GO:0001567 cholesterol 25-hydroxylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0001567 cholesterol 25-hydroxylase activity describes the enzymatic conversion of cholesterol and O2 into 25-hydroxycholesterol (25-HC) using a hydrogen donor.
• The enzyme cholesterol 25-hydroxylase (CH25H) is the primary gene product annotated to this activity and is rapidly induced by interferons and Toll-like receptor signaling.
• 25-HC produced by CH25H acts as a signaling lipid that regulates immune cell function, including macrophage polarization and immunosuppression.
• CH25H and 25-HC are implicated in diverse pathologies such as tauopathy-associated neurodegeneration, diabetic kidney disease, atherosclerosis, and colorectal cancer.
• Loss-of-function and gain-of-function studies in mice and cell models show that CH25H can be protective or detrimental depending on tissue context.
• CRISPR-based knockout, point mutation, and knock-in models are essential tools for dissecting the causal roles of CH25H and 25-HC in disease.
Description
Cholesterol 25-hydroxylase activity (GO:0001567) is a molecular function that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25-HC), a bioactive oxysterol involved in lipid metabolism and immune regulation. This activity is encoded by the CH25H gene in humans and mice, and its product, 25-HC, serves as a ligand for nuclear receptors and a modulator of inflammatory signaling. Researchers study this term to understand how a single enzymatic step can influence diverse processes ranging from antiviral defense to neurodegeneration. The importance of GO:0001567 extends beyond basic lipid biochemistry; genome-wide association and functional studies have linked CH25H and 25-HC to metabolic, cardiovascular, and neoplastic diseases. As a result, precise experimental models that manipulate this activity are critical for validating its causal roles and for developing targeted therapeutics.
cholesterol 25-hydroxylase activity At A Glance
| GO ID | GO:0001567 |
|---|---|
| GO term | cholesterol 25-hydroxylase activity |
| Ontology | molecular_function |
| Synonym | cholesterol 25-monooxygenase activity; cholesterol,hydrogen-donor:oxygen oxidoreductase (25-hydroxylating) activity |
| Definition | Catalysis of the reaction: AH2 + cholesterol + O2 = 25-hydroxycholesterol + A + H2O. |
| Major function | Production of 25-hydroxycholesterol, a bioactive oxysterol involved in immune regulation and lipid metabolism. |
| Representative gene | CH25H (cholesterol 25-hydroxylase). |
| Cofactors | Molecular oxygen (O2) and a hydrogen donor (AH2). |
| Subcellular location | Endoplasmic reticulum membrane (inferred from enzyme family). |
What Is GO:0001567?
According to the Gene Ontology, GO:0001567 cholesterol 25-hydroxylase activity is defined as the catalysis of the reaction: AH2 + cholesterol + O2 = 25-hydroxycholesterol + A + H2O. In simpler terms, it is the enzyme-driven addition of a hydroxyl group to the 25th carbon of cholesterol, using molecular oxygen and a reducing agent (AH2). This reaction produces 25-hydroxycholesterol, a potent oxysterol that can diffuse across membranes and act as a signaling molecule.
Why Is cholesterol 25-hydroxylase activity Important in Cell Biology?
GO:0001567 is important because the product of this enzymatic activity, 25-hydroxycholesterol, is a multifunctional lipid mediator that bridges cholesterol metabolism with innate and adaptive immunity. Dysregulation of CH25H and 25-HC has been observed in neurodegenerative, metabolic, and inflammatory diseases, making this activity a potential therapeutic target. Understanding its regulation and downstream effects can reveal new strategies for modulating immune responses and treating chronic diseases.
• CH25H is an interferon-stimulated gene, linking this activity to antiviral defense.
• 25-HC regulates macrophage polarization and immunosuppressive functions in tumors.
• CH25H deficiency aggravates diet-induced hepatic steatosis, indicating a role in lipid homeostasis.
• Macrophage-derived 25-HC promotes vascular inflammation and atherogenesis.
• In tauopathy models, CH25H mediates neuroinflammation and neurodegeneration.
• CH25H protects against diabetic kidney disease by regulating ARF4.
• 25-HC can inhibit viral entry, as shown for porcine deltacoronavirus.
• The activity is a node for crosstalk between cholesterol metabolism and immune signaling.
• Genetic and pharmacological modulation of CH25H is feasible in preclinical models.
• CRISPR screens can identify modifiers of 25-HC production and function.
Molecular Mechanism of cholesterol 25-hydroxylase activity
Substrate binding and catalysis
In simple terms: The enzyme grabs cholesterol and oxygen, then adds a hydroxyl group to the 25th carbon.
