CH25H

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0000254 C-4 methylsterol oxidase activity	IBA GO_REF:0000033	REMOVE	Summary: Family-root IBA (PTN000221246, Eukaryota) seeded by the C-4 methylsterol oxidases MSMO1/ERG25 and plant SMO1 paralogs. CH25H is a side-chain C-25 hydroxylase of cholesterol; no member of the CH25H clade has been shown to oxidise 4-methylsterols. Reason: The IBD sits at the root of the heterogeneous sterol desaturase family (PTHR11863), whose branches catalyse different reactions (C-4 methyl oxidation, C-5 desaturation, sphingolipid C-4 hydroxylation, C-25 hydroxylation). Every donor is a methylsterol oxidase (human MSMO1 Q15800, mouse Msmo1, yeast ERG25, Arabidopsis SMO1 paralogs); the CH25H subfamily (PANTHER SF213, with its own PAINT node PTN002627316 for cholesterol 25-hydroxylase activity) is a divergent branch whose characterised members hydroxylate cholesterol at C-25 instead. The PTHR11863 family review likewise concludes that C-4 methylsterol oxidase activity is not a family-wide grant and is not supported for CH25H. Horse CH25H is therefore inside the descendant set only because of the node placement, and there is substrate-specific evidence against the function in its clade. The human ortholog review marks the same row over-annotated; here REMOVE is used because the claim is a specific reaction the enzyme is not known to perform, not a broad true term. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000221246 SUPPORTS SOURCE BUT NOT TARGET Eukaryote-wide family node; the methylsterol oxidase activity is right for the MSMO1/ERG25/SMO1 branches but CH25H diverged to cholesterol C-25 hydroxylation. Supporting Evidence: PMID:9852097 Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but rather it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates. file:human/CH25H/CH25H-uniprot.txt Reaction=cholesterol + AH2 + O2 = 25-hydroxycholesterol + A + H2O;
GO:0001567 cholesterol 25-hydroxylase activity	IBA GO_REF:0000033	ACCEPT	Summary: Core activity. The IBD node PTN002627316 (Euteleostomi) is the CH25H-clade node, seeded by experimentally characterised human CH25H and mouse Ch25h; horse CH25H is a one-to-one eutherian ortholog in this clade and keeps all three diiron histidine boxes. Reason: Human and mouse CH25H were cloned and shown to synthesise 25-hydroxycholesterol, with the histidine clusters required for activity (PMID:9852097); human CH25H activity was reconfirmed by mass spectrometry with a His242/His243 mutant control (PMID:33239446). The PAINT node is the specific CH25H subfamily node, not the heterogeneous family root, so the placement is sound. The horse protein aligns over 100% of its length with human CH25H and conserves every histidine of boxes 1-3 (file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md). Supporting Evidence: PMID:9852097 The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity. PMID:33239446 Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md Histidine box-3: human 238-244 HHDLHHS -> horse 238-244 HHDLHHS; all histidines conserved: True
GO:0001567 cholesterol 25-hydroxylase activity	IEA GO_REF:0000120	ACCEPT	Summary: Same claim as the IBA row, here from combined ortholog-based IEA methods using human and mouse CH25H; correct core activity. Reason: Consistent with the IBA row and with the conserved diiron histidine boxes of the horse protein; the donors are the experimentally characterised human and mouse enzymes. Supporting Evidence: file:human/CH25H/CH25H-uniprot.txt Catalyzes the formation of 25-hydroxycholesterol from file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md Histidine box-1: human 142-146 WHLLH -> horse 142-146 WHVLH; all histidines conserved: True
GO:0005506 iron ion binding	IEA GO_REF:0000002	ACCEPT	Summary: CH25H is a non-heme diiron enzyme; the iron is coordinated by the three histidine boxes, all conserved in horse. Iron binding is a genuine cofactor-binding property required for catalysis. Reason: Membership of the sterol desaturase / fatty acid hydroxylase family (InterPro IPR050307, Pfam PF04116) and conservation of all histidine-box residues support a diiron centre. The cloning paper places CH25H among diiron-cofactor hydroxylases, and UniProt records Fe as cofactor for the human enzyme by similarity to mouse. No more specific term (e.g. a diiron cluster term in MF) is warranted from the available data. Supporting Evidence: PMID:9852097 it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md Histidine box-2: human 157-161 HKVHH -> horse 157-161 HKMHH; all histidines conserved: True
GO:0005789 endoplasmic reticulum membrane	IBA GO_REF:0000033	ACCEPT	Summary: ER-membrane localisation is shared across the sterol desaturase family and is established for mouse Ch25h (one of the node's donors); horse CH25H has four predicted transmembrane helices. Reason: Unlike the reaction-specific MF term at the same node, ER membrane residence is a family-wide property of these polytopic diiron sterol enzymes, and the donor list includes mouse Ch25h itself. UniProt records the human protein in the ER membrane by similarity to mouse. Phobius predicts four TM helices in the horse sequence. Supporting Evidence: file:human/CH25H/CH25H-uniprot.txt CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane file:HORSE/CH25H/CH25H-uniprot.txt TRANSMEM 38..62
GO:0008203 cholesterol metabolic process	IEA GO_REF:0000107	ACCEPT	Summary: CH25H directly converts cholesterol to 25-hydroxycholesterol, so it catalyses a step of cholesterol metabolism; Ensembl Compara projection from human CH25H. Reason: The enzyme performs a step of the process (cholesterol is its substrate), satisfying the participation test. No more specific cholesterol-catabolism or oxysterol-biosynthesis term exists that captures the 25-hydroxylation alone. Supporting Evidence: PMID:32944968 CH25H converts cholesterol to 25-hydrocholesterol (25HC)
