal mechanistic validation described
    • Direct link between cytochrome b5 electron-transfer function and autophagy not established
    • Role of TRAF6 not mechanistically resolved
  5. 2015 Medium

    Quantitatively established CYB5A as a dose-dependent augmenter of CYP17A1 17,20-lyase activity by linking an expression-increasing SNP to elevated lyase output.

    Evidence Radiolabeled steroid conversion assay in synovial fibroblasts with qRT-PCR and immunohistochemistry

    PMID:25890314

    Open questions at the time
    • Correlative between mRNA level and lyase activity
    • Did not map the interaction interface with CYP17A1
  6. 2019 High

    Resolved the molecular mechanism by mapping specific CYB5A surface residues that mediate physical interaction with CYP17A1 to stimulate the 17,20-lyase and delta-16-synthase reactions.

    Evidence Site-directed mutagenesis of CYB5A/CYP17A1 reconstituted with POR and CYB5R3 in HEK293, radiolabeled pregnenolone conversion by HPLC

    PMID:31509771

    Open questions at the time
    • No high-resolution structure of the CYB5A-CYP17A1 complex
    • Electron transfer versus allosteric contribution to lyase stimulation not fully separated
  7. 2019 Medium

    Demonstrated the in vivo requirement for CYB5A in human steroidogenesis, showing loss-of-function causes isolated 17,20-lyase deficiency with methemoglobinemia.

    Evidence Clinical case with genetic sequencing, hormonal studies, and methemoglobin measurement

    PMID:32051920

    Open questions at the time
    • Single case
    • Did not dissect tissue-specific contributions or developmental dependence
  8. 2021 Medium

    Refined the in vivo model by showing CYB5A dependence of DHEA synthesis is developmentally staged and that a separate, CYB5A-independent lyase activity handles cortisol side-chain cleavage.

    Evidence Urine steroid metabolomics across age groups in CYB5A-deficient patients with gonadotropin stimulation testing

    PMID:33626548

    Open questions at the time
    • Small case series
    • Identity of the CYB5A-independent lyase activity not determined
    • Mechanism of reduced neonatal DHEAS dependence unresolved
  9. 2024 High

    Defined a second metabolic role by showing CYB5A and mitochondrial CYB5B act redundantly to support the sterol-C4 demethylation step of cholesterol biosynthesis.

    Evidence CRISPR knockout of CYB5B in HeLa, liver-specific Cyb5b knockout mice with Cyb5a shRNA, sterol profiling and SREBP/PPARgamma analysis

    PMID:39489939

    Open questions at the time
    • Relative contribution of CYB5A versus CYB5B in steroidogenic tissues not addressed
    • Direct enzyme target of electron transfer in C4-demethylation not specified
  10. 2025 Medium

    Extended the autophagy role to a defined signaling axis, showing CYB5A promotes osteogenic differentiation by stimulating autophagy through AKT/mTOR/ULK1.

    Evidence Overexpression and siRNA in MC3T3-E1 cells with LC3/P62/phospho-mTOR/ULK1 Western blots and pharmacological autophagy modulation

    PMID:40246926

    Open questions at the time
    • How the cytochrome b5 electron-transfer function connects to AKT/mTOR signaling is unknown
    • Direct molecular partners upstream of AKT not identified
    • Single cell-line system

Open questions

Synthesis pass · forward-looking unresolved questions
  • How CYB5A's canonical electron-transfer biochemistry mechanistically links to its autophagy-promoting activity, and what governs its tissue- and developmental-stage-specific deployment across steroidogenesis and cholesterol biosynthesis, remains unresolved.
  • No structure of the CYB5A-CYP17A1 complex
  • Mechanistic bridge between electron transfer and AKT/mTOR/ULK1 signaling undefined
  • Identity of the CYB5A-independent cortisol lyase activity unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016491 oxidoreductase activity 3 GO:0098772 molecular function regulator activity 2
Localization
GO:0005783 endoplasmic reticulum 1
Pathway
R-HSA-1430728 Metabolism 3 R-HSA-9612973 Autophagy 1
Partners
CYB5BCYB5R3CYP17A1HSD3B2POR