CH25H is a member of the sterol desaturase family and uses a diiron center to activate molecular oxygen, incorporating one oxygen atom into cholesterol to form 25-hydroxycholesterol. The reaction requires a hydrogen donor (AH2) to complete the reduction of the second oxygen atom to water. This catalytic mechanism is inferred from the GO definition and enzyme family homology.
Cofactors and redox partners
In simple terms: The enzyme needs oxygen and a helper molecule to donate electrons.
The reaction consumes O2 and a hydrogen donor (AH2), which is oxidized to A. While the exact physiological electron donor for CH25H is not fully defined, the requirement for a reducing agent is a hallmark of this activity. Cellular redox status may therefore influence enzymatic turnover.
Regulation by interferons and TLR signaling
In simple terms: Immune signals turn up the enzyme to make more 25-HC.
CH25H expression is strongly induced by type I and II interferons and by Toll-like receptor activation, leading to increased 25-HC production. This transcriptional regulation places GO:0001567 at the interface of innate immunity and lipid metabolism.
Post-transcriptional and post-translational control
In simple terms: The enzyme can be fine-tuned after the gene is turned on.
Interferon-induced miR-7705 modulates the antiviral activity of CH25H, suggesting post-transcriptional regulation of this activity. Additionally, 25-HC itself can feedback on cholesterol synthesis pathways, indirectly affecting substrate availability.
Downstream signaling by 25-HC
In simple terms: The product acts as a messenger to change cell behavior.
25-HC regulates lysosomal AMP kinase activation and metabolic reprogramming in macrophages, and it can activate STING-TBK1-RIPK3 signaling in myeloid-derived suppressor cells. These downstream effects link the enzymatic activity to immune suppression and inflammation.
Key Genes Involved in GO:0001567 cholesterol 25-hydroxylase activity
The following genes and proteins are directly or indirectly involved in cholesterol 25-hydroxylase activity and its downstream effects.
| Gene | Major Role | Research Relevance |
|---|---|---|
| CH25H | Encodes the enzyme catalyzing 25-HC production | Central to GO:0001567; knockout and overexpression models available |
| CYP46A1 | Cholesterol 24-hydroxylase, related oxysterol pathway | May compensate or interact in cholesterol turnover |
| ABCA1 | Cholesterol efflux transporter | Affects substrate availability for CH25H |
| APOE | Lipoprotein involved in cholesterol transport | Modifies 25-HC effects in neurodegeneration |
| ARF4 | ADP ribosylation factor 4 | Mediates CH25H protective effects in diabetic kidney disease |
| STING | Stimulator of interferon genes | Downstream of 25-HC in MDSC immunosuppression |
| TBK1 | TANK-binding kinase 1 | Part of STING-TBK1-RIPK3 axis activated by 25-HC |
| RIPK3 | Receptor-interacting protein kinase 3 | Mediates 25-HC effects in colorectal cancer |
| AMPK | AMP-activated protein kinase | Regulated by 25-HC in lysosomes |
| mTOR | Mechanistic target of rapamycin | Metabolic reprogramming downstream of 25-HC |
| IFNAR1 | Type I interferon receptor | Induces CH25H expression |
| STAT1 | Signal transducer and activator of transcription 1 | Transcription factor for CH25H |
| NF-kB | Nuclear factor kappa B | Inflammatory signaling modulated by 25-HC |
| NLRP3 | NLR family pyrin domain containing 3 | Inflammasome affected by 25-HC |
| SREBP2 | Sterol regulatory element-binding protein 2 | Regulates cholesterol synthesis, affecting substrate pool |
| LXR | Liver X receptor | Oxysterol receptor, may bind 25-HC |
| miR-7705 | MicroRNA induced by interferon | Modulates CH25H antiviral activity |
How Is cholesterol 25-hydroxylase activity Regulated?
CH25H expression and therefore GO:0001567 activity are primarily regulated at the transcriptional level by interferon signaling through STAT1 and by Toll-like receptor pathways. Post-transcriptional control by miR-7705 further tunes the antiviral function of CH25H. Additionally, substrate availability from cholesterol synthesis and efflux pathways can influence the reaction rate. Downstream, 25-HC can feedback on lipid metabolism and immune signaling, creating complex regulatory loops.
cholesterol 25-hydroxylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| CH25H | Tauopathy-associated neurodegeneration | Ch25h knockout mice crossed with tau transgenic mice |
| CH25H | Diabetic kidney disease | Ch25h knockout or overexpression in diabetic mouse models |
| CH25H | Colorectal cancer immunosuppression | Ch25h knockout mice with MC38 or CT26 tumor implants |
| CH25H | Diet-induced steatosis | Liver-specific Ch25h knockout mice on high-fat diet |
| CH25H | Atherosclerosis | Ldlr-/- mice with macrophage-specific Ch25h deletion |
Neurodegeneration and tauopathy
In a mouse model of tauopathy, CH25H mediates neuroinflammation and neurodegeneration, and its deletion or inhibition reduces pathology. This suggests that 25-HC production contributes to chronic neuroinflammation in tauopathies.
