CYP7B1

Summary: Refine the valid broad steroid hydroxylase inference to the directly established 7-alpha activity.

Reason: The actual PTHR24304 tree places human O75881 at PTN002518411 below the GO:0008395 IBD at PTN001211310. The ungapped target MSA sequence matches all 506 residues, and no loss annotation occurs on the target path. The current IBD includes human CYP7B1 among its experimental descendants; that legitimate self-grounding is not circular. No exact broad-term experimental row remains in the retrieved descendant records, but the positive specific hydroxylase assays and current human EXP annotations provide the underlying chemistry. Human assays support the more informative GO:0008387 steroid 7-alpha-hydroxylase activity across oxysterol and other 3-hydroxy steroid substrates. The secondary product discussed in PMID:24491228 is not used here as independent proof of a particular additional hydroxylation position. This target-specific refinement does not challenge the IBD node or transfer every substrate specificity of CYP7A1 or CYP39A1 to CYP7B1. GO:0008387 is also present in current GOA via GO_REF:0000117 and ARBA:ARBA00092070; its current rule-premise mismatch is evaluated separately from the independently demonstrated chemistry.

Proposed replacements: steroid 7-alpha-hydroxylase activity

Summary: CYP7B1 performs an oxysterol hydroxylation step of alternative bile acid synthesis.

Reason: Human CYP7B1 lies below the actual GO:0006699 IBD at PTN001548757. Its literal descendants resolve to mouse Cyp7b1 and Cyp39a1, rat Cyp7a1, and human CYP7A1 and CYP39A1. Five current experimental same-term records are recovered, including the positive mouse Cyp7b1 record linked to PMID:10748047. The original paper assays CYP7B1 as well as the CYP39A1 emphasized in its title; these are distinct substrate routes within bile acid synthesis. Independent human CYP7B1 liver-membrane and recombinant assays in PMID:9802883 and PMID:10588945 establish that the target performs the hydroxylation step. The inference does not rest on disease necessity alone.

Summary: Oxysterol clearance and entry into bile acid synthesis provide a defensible contextual contribution to cholesterol homeostasis.

Reason: The GO:0042632 IBD at PTN001548757 is on the reconstructed target path. Its sole literal donor RGD:2482 resolves to rat Cyp7a1/P18125, which has a current IDA record from PMID:1694852. The complete abstract reports regulation of actual hepatic enzyme activity as well as protein and mRNA by cholesterol, cholestyramine and bile-acid feedback. A single donor does not invalidate a PAINT inference. Human CYP7B1 independently clears oxygenated cholesterol substrates into the alternative bile-acid pathway, supporting the broader homeostatic role without assigning CYP7A1's direct cholesterol reaction or rat transcriptional responses to CYP7B1. The physiological scope is retained as non-core; the rat original beyond its complete abstract was not recovered.

Summary: CYP7B1 performs steroid 7-alpha-hydroxylation.

Reason: The InterPro-derived monooxygenase class is biologically sound, but steroid 7-alpha-hydroxylase activity is a more informative description of the directly demonstrated human 25/26-hydroxycholesterol, DHEA and pregnenolone transformations (PMID:9802883, PMID:10588945, PMID:24491228). This refinement describes the established major chemistry; it does not deny additional substrate-dependent hydroxylation positions. GO:0008387 is also present in current GOA via GO_REF:0000117 and ARBA:ARBA00092070; its current rule-premise mismatch is evaluated separately from the independently demonstrated chemistry.

Proposed replacements: steroid 7-alpha-hydroxylase activity

Summary: The catalytic heme contains bound iron.

Reason: Purified human CYP7B1 has heme-dependent absorbance and a ferrous-CO P450 spectrum; heme content was measured by the pyridine hemochromogen method (PMID:24491228). The iron-binding assertion describes the catalytic heme, not a separate metal-storage or regulatory activity. The exact axial residue is supported by official sequence-family annotation rather than a new residue-specific assay here.