GO:0008395 steroid hydroxylase activity	IEA GO_REF:0000107	MODIFY	Summary: True but general; GO:0001567 cholesterol 25-hydroxylase activity is an is_a descendant (verified in OLS) and is already carried by this gene. Reason: Correct-but-general terms are refined rather than kept. The specific supported activity is cholesterol 25-hydroxylase activity, which this gene already carries by IBA and IEA. Proposed replacements: cholesterol 25-hydroxylase activity Supporting Evidence: file:human/CH25H/CH25H-uniprot.txt Catalyzes the formation of 25-hydroxycholesterol from
GO:0008610 lipid biosynthetic process	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: CH25H synthesises the oxysterol 25-HC, a lipid, so the broad term is literally true. GO has no oxysterol/25-hydroxycholesterol biosynthetic process term, so nothing more specific can replace it. Reason: The InterPro2GO mapping is broad and was written for the whole sterol desaturase family. For CH25H it holds as the formation of 25-HC, but the informative description of the process is cholesterol metabolic process plus the specific MF; using sterol biosynthetic process instead would wrongly suggest the cholesterol synthesis pathway, which CH25H represses rather than performs. Supporting Evidence: PMID:9852097 Among the most potent of regulatory oxysterols is 25-hydroxycholesterol, whose biosynthetic enzyme has not yet been isolated.
GO:0016020 membrane	IEA GO_REF:0000044	MODIFY	Summary: Correct but general; the specific supported location is the ER membrane. Reason: The horse record's SubCell "Membrane" comes from an ARBA rule plus Phobius TM prediction. The ortholog evidence places CH25H in the endoplasmic reticulum membrane, a term this gene already carries by IBA. Proposed replacements: endoplasmic reticulum membrane Supporting Evidence: file:human/CH25H/CH25H-uniprot.txt CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane
GO:0034340 response to type I interferon	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: CH25H is an interferon-stimulated gene in human and mouse; the term captures its induction by type I IFN and its role as an antiviral effector. Projected from human. Reason: Induction by interferon is a regulatory property of the gene, and the antiviral effect is mediated by the 25-HC product. IFN inducibility of the horse gene has not been tested, so the term is kept as a projected, non-core process rather than a core function. Supporting Evidence: file:human/CH25H/CH25H-uniprot.txt CC -!- INDUCTION: Induced by interferon (IFN) upon infection by virus like PMID:32944968 Cholesterol‐25‐hydroxylase (CH25H) is one of the ISGs (Schneider et al, 2014)
GO:0035754 B cell chemotaxis	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Projected from mouse Ch25h. CH25H makes 25-HC, the precursor that CYP7B1 converts into 7-alpha,25-OHC, the chemoattractant ligand of GPR183/EBI2 that positions B cells in lymphoid follicles. Reason: CH25H performs the first enzymatic step in making the chemoattractant, so it contributes work to generating the guidance cue rather than being merely necessary; this is the basis on which the human and mouse orthologs carry the term. It is peripheral to the core enzymatic function and untested in horse. Supporting Evidence: file:human/CH25H/CH25H-uniprot.txt Plays a key role in cell positioning and movement in
GO:0036197 zymosterol biosynthetic process	IBA GO_REF:0000033	REMOVE	Summary: IBA from the Eumetazoa node PTN002275159 seeded only by mouse Msmo1, a C-4 methylsterol oxidase of post-squalene cholesterol synthesis. CH25H does not act on zymosterol or its 4-methylsterol precursors and is not a cholesterol-synthesis enzyme. Reason: Zymosterol formation requires C-4 demethylation of lanosterol-derived methylsterols, the MSMO1 reaction. CH25H hydroxylates finished cholesterol and its product represses, rather than contributes to, sterol synthesis via SREBP. This is the same functional divergence as the C-4 methylsterol oxidase row; the IBA reaches CH25H only through node placement. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN002275159 SUPPORTS SOURCE BUT NOT TARGET Node seeded by mouse Msmo1 (methylsterol oxidase); correct for MSMO1 but CH25H diverged to cholesterol side-chain hydroxylation. Supporting Evidence: PMID:9852097 Expression of cholesterol 25-hydroxylase in transfected cells reduces the biosynthesis of cholesterol from acetate and suppresses the cleavage of sterol regulatory element binding protein-1 and -2.
GO:0090206 negative regulation of cholesterol metabolic process	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: 25-HC is a co-repressor that blocks SREBP processing, reducing cholesterol synthesis; CH25H expression in cells reduces cholesterol biosynthesis. Projected from mouse. Reason: The regulatory effect is exerted by the enzyme's product on the SCAP/INSIG/SREBP system; CH25H's own contribution is synthesising that regulator. Supported by the ortholog data and kept as a downstream, non-core process. Supporting Evidence: PMID:9852097 regulating lipid metabolism by synthesizing a co-repressor that blocks sterol regulatory element binding protein processing
GO:1903914 negative regulation of fusion of virus membrane with host plasma membrane	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Human CH25H and 25-HC block SARS-CoV-2 and other coronavirus spike-mediated fusion at the plasma membrane by depleting accessible cholesterol via ACAT; projected from human. Reason: Well supported for the human ortholog, including a catalytically dead CH25H control, so the antiviral effect depends on 25-HC production. It is a downstream consequence of the core enzymatic activity, and no equine virus study was found, so it is kept as non-core. Supporting Evidence: PMID:32944968 These results strongly support a function for 25HC in blocking human coronavirus spike‐mediated fusion with the plasma membrane, thereby suppressing virus entry. PMID:33239446 Collectively, our data support a model that 25HC suppresses SARS-CoV-2 S protein-mediated fusion, which inhibits virus replication, possibly by altering cholesterol levels.