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2010 CYB5A is required for androstenedione production in human adrenal cells: siRNA knockdown of CYB5A in H295R adrenal cells significantly inhibited androstenedione production, and co-expression of CYB5A with HSD3B2 was identified at the ZF/ZR border as the cellular basis for adrenal androstenedione synthesis. siRNA knockdown in H295R cells with androstenedione measurement; co-immunolocalization of HSD3B2 and CYB5A in human adrenal tissue The Journal of steroid biochemistry and molecular biology Medium 21185375
2015 A CYB5A intronic SNP (rs1790834) doubles CYB5A mRNA expression and results in 2–3-fold activation of steroid 17,20-lyase activity, establishing CYB5A as a critical cofactor that augments CYP17A1 17,20-lyase activity in human steroidogenesis. Radiolabeled steroid conversion assay in synovial fibroblasts; quantitative RT-PCR; immunohistochemistry of synovial tissue Arthritis research & therapy Medium 25890314
2005 A SNP in the 5' UTR of porcine CYB5A (G→T at position −8 of ATG) reduces CYB5A enzymatic activity and is associated with lower fat androstenone levels, demonstrating that CYB5A activity directly drives 16-androstene (androstenone) biosynthesis. Functional analysis of CYB5A enzyme activity in individuals of different genotypes; androstenone measurement in 229 testis samples Mammalian genome Medium 16104384
2019 Specific surface residues of porcine CYB5A mediate its physical interaction with CYP17A1 to stimulate 17,20-lyase and delta-16-synthase reactions: mutations at CYB5A surface residues (including N62S, N21K, L28V, R52M) altered production of DHEA and 16-androstene (16A) from pregnenolone, and the N62S mutation increased both 16A and DHEA production when combined with wild-type CYP17A1. Site-directed mutagenesis of CYB5A and CYP17A1; co-expression with POR and CYB5R3 in HEK293 cells; radiolabeled pregnenolone conversion measured by HPLC The Journal of steroid biochemistry and molecular biology High 31509771
2024 CYB5A and mitochondrial CYB5B redundantly support sterol C4-demethylation in cholesterol biosynthesis: knockout of CYB5B alone blocks cholesterol biosynthesis at the sterol-C4 oxidation step in HeLa cells (accumulating T-MAS and dihydro-T-MAS), whereas liver-specific Cyb5b knockout mice are rescued by residual Cyb5a; combined knockdown of both in liver causes marked T-MAS/dihydro-T-MAS accumulation, demonstrating that either CYB5A or CYB5B is sufficient for sterol C4-demethylation. CRISPR knockout of CYB5B in HeLa cells; liver-specific Cyb5b knockout mice with shRNA knockdown of Cyb5a; sterol profiling; SREBP/PPARγ pathway analysis Cell reports High 39489939
2019 Loss-of-function mutations in CYB5A cause isolated 17,20-lyase deficiency in humans: a homozygous nonsense mutation (p.Tyr35Ter) in CYB5A results in methemoglobinemia and biochemically confirmed isolated 17,20-lyase deficiency, demonstrating that CYB5A is required for CYP17A1 17,20-lyase activity in vivo. Clinical case with genetic sequencing; hormonal studies confirming isolated 17,20-lyase deficiency; methemoglobin measurement Journal of the Endocrine Society Medium 32051920
2021 Steroid metabolomics in patients with novel CYB5A loss-of-function variants reveals that CYB5A is required for DHEA synthesis postnatally but that neonatal DHEAS production is less dependent on CYB5A, and that a separate lyase activity independent of CYB5A handles cortisol side-chain cleavage. Urine steroid metabolomics (mass spectrometry) across age groups in CYB5A-deficient patients; gonadotropin stimulation testing Hormone research in paediatrics Medium 33626548
2014 CYB5A inhibits oncogenic phenotypes in pancreatic cancer cells through induction of autophagy, and its inhibitory activity involves downstream pathways including TRAF6. In vitro and in vivo studies (loss-of-function) with autophagy readouts; pathway analysis involving TRAF6 Autophagy Low 24448000
2025 CYB5A promotes osteogenic differentiation of MC3T3-E1 cells by stimulating autophagy through the AKT/mTOR/ULK1 signaling pathway: CYB5A overexpression increased LC3-II/LC3-I ratio, decreased P62, activated ULK1, and reduced mTOR phosphorylation; autophagy inhibitors and activators confirmed pathway mediation. Overexpression and siRNA knockdown in MC3T3-E1 cells; Western blotting for LC3-II/LC3-I, P62, phospho-mTOR, ULK1; autophagy inhibitor/activator pharmacological validation Scientific reports Medium 40246926
2014 SNPs in the porcine CYB5A promoter region affect transcription factor binding and CYB5A expression levels: haplotype B (containing specific SNPs) showed higher reporter activity in cell transfection assays, and EMSA demonstrated that the SNP at position 165901767 affects stable binding of transcription factors (including predicted MAZ binding site) to the CYB5A promoter, translating into differential androstenone accumulation between Chinese and European pig breeds. Luciferase reporter assay; electrophoretic mobility shift assay (EMSA); quantitative PCR of liver tissues; in silico transcription factor prediction Animal genetics Medium 25516134