Summary: CYP7B1 is a microsomal enzyme associated with the endoplasmic reticulum membrane.

Reason: This UniProt location mapping is consistent with the human liver membrane enzyme measurements and the membrane-associated P450 protein in the primary studies. Official UniProt places CYP7B1 at ER and microsome membranes using curator inference from PMID:9802883. Its two transmembrane segments (17–37 and 289–309) are sequence predictions, not an experimentally mapped topology. The HPA response supplies no subcellular localization measurement. Retain the ER-membrane location without claiming a measured helix count, sidedness, or a direct human immunolocalization assay.

Summary: CYP7B1 directly metabolizes steroid substrates through hydroxylation.

Reason: Both complete predicates of the matching ARBA00027309 branches 3 and 36 are satisfied by the saved human taxonomy and official target family features. This broad process also has direct biochemical support: recombinant human CYP7B1 hydroxylates oxysterols, DHEA, pregnenolone and additional 3-hydroxy steroids in the primary assays. Steroid metabolism is retained without converting all of these turnover reactions into steroid hormone biosynthesis.

Summary: CYP7B1 receives reducing equivalents through a flavoprotein reductase.

Reason: The paired-donor oxidoreductase annotation is correct but can specify reduced flavin or flavoprotein as an electron donor and incorporation of one oxygen atom. The human enzyme was reconstituted with rat NADPH-cytochrome P450 reductase and NADPH in PMID:24491228. NADPH feeds the reductase; this does not assign CYP7B1 independent NADPH reductase activity. GO:0016712 is also present in current GOA via GO_REF:0000117 and ARBA:ARBA00027735; its current rule-premise mismatch is evaluated separately from the independently demonstrated chemistry.

Proposed replacements: oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen

Summary: Heme binding is integral to CYP7B1 catalysis.

Reason: The purified human enzyme has characteristic P450/ferrous-CO spectra, and the G57R and F216S preparations contain only trace heme compared with wild type (PMID:24491228). These measurements support heme binding, with the mutant folding failure kept separate from wild-type function.

Summary: The human 24S-specific activity remains unresolved across assay and transfer contexts.

Reason: The official 24S reaction is an inherited mouse-based catalytic assertion. Positive mouse CYP7B1 24-hydroxycholesterol conversion is genuinely reported in PMID:11067870 and PMID:10748047, although the inspected tracer descriptions do not resolve its 24 stereochemistry. In contrast, the tested N-terminally shortened human protein in PMID:24491228 showed spectral responses only to 25- and 27-hydroxycholesterol among a panel explicitly including 24S. Its quantitative Table 1 and supplements were not recovered. Preserve uncertainty about the specific human 24S claim rather than infer a wrong paralog, universal nonactivity, or a verified human turnover rate.

Summary: Human CYP7B1 directly hydroxylates both 25- and 26(27)-hydroxycholesterol.

Reason: GO:0033783 explicitly covers 25-hydroxycholesterol and (25R)-cholest-5-ene-3beta,26-diol. Human 25-substrate activity is reported in PMID:9802883 and directly characterized with purified enzyme in PMID:24491228; the human 27-substrate assay in PMID:10588945 supports the second reaction despite the shorter GO label. Preserve the literal electronic provenance while grounding the activity in these experiments.

Summary: BioPlex records a positive association of CYP7B1 with ubiquitin B in HEK293T cells.

Reason: The exact positive IntAct record EBI-21841310/IM-25778-10085 pairs O75881 with P0CG47 (UBB, not UBC). The original Methods use C-terminal HA-FLAG-tagged human bait proteins and anti-tag affinity purification followed by mass spectrometry. The literal record identifies CYP7B1 as bait in HEK293T cells; both sequence-derived ROGIDs match the official accessions. P0CG47 is the 229-residue ubiquitin B precursor, so its curated accession does not establish that an intact precursor, a particular processed ubiquitin unit, or a noncovalent interface was measured. Preserve this supported association under the standing project policy. It does not establish a specific ubiquitin binding activity, a ubiquitination site, or a core physiological interaction.