GO Term

Evidence

Action

Reason

GO:0000254 C-4 methylsterol oxidase activity

IBA
GO_REF:0000033

REMOVE

Summary: Family-root IBA (PTN000221246, Eukaryota) seeded by the C-4 methylsterol oxidases MSMO1/ERG25 and plant SMO1 paralogs. CH25H is a side-chain C-25 hydroxylase of cholesterol; no member of the CH25H clade has been shown to oxidise 4-methylsterols.

Reason: The IBD sits at the root of the heterogeneous sterol desaturase family (PTHR11863), whose branches catalyse different reactions (C-4 methyl oxidation, C-5 desaturation, sphingolipid C-4 hydroxylation, C-25 hydroxylation). Every donor is a methylsterol oxidase (human MSMO1 Q15800, mouse Msmo1, yeast ERG25, Arabidopsis SMO1 paralogs); the CH25H subfamily (PANTHER SF213, with its own PAINT node PTN002627316 for cholesterol 25-hydroxylase activity) is a divergent branch whose characterised members hydroxylate cholesterol at C-25 instead. The PTHR11863 family review likewise concludes that C-4 methylsterol oxidase activity is not a family-wide grant and is not supported for CH25H. Horse CH25H is therefore inside the descendant set only because of the node placement, and there is substrate-specific evidence against the function in its clade. The human ortholog review marks the same row over-annotated; here REMOVE is used because the claim is a specific reaction the enzyme is not known to perform, not a broad true term.