Source papers

Stage 0 corpus · 26 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2010 Human adrenal cells that express both 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) contribute to adrenal androstenedione production. The Journal of steroid biochemistry and molecular biology 28 21185375
2015 CYB5A polymorphism increases androgens and reduces risk of rheumatoid arthritis in women. Arthritis research & therapy 24 25890314
2014 3βHSD and CYB5A double positive adrenocortical cells during adrenal development/aging. Endocrine research 20 24832628
2005 A novel polymorphism in the 5' untranslated region of the porcine cytochrome b5 (CYB5) gene is associated with decreased fat androstenone level. Mammalian genome : official journal of the International Mammalian Genome Society 20 16104384
2012 Evaluation of polymorphisms in the sulfonamide detoxification genes NAT2, CYB5A, and CYB5R3 in patients with sulfonamide hypersensitivity. Pharmacogenetics and genomics 15 22850190
2014 Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women. Cancer causes & control : CCC 13 25225034
2014 Differential expression of CYB5A in Chinese and European pig breeds due to genetic variations in the promoter region. Animal genetics 13 25516134
2014 CYB5A and autophagy-mediated cell death in pancreatic cancer. Autophagy 12 24448000
2019 Isolated 17,20-Lyase Deficiency in a CYB5A Mutated Female With Normal Sexual Development and Fertility. Journal of the Endocrine Society 11 32051920
2004 Disruption of the Candida albicans CYB5 gene results in increased azole sensitivity. Antimicrobial agents and chemotherapy 11 15328107
2024 Defects in CYB5A and CYB5B impact sterol-C4 oxidation in cholesterol biosynthesis and demonstrate regulatory roles of dimethyl sterols. Cell reports 8 39489939
2019 Effect of mutations in porcine CYB5A and CYP17A1 on the metabolism of pregnenolone. The Journal of steroid biochemistry and molecular biology 8 31509771
2012 Evaluation of polymorphisms in the sulfonamide detoxification genes CYB5A and CYB5R3 in dogs with sulfonamide hypersensitivity. Journal of veterinary internal medicine 7 22816446
2022 Functional variants in cytochrome b5 type A (CYB5A) are enriched in Southwest American Indian individuals and associate with obesity. Obesity (Silver Spring, Md.) 6 35043601
2016 2,3,7,8 Tetrachlorodibenzo-p-dioxin-induced RNA abundance changes identify Ackr3, Col18a1, Cyb5a and Glud1 as candidate mediators of toxicity. Archives of toxicology 6 27136898
2008 Effect of polymorphism in the porcine cytochrome b5 ( CYB5A) gene on androstenone and skatole concentrations and sexual development in Swedish pig populations. Animal : an international journal of animal bioscience 6 22445012
2025 CYB5A promotes osteogenic differentiation of MC3T3-E1 cells through autophagy mediated by the AKT/mTOR/ULK1 signaling pathway. Scientific reports 5 40246926
2021 Isolated 17, 20 Lyase Deficiency Secondary to a Novel CYB5A Variant: Comparison of Steroid Metabolomic Findings with Published Cases Provides Diagnostic Guidelines and Greater Insight into Its Biological Role. Hormone research in paediatrics 4 33626548
2021 Lack of association between CYB5A gene rs1790834 polymorphism and the response to leflunomide in women with rheumatoid arthritis. European journal of clinical pharmacology 3 34160668
2025 Two families, two pathways: a case series of 46, XY DSD with 17α-hydroxylase deficiency and isolated 17,20-lyase deficiency due to novel CYB5A variant. Journal of pediatric endocrinology & metabolism : JPEM 2 40123165
2023 The association between CYB5A gene rs1790834 polymorphism and clinical improvement after 6 months of leflunomide treatment in women with rheumatoid arthritis. Clinical rheumatology 1 37289314
2022 An alternative CYB5A transcript is expressed in aneuploid ALL and enriched in relapse. BMC genomic data 1 35436854
2016 Development of CYB5-fusion monitoring system for efficient periplasmic expression of multimeric proteins in Escherichia coli. Protein expression and purification 1 27524697
2026 The azole-resistance phenotype of a Nakaseomyces glabratus clinical strain encoding a wild-type PDR1 allele involves the efflux pumps Aus1 and Pdh1 and Cyb5, an alternative reductase required for ergosterol biosynthesis. Microbiology spectrum 0 41891695
2025 Haplotype-based association of CYB5A gene polymorphisms (rs1790834 and rs1790858) with polycystic ovary syndrome in a south Indian cohort. Gene 0 41052660
2025 Micro RNA miR-726-3p targets CYB5A in Hen ovaries to modulate granulosa cell proliferation and differentiation. Scientific reports 0 41453937

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