Summary: BioPlex 3.0 retains a positive CYP7B1–ubiquitin B physical association in HEK293T cells.

Reason: The exact positive IntAct record EBI-54746600/IM-29278-10863 pairs O75881 and UBB/P0CG47, with CYP7B1 as the tagged bait and HEK293T as the literal host. Official sequences reconstruct both recorded ROGIDs. Independently read original Methods describe anti-HA enrichment of C-terminal FLAG-HA constructs and protein-level mass-spectrometry identification. BioPlex 3.0 reanalyzes earlier BioPlex data, so these two publications are not claimed as independent repetitions of this particular pair. The normal cache's full-text flag exceeds its actual abstract/introduction content; the Methods were read from separately saved original PMC HTML. Retain the positive generic association as non-core without inferring direct noncovalent ubiquitin binding or a specific processed ubiquitin product.

Summary: The mouse-derived pathway mapping agrees with independently measured human CYP7B1 bile-acid chemistry.

Reason: The historical combined mapping to bile acid biosynthesis is preserved. Its current source context includes mouse Cyp7b1/Q60991, whose exact GO:0006699 IDA record points to PMID:10748047. The original compares CYP7B1 with CYP39A1 rather than excluding CYP7B1 from the experiments. This pathway role is independently supported by the human oxysterol reactions and native liver evidence in PMID:9802883 and PMID:10588945. No CYP39A1-specific 24-hydroxycholesterol preference is transferred merely because the enzymes share a downstream bile-acid pathway.

Summary: CYP7B1 supplies a chemotactic oxysterol ligand in the mouse pathway supporting the human orthology inference.

Reason: The actual mouse Q60991 donor carries an IMP B-cell-chemotaxis annotation from PMID:22999953. Its original Results measure loss of EBI2 ligand activity in Cyp7b1-deficient lymphoid tissues, restoration of ligand production by CH25H/CYP7B1 expression, and altered positioning of transferred activated B cells in deficient recipients. Bone-marrow chimeras identify a major stromal enzyme contribution. CYP7B1 performs the ligand-producing chemical step; it need not be the B-cell receptor or a motor to support this contextual process. Retain the Ensembl-Compara transfer as non-core, explicitly as inference from mouse physiology and conserved human hydroxylation chemistry, not a direct human chemotaxis experiment.

Summary: CYP7B1 is an enzyme in the modeled alternative routes to bile acids.

Reason: The inspected Reactome pathway R-HSA-192105 reaches the target through reaction 192065 and through subpathway 193807/reaction 191972. Both reactions use catalyst 192095 and candidate set 2975806, whose actual member 192117 resolves to O75881. This is a catalyst role, not mere membership as a consumed substrate. Primary human assays independently establish the 25- and 26(27)-hydroxycholesterol hydroxylation steps. CYP7B1 is not assigned all reactions or the CYP7A1-initiated classical pathway by this annotation.

Summary: CYP7B1 hydroxylates 26(27)-hydroxycholesterol within the alternative bile-acid route.

Reason: The actual path R-HSA-193807 → reaction 191972 → catalyst 192095 → set 2975806 → member 192117 → reference O75881 places CYP7B1 at the performed oxysterol hydroxylation step. The primary human enzyme assays support this chemistry. The historical 27-hydroxycholesterol name corresponds to the (25R)-26-hydroxycholesterol substrate in the current reaction description; this annotation does not imply that CYP7B1 carries out later side-chain shortening or bile-acid conjugation.

Summary: CYP7B1 directly transforms endogenous sterols.

Reason: Reactome R-HSA-211976 includes the target-bearing 25-hydroxycholesterol reaction 192065 through the verified catalyst/member path to O75881. Hydroxylation of 25- and 26(27)-hydroxycholesterol is directly demonstrated in human CYP7B1 assays, satisfying sterol metabolism. The broad pathway description also discusses cholesterol synthesis and elimination by other CYPs; those other enzymes' individual reactions are not assigned to CYP7B1.