Propagation Review

Root cause: PROPAGATION BAD

Failure modes: FUNCTIONAL DIVERGENCE

Sources checked:

PANTHER:PTN000221246 SUPPORTS SOURCE BUT NOT TARGET

Eukaryote-wide family node; the methylsterol oxidase activity is right for the MSMO1/ERG25/SMO1 branches but CH25H diverged to cholesterol C-25 hydroxylation.

Supporting Evidence:

PMID:9852097

Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but rather it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates.

file:human/CH25H/CH25H-uniprot.txt

Reaction=cholesterol + AH2 + O2 = 25-hydroxycholesterol + A + H2O;

GO:0001567 cholesterol 25-hydroxylase activity

IBA
GO_REF:0000033

Summary: Core activity. The IBD node PTN002627316 (Euteleostomi) is the CH25H-clade node, seeded by experimentally characterised human CH25H and mouse Ch25h; horse CH25H is a one-to-one eutherian ortholog in this clade and keeps all three diiron histidine boxes.

Reason: Human and mouse CH25H were cloned and shown to synthesise 25-hydroxycholesterol, with the histidine clusters required for activity (PMID:9852097); human CH25H activity was reconfirmed by mass spectrometry with a His242/His243 mutant control (PMID:33239446). The PAINT node is the specific CH25H subfamily node, not the heterogeneous family root, so the placement is sound. The horse protein aligns over 100% of its length with human CH25H and conserves every histidine of boxes 1-3 (file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md).

Supporting Evidence:

PMID:9852097

The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity.

PMID:33239446

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses.

file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md

Histidine box-3: human 238-244 HHDLHHS -> horse 238-244 HHDLHHS; all histidines conserved: True

GO:0001567 cholesterol 25-hydroxylase activity

IEA
GO_REF:0000120

Summary: Same claim as the IBA row, here from combined ortholog-based IEA methods using human and mouse CH25H; correct core activity.

Reason: Consistent with the IBA row and with the conserved diiron histidine boxes of the horse protein; the donors are the experimentally characterised human and mouse enzymes.

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt

Catalyzes the formation of 25-hydroxycholesterol from

file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md

Histidine box-1: human 142-146 WHLLH -> horse 142-146 WHVLH; all histidines conserved: True

GO:0005506 iron ion binding

IEA
GO_REF:0000002

Summary: CH25H is a non-heme diiron enzyme; the iron is coordinated by the three histidine boxes, all conserved in horse. Iron binding is a genuine cofactor-binding property required for catalysis.

Reason: Membership of the sterol desaturase / fatty acid hydroxylase family (InterPro IPR050307, Pfam PF04116) and conservation of all histidine-box residues support a diiron centre. The cloning paper places CH25H among diiron-cofactor hydroxylases, and UniProt records Fe as cofactor for the human enzyme by similarity to mouse. No more specific term (e.g. a diiron cluster term in MF) is warranted from the available data.

Supporting Evidence:

PMID:9852097

it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates

file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md

Histidine box-2: human 157-161 HKVHH -> horse 157-161 HKMHH; all histidines conserved: True

GO:0005789 endoplasmic reticulum membrane

IBA
GO_REF:0000033

Summary: ER-membrane localisation is shared across the sterol desaturase family and is established for mouse Ch25h (one of the node's donors); horse CH25H has four predicted transmembrane helices.

Reason: Unlike the reaction-specific MF term at the same node, ER membrane residence is a family-wide property of these polytopic diiron sterol enzymes, and the donor list includes mouse Ch25h itself. UniProt records the human protein in the ER membrane by similarity to mouse. Phobius predicts four TM helices in the horse sequence.

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt

CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane

file:HORSE/CH25H/CH25H-uniprot.txt

TRANSMEM 38..62

GO:0008203 cholesterol metabolic process

IEA
GO_REF:0000107

Summary: CH25H directly converts cholesterol to 25-hydroxycholesterol, so it catalyses a step of cholesterol metabolism; Ensembl Compara projection from human CH25H.

Reason: The enzyme performs a step of the process (cholesterol is its substrate), satisfying the participation test. No more specific cholesterol-catabolism or oxysterol-biosynthesis term exists that captures the 25-hydroxylation alone.