Summary: The positive 27-hydroxycholesterol reaction has a chemically matching specific term.

Reason: The mapped 27-hydroxycholesterol hydroxylation is supported by human CYP7B1 assays in PMID:10588945 and PMID:24491228. GO:0033783 explicitly includes the 26(27)-hydroxy substrate. The present GO:0008396 definition instead specifies NADPH-dependent reduction of 7-oxocholesterol, inconsistent with this P450 hydroxylation. Refine the reviewed term without changing the literal Reactome source or claiming that oxysterol hydroxylation is absent. GO:0033783 is already asserted in three historical GOA annotations, including the human experimental sources; this is a refinement to existing supported chemistry.

Proposed replacements: 25-hydroxycholesterol 7-alpha-hydroxylase activity

Summary: The positive 25-hydroxycholesterol reaction is directly supported.

Reason: Human 25-hydroxycholesterol 7-alpha-hydroxylation is supported by PMID:9802883 and PMID:24491228. GO:0033783 matches that reaction, whereas the current GO:0008396 definition describes 7-oxocholesterol reduction with NADPH. The action corrects the reaction representation and preserves the positive source biology and literal Reactome assertion. GO:0033783 is already asserted in three historical GOA annotations, including the human experimental sources; this is a refinement to existing supported chemistry.

Proposed replacements: 25-hydroxycholesterol 7-alpha-hydroxylase activity

Summary: Normal CYP7B1 catalysis is supported separately from the defective-mutant event.

Reason: The original Reactome record concerns defective CYP7B1, so it must not be presented as a successful mutant hydroxylation assay. Independent human wild-type activity in PMID:9802883 and PMID:24491228 supports the gene-level 25-hydroxycholesterol reaction. Use GO:0033783, which matches that chemistry, rather than the current GO:0008396 ketone-reduction equation. In PMID:9802883 the plotted R388Stop comparison itself used mouse cDNA, whereas the human clone and human liver membrane measurements are separate positive evidence. GO:0033783 is already asserted in three historical GOA annotations, including the human experimental sources; this is a refinement to existing supported chemistry.

Proposed replacements: 25-hydroxycholesterol 7-alpha-hydroxylase activity

Summary: The positive mouse source does not fully settle the specific human 24S transfer.

Reason: Q60991 is a genuine mouse CYP7B1 donor, and PMID:11067870 actually uses mouse CYP7B1 to produce 7-alpha-hydroxylated 24-, 25- and 27-hydroxycholesterol intermediates. PMID:10748047 independently reports mouse 24-hydroxycholesterol turnover as the weakest tested substrate. These are positive source assays, not title-based paralog errors. However, the inspected tracer descriptions do not establish 24S configuration, and the human construct in PMID:24491228 did not show the corresponding 24S spectral response. Retain the original ISS donor and use UNDECIDED for the exact human 24S assertion.

Summary: The original human 27-substrate assay satisfies the complete term definition.

Reason: PMID:10588945 measures conversion of 27-hydroxycholesterol to its 7-alpha product by full-length and N-terminally shortened human CYP7B1 in 293/T cells, with a reverse-orientation construct as a negative control. GO:0033783 explicitly includes the 26(27)-hydroxycholesterol reaction, so acceptance does not require pretending that this paper directly tested 25-hydroxycholesterol.

Summary: The cited study includes direct human 25-hydroxycholesterol activity.

Reason: PMID:9802883 reports a threefold increase over background following transfection of the human 506-aa cDNA into 293 cells using 25-hydroxycholesterol or DHEA, and it measures 25-substrate conversion in control human liver microsomal membranes. Human-clone results are reported as data not shown. Figure 12 instead tests mouse WT/R388Stop proteins; that distinction does not erase the separate positive human evidence.

Summary: Manual transfer from mouse supports a contextual ligand-producing contribution to B-cell chemotaxis.