Supporting Evidence:

PMID:32944968

CH25H converts cholesterol to 25-hydrocholesterol (25HC)

GO:0008395 steroid hydroxylase activity

IEA
GO_REF:0000107

MODIFY

Summary: True but general; GO:0001567 cholesterol 25-hydroxylase activity is an is_a descendant (verified in OLS) and is already carried by this gene.

Reason: Correct-but-general terms are refined rather than kept. The specific supported activity is cholesterol 25-hydroxylase activity, which this gene already carries by IBA and IEA.

Proposed replacements: cholesterol 25-hydroxylase activity

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt

Catalyzes the formation of 25-hydroxycholesterol from

GO:0008610 lipid biosynthetic process

IEA
GO_REF:0000002

KEEP AS NON CORE

Summary: CH25H synthesises the oxysterol 25-HC, a lipid, so the broad term is literally true. GO has no oxysterol/25-hydroxycholesterol biosynthetic process term, so nothing more specific can replace it.

Reason: The InterPro2GO mapping is broad and was written for the whole sterol desaturase family. For CH25H it holds as the formation of 25-HC, but the informative description of the process is cholesterol metabolic process plus the specific MF; using sterol biosynthetic process instead would wrongly suggest the cholesterol synthesis pathway, which CH25H represses rather than performs.

Supporting Evidence:

PMID:9852097

Among the most potent of regulatory oxysterols is 25-hydroxycholesterol, whose biosynthetic enzyme has not yet been isolated.

GO:0016020 membrane

IEA
GO_REF:0000044

MODIFY

Summary: Correct but general; the specific supported location is the ER membrane.

Reason: The horse record's SubCell "Membrane" comes from an ARBA rule plus Phobius TM prediction. The ortholog evidence places CH25H in the endoplasmic reticulum membrane, a term this gene already carries by IBA.

Proposed replacements: endoplasmic reticulum membrane

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt

CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane

GO:0034340 response to type I interferon

IEA
GO_REF:0000107

KEEP AS NON CORE

Summary: CH25H is an interferon-stimulated gene in human and mouse; the term captures its induction by type I IFN and its role as an antiviral effector. Projected from human.

Reason: Induction by interferon is a regulatory property of the gene, and the antiviral effect is mediated by the 25-HC product. IFN inducibility of the horse gene has not been tested, so the term is kept as a projected, non-core process rather than a core function.

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt

CC -!- INDUCTION: Induced by interferon (IFN) upon infection by virus like

PMID:32944968

Cholesterol‐25‐hydroxylase (CH25H) is one of the ISGs (Schneider et al, 2014)

GO:0035754 B cell chemotaxis

IEA
GO_REF:0000107

KEEP AS NON CORE

Summary: Projected from mouse Ch25h. CH25H makes 25-HC, the precursor that CYP7B1 converts into 7-alpha,25-OHC, the chemoattractant ligand of GPR183/EBI2 that positions B cells in lymphoid follicles.

Reason: CH25H performs the first enzymatic step in making the chemoattractant, so it contributes work to generating the guidance cue rather than being merely necessary; this is the basis on which the human and mouse orthologs carry the term. It is peripheral to the core enzymatic function and untested in horse.

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt

Plays a key role in cell positioning and movement in

GO:0036197 zymosterol biosynthetic process

IBA
GO_REF:0000033

REMOVE

Summary: IBA from the Eumetazoa node PTN002275159 seeded only by mouse Msmo1, a C-4 methylsterol oxidase of post-squalene cholesterol synthesis. CH25H does not act on zymosterol or its 4-methylsterol precursors and is not a cholesterol-synthesis enzyme.

Reason: Zymosterol formation requires C-4 demethylation of lanosterol-derived methylsterols, the MSMO1 reaction. CH25H hydroxylates finished cholesterol and its product represses, rather than contributes to, sterol synthesis via SREBP. This is the same functional divergence as the C-4 methylsterol oxidase row; the IBA reaches CH25H only through node placement.

Propagation Review

Root cause: PROPAGATION BAD

Failure modes: FUNCTIONAL DIVERGENCE

Sources checked:

PANTHER:PTN002275159 SUPPORTS SOURCE BUT NOT TARGET

Node seeded by mouse Msmo1 (methylsterol oxidase); correct for MSMO1 but CH25H diverged to cholesterol side-chain hydroxylation.