Reason: This ISS source is mouse Cyp7b1/Q60991, with an exact current IMP record from PMID:22999953. The original study measures CYP7B1-dependent EBI2 ligand production and the loss of normal outer-follicular B-cell positioning in deficient recipients, including stromal-compartment experiments. Thus there is a performed biochemical contribution to the guidance system, beyond an unexplained knockout phenotype. Conserved human 25-hydroxycholesterol hydroxylation supports the transfer, while the cellular migration assays remain mouse evidence. Retain as non-core without claiming that CYP7B1 itself moves B cells or binds EBI2.

Summary: The disease model places CYP7B1 variants in the ER membrane, consistent with the independent wild-type localization.

Reason: Reactome 5602885 is explicitly a FailedReaction. Its catalyst 9631820 points to the nine-member CYP7B1 variant set 5602941, whose members resolve to O75881 and have ER-membrane compartments. That modeled location does not demonstrate catalytic competence, equal stability, or experimentally measured trafficking of every mutant. The gene-level ER-membrane location is independently supported by native human microsomal activity and the official location assessment. Retain the location while keeping the failed-reaction and unmeasured topology limits explicit.

Summary: The 26(27)-hydroxycholesterol reaction places the CYP7B1 catalyst in the ER membrane.

Reason: Reaction 191972 reaches O75881 through catalyst 192095, set 2975806 and actual member 192117. The target protein's compartment is GO:0005789. This agrees with native microsomal activity and the independently curated ER localization; the presence of cytosolic small-molecule participants in the same reaction does not relocate the protein to the cytosol. No catalytic sidedness or experimental transmembrane topology is inferred from the model.

Summary: The 25-hydroxycholesterol reaction contains ER-membrane CYP7B1.

Reason: Reaction 192065 uses the same inspected catalyst 192095/set 2975806/member 192117 path to human O75881 in the ER membrane. Primary liver-membrane evidence and the official location record corroborate the assignment. This is a protein-location assertion, not a claim that the model directly measured membrane orientation or that all reaction participants occupy the same compartment.

Summary: CYP7B1 catalyzes a step in the alternative bile-acid pathway.

Reason: The cited study combines human oxysterol hydroxylation assays with a CYP7B1 defect, loss of liver microsomal activity, accumulation of pathway intermediates and loss of normal primary bile-acid production (PMID:9802883). Participation is established by the performed catalytic step, not by disease necessity alone. This does not assign CYP7B1 every downstream reaction or the entire pathway flux.

Summary: The cited human activity is 25-hydroxycholesterol 7-alpha-hydroxylation.

Reason: PMID:9802883 directly supports the normal 25-hydroxycholesterol hydroxylation represented by GO:0033783. The current GO:0008396 definition is instead 7-oxocholesterol reduction with NADPH. Retain the original TAS/reference fields and refine the reviewed chemistry; preserve the human-clone, human liver and mouse mutant assay boundaries. GO:0033783 is already asserted in three historical GOA annotations, including the human experimental sources; this is a refinement to existing supported chemistry.

Proposed replacements: 25-hydroxycholesterol 7-alpha-hydroxylase activity

Summary: Steroid 7-alpha-hydroxylation is directly supported despite the current rule-premise issue.

Reason: Human CYP7B1 catalyzes 7-alpha-hydroxylation of DHEA and oxysterols in PMID:9802883 and PMID:10588945, with purified-enzyme substrate characterization in PMID:24491228. This broader steroid term preserves activity beyond C27 oxysterols. None of the two current ARBA00092070 condition branches matches the saved official target features. The biological activity is independently supported; the current rule premise remains unresolved and its literal provenance is retained.

Summary: The flavoprotein-dependent monooxygenase class matches the measured catalytic system.

Reason: The reconstituted human CYP7B1 assay in PMID:24491228 includes rat NADPH-cytochrome P450 reductase and NADPH, supporting this redox class. None of the 28 current ARBA00027735 condition branches matches the saved official target features. The rule premise remains unresolved, separately from the directly demonstrated chemistry. No independent CYP7B1 flavin-binding or reductase activity is inferred.