Supporting Evidence:

PMID:9852097

Expression of cholesterol 25-hydroxylase in transfected cells reduces the biosynthesis of cholesterol from acetate and suppresses the cleavage of sterol regulatory element binding protein-1 and -2.

GO:0090206 negative regulation of cholesterol metabolic process

IEA
GO_REF:0000107

KEEP AS NON CORE

Summary: 25-HC is a co-repressor that blocks SREBP processing, reducing cholesterol synthesis; CH25H expression in cells reduces cholesterol biosynthesis. Projected from mouse.

Reason: The regulatory effect is exerted by the enzyme's product on the SCAP/INSIG/SREBP system; CH25H's own contribution is synthesising that regulator. Supported by the ortholog data and kept as a downstream, non-core process.

Supporting Evidence:

PMID:9852097

regulating lipid metabolism by synthesizing a co-repressor that blocks sterol regulatory element binding protein processing

GO:1903914 negative regulation of fusion of virus membrane with host plasma membrane

IEA
GO_REF:0000107

KEEP AS NON CORE

Summary: Human CH25H and 25-HC block SARS-CoV-2 and other coronavirus spike-mediated fusion at the plasma membrane by depleting accessible cholesterol via ACAT; projected from human.

Reason: Well supported for the human ortholog, including a catalytically dead CH25H control, so the antiviral effect depends on 25-HC production. It is a downstream consequence of the core enzymatic activity, and no equine virus study was found, so it is kept as non-core.

Supporting Evidence:

PMID:32944968

These results strongly support a function for 25HC in blocking human coronavirus spike‐mediated fusion with the plasma membrane, thereby suppressing virus entry.

PMID:33239446

Collectively, our data support a model that 25HC suppresses SARS-CoV-2 S protein-mediated fusion, which inhibits virus replication, possibly by altering cholesterol levels.

Core Functions

Cholesterol 25-hydroxylase: an ER-membrane, non-heme diiron oxygenase that hydroxylates cholesterol at C-25 to produce the regulatory oxysterol 25-hydroxycholesterol.

Molecular Function:

cholesterol 25-hydroxylase activity

Directly Involved In:

cholesterol metabolic process

Cellular Locations:

endoplasmic reticulum membrane

Supporting Evidence:

PMID:9852097
The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity.
file:human/CH25H/CH25H-uniprot.txt
Reaction=cholesterol + AH2 + O2 = 25-hydroxycholesterol + A + H2O;
file:HORSE/CH25H/CH25H-bioinformatics/RESULTS-rereview.md
Histidine box-3: human 238-244 HHDLHHS -> horse 238-244 HHDLHHS; all histidines conserved: True

External Prediction Reviews

These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.

ProtNLM2 External predictions

View prediction review YAML · CH25H-protnlm-predictions-review.yaml · Review status: COMPLETE

Assessment of 3 ProtNLM2 GO predictions for horse CH25H (F6T000), based on experimental findings for the human and mouse orthologs and a horse-human sequence comparison. All three are correct and none is novel: oxidoreductase activity and membrane are less specific than the cholesterol 25-hydroxylase activity and endoplasmic reticulum membrane annotations the gene already carries (LSP), and lipid biosynthetic process matches an existing GOA term (CNN). A PubMed search found no horse-specific CH25H studies, so support is by orthology.

Source documents: genes/human/CH25H/CH25H-uniprot.txt · genes/HORSE/CH25H/CH25H-bioinformatics/RESULTS.md · projects/PROTNLM_EVALUATION/mammal-benchmark/horse40-predictions.csv

Review score: 2 = concordant with evidence; 1 = uncertain; 0 = discordant with evidence. This is an assessment score, not a model probability.

GO:0016491 oxidoreductase activity GO_MF

LSP — Less precise than existing annotation Review score: 2/2

Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-09-08

Review rationale: Cholesterol 25-hydroxylation is a specific oxidoreductase reaction established for cloned human and mouse enzymes in PMID:9852097. The horse protein retains the conserved enzyme sequence and histidine-rich architecture. The prediction is biologically correct but less specific than cholesterol 25-hydroxylase activity already present in horse GOA. Training-set membership is unknown.

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt: "CC -!- FUNCTION: Catalyzes the formation of 25-hydroxycholesterol from CC cholesterol, leading to repress cholesterol biosynthetic enzymes CC (PubMed:9852097). Plays a key role in cell positioning and movement in CC lymphoid tissues: 25-hydroxycholesterol is an intermediate in CC biosynthesis of 7-alpha,25-dihydroxycholesterol (7-alpha,25-OHC), an CC oxysterol that acts as a ligand for the G protein-coupled receptor CC GPR183/EBI2, a chemotactic receptor for a number of lymphoid cells (By CC similarity). May play an important role in regulating lipid metabolism CC by synthesizing a corepressor that blocks sterol regulatory element CC binding protein (SREBP) processing (PubMed:9852097). As an interferon- CC stimulated gene, has broad antiviral activities against a wide range of CC enveloped viruses, such as vesicular stomatitis virus (VSV) and SARS CC coronavirus-2 (SARS-CoV-2). Its product, 25-hydroxycholesterol, CC activates the ER-localized enzyme ACAT to induce internalization of CC accessible cholesterol on the plasma membrane and restricts SARS-CoV-2 CC S protein-mediated fusion which inhibits virus replication CC (PubMed:32944968, PubMed:33239446). In testis, production of 25- CC hydroxycholesterol by macrophages plays a role in Leydig cell CC differentiation (By similarity). Required to restrain inflammation in CC macrophages: production of 25-hydroxycholesterol protects macrophages CC from cholesterol overload, thereby preventing mitochondrial DNA release CC and subsequent activation of the AIM2 inflammasome (By similarity). CC {ECO:0000250|UniProtKB:Q4QQV7, ECO:0000250|UniProtKB:Q9Z0F5, CC ECO:0000269|PubMed:32944968, ECO:0000269|PubMed:33239446, CC ECO:0000269|PubMed:9852097}."
file:HORSE/CH25H/CH25H-bioinformatics/RESULTS.md: "The horse sequence F6T000 (270 residues) aligns to human O95992 (272 residues) with 83.33% identity across 270 paired residues. Paired coverage is 99.26% of the human sequence and 100.0% of the horse sequence."
PMID:9852097: "Here, we report the cloning of cholesterol 25-hydroxylase cDNAs from the mouse and human. The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity. Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but rather it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates. The cholesterol 25-hydroxylase gene lacks introns, and in the human it is located on chromosome 10q23. The murine gene is expressed at low levels in multiple tissues. Expression of cholesterol 25-hydroxylase in transfected cells reduces the biosynthesis of cholesterol from acetate and suppresses the cleavage of sterol regulatory element binding protein-1 and -2. The data suggest that cholesterol 25-hydroxylase has the capacity to play an important role in regulating lipid metabolism by synthesizing a co-repressor that blocks sterol regulatory element binding protein processing and ultimately leads to inhibition of gene transcription."

GO:0008610 lipid biosynthetic process GO_BP

CNN — Correct but not novel Review score: 2/2

Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-09-08

Review rationale: CH25H produces the oxysterol 25-hydroxycholesterol from cholesterol. That product-forming reaction supports lipid biosynthesis even though the product also represses cholesterol biosynthesis; inhibition of the latter pathway does not make this broad BP false. The exact target GOA term is present. This does not validate the separate zymosterol or C-4 methylsterol assertions. Training-set membership is unknown.

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt: "CC -!- FUNCTION: Catalyzes the formation of 25-hydroxycholesterol from CC cholesterol, leading to repress cholesterol biosynthetic enzymes CC (PubMed:9852097). Plays a key role in cell positioning and movement in CC lymphoid tissues: 25-hydroxycholesterol is an intermediate in CC biosynthesis of 7-alpha,25-dihydroxycholesterol (7-alpha,25-OHC), an CC oxysterol that acts as a ligand for the G protein-coupled receptor CC GPR183/EBI2, a chemotactic receptor for a number of lymphoid cells (By CC similarity). May play an important role in regulating lipid metabolism CC by synthesizing a corepressor that blocks sterol regulatory element CC binding protein (SREBP) processing (PubMed:9852097). As an interferon- CC stimulated gene, has broad antiviral activities against a wide range of CC enveloped viruses, such as vesicular stomatitis virus (VSV) and SARS CC coronavirus-2 (SARS-CoV-2). Its product, 25-hydroxycholesterol, CC activates the ER-localized enzyme ACAT to induce internalization of CC accessible cholesterol on the plasma membrane and restricts SARS-CoV-2 CC S protein-mediated fusion which inhibits virus replication CC (PubMed:32944968, PubMed:33239446). In testis, production of 25- CC hydroxycholesterol by macrophages plays a role in Leydig cell CC differentiation (By similarity). Required to restrain inflammation in CC macrophages: production of 25-hydroxycholesterol protects macrophages CC from cholesterol overload, thereby preventing mitochondrial DNA release CC and subsequent activation of the AIM2 inflammasome (By similarity). CC {ECO:0000250|UniProtKB:Q4QQV7, ECO:0000250|UniProtKB:Q9Z0F5, CC ECO:0000269|PubMed:32944968, ECO:0000269|PubMed:33239446, CC ECO:0000269|PubMed:9852097}."
file:HORSE/CH25H/CH25H-bioinformatics/RESULTS.md: "The horse sequence F6T000 (270 residues) aligns to human O95992 (272 residues) with 83.33% identity across 270 paired residues. Paired coverage is 99.26% of the human sequence and 100.0% of the horse sequence."
PMID:9852097: "Here, we report the cloning of cholesterol 25-hydroxylase cDNAs from the mouse and human. The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity. Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but rather it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates. The cholesterol 25-hydroxylase gene lacks introns, and in the human it is located on chromosome 10q23. The murine gene is expressed at low levels in multiple tissues. Expression of cholesterol 25-hydroxylase in transfected cells reduces the biosynthesis of cholesterol from acetate and suppresses the cleavage of sterol regulatory element binding protein-1 and -2. The data suggest that cholesterol 25-hydroxylase has the capacity to play an important role in regulating lipid metabolism by synthesizing a co-repressor that blocks sterol regulatory element binding protein processing and ultimately leads to inhibition of gene transcription."

GO:0016020 membrane GO_CC

LSP — Less precise than existing annotation Review score: 2/2

Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-09-08

Review rationale: The cloned human and mouse enzymes are polytopic membrane proteins (PMID:9852097), and UniProt places human CH25H in the endoplasmic reticulum membrane as a multi-pass protein by similarity to mouse Ch25h. The horse sequence aligns almost full length to human and keeps the membrane-enzyme architecture. Membrane is therefore correct, but it is less specific than the endoplasmic reticulum membrane annotation the horse gene already carries by IBA, which the gene review accepts as the core location (the review refines the IEA membrane row to it). Training-set membership is unknown.

Supporting Evidence:

file:human/CH25H/CH25H-uniprot.txt: "CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane CC {ECO:0000250|UniProtKB:Q9Z0F5}; Multi-pass membrane protein"
file:HORSE/CH25H/CH25H-bioinformatics/RESULTS.md: "The horse sequence F6T000 (270 residues) aligns to human O95992 (272 residues) with 83.33% identity across 270 paired residues. Paired coverage is 99.26% of the human sequence and 100.0% of the horse sequence."
PMID:9852097: "Here, we report the cloning of cholesterol 25-hydroxylase cDNAs from the mouse and human. The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity. Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but rather it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates. The cholesterol 25-hydroxylase gene lacks introns, and in the human it is located on chromosome 10q23. The murine gene is expressed at low levels in multiple tissues. Expression of cholesterol 25-hydroxylase in transfected cells reduces the biosynthesis of cholesterol from acetate and suppresses the cleavage of sterol regulatory element binding protein-1 and -2. The data suggest that cholesterol 25-hydroxylase has the capacity to play an important role in regulating lipid metabolism by synthesizing a co-repressor that blocks sterol regulatory element binding protein processing and ultimately leads to inhibition of gene transcription."

📚 Additional Documentation

Bioinformatics Results

(RESULTS.md)

Loading supporting content…

Download this section (compressed HTML)

Gene Description

Existing Annotations Review

Core Functions

Cholesterol 25-hydroxylase: an ER-membrane, non-heme diiron oxygenase that hydroxylates cholesterol at C-25 to produce the regulatory oxysterol 25-hydroxycholesterol.

References

Suggested Questions for Experts

Suggested Experiments

External Prediction Reviews

ProtNLM2 External predictions

Deep Research

Cyberian

📚 Additional Documentation

Notes

Bioinformatics Results

📄 View Raw YAML