Metabolic and kidney diseases
Hepatic reduction of CH25H aggravates diet-induced steatosis, indicating a protective role in lipid homeostasis. Conversely, CH25H protects against diabetic kidney disease by regulating ARF4, highlighting tissue-specific effects.
Cancer and immunosuppression
25-HC produced by CH25H enhances myeloid-derived suppressor cell immunosuppression via the STING-TBK1-RIPK3 pathway in colorectal cancer, promoting tumor immune evasion. In macrophages, 25-HC regulates lysosomal AMPK and metabolic reprogramming to educate immunosuppressive phenotypes.
Atherosclerosis and vascular inflammation
Macrophage-derived 25-HC promotes vascular inflammation, atherogenesis, and lesion remodeling, linking this enzymatic activity to cardiovascular disease.
From cholesterol 25-hydroxylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does CH25H loss affect 25-HC levels and immune cell function? | CH25H knockout cell lines or mice |
| Which amino acid residues are essential for catalytic activity? | Point mutation of predicted iron-binding residues in CH25H |
| Can a tagged CH25H be used to track subcellular localization? | Knock-in of FLAG- or GFP-tagged CH25H |
| Does CH25H overexpression alter lipid metabolism or viral entry? | Stable CH25H overexpression in cell lines |
| What genes modify 25-HC production? | CRISPR library screening in reporter cells |
| How does 25-HC affect macrophage polarization? | Bone marrow-derived macrophages from Ch25h-/- mice |
How to Study the cholesterol 25-hydroxylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| LC-MS/MS | 25-HC levels | Quantify enzymatic activity in cells/tissues |
| CRISPR knockout | Gene function loss | Test causal role of CH25H in disease models |
| CRISPR activation | Gene overexpression | Enhance CH25H expression to study downstream effects |
| RNA-seq | Transcriptome changes | Identify pathways regulated by 25-HC |
| Western blot | Protein expression | Validate CH25H knockout or overexpression |
| Immunofluorescence | Subcellular localization | Confirm ER localization of tagged CH25H |
| Viral entry assay | Infection efficiency | Test 25-HC antiviral activity |
| Seahorse assay | Metabolic flux | Measure metabolic reprogramming by 25-HC |
Lipidomics and mass spectrometry
Quantification of 25-hydroxycholesterol by LC-MS/MS is the gold standard to measure CH25H enzymatic activity in cells and tissues.
CRISPR-based genetic screens
Genome-wide CRISPR knockout or activation screens can identify modifiers of CH25H expression or 25-HC production, as demonstrated in cancer immunity studies.
Transcriptional and post-transcriptional analysis
RNA-seq and miRNA profiling reveal regulation of CH25H by interferons and miR-7705.
Imaging and subcellular localization
Fluorescent tagging of CH25H allows visualization of its endoplasmic reticulum localization and trafficking.
How CRISPR Can Be Used to Study GO:0001567 cholesterol 25-hydroxylase activity
Knockout
CRISPR-Cas9 knockout of CH25H eliminates 25-HC production, providing a clean background to test its role in immune responses, neurodegeneration, and cancer. Knockout mice or cell lines are widely used to validate target engagement.
Point Mutation
Introducing point mutations in the catalytic residues of CH25H can dissect enzyme-dependent versus independent functions. Such models help confirm that observed phenotypes are due to loss of 25-hydroxylase activity.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) or fluorescent proteins allows tracking of endogenous CH25H localization and interaction partners without overexpression artifacts.
Overexpression
CRISPR activation or lentiviral overexpression of CH25H increases 25-HC levels, enabling gain-of-function studies in antiviral defense, lipid metabolism, and immunosuppression.
How EDITGENE Supports cholesterol 25-hydroxylase activity Research
Researchers studying cholesterol 25-hydroxylase activity-related genes often need to determine whether a candidate gene is causally involved in 25-HC production or downstream signaling. EDITGENE provides custom CRISPR cell models and screening services to accelerate this discovery.
Contact EDITGENE today to design your custom CRISPR model for cholesterol 25-hydroxylase activity research.
Frequently Asked Questions About cholesterol 25-hydroxylase activity
What is cholesterol 25-hydroxylase activity?
It is the enzymatic activity (GO:0001567) that converts cholesterol to 25-hydroxycholesterol using oxygen and a hydrogen donor.
What gene encodes cholesterol 25-hydroxylase?
The CH25H gene encodes the enzyme responsible for this activity in humans and mice.
What is the function of 25-hydroxycholesterol?
25-HC is a bioactive oxysterol that regulates immune responses, lipid metabolism, and antiviral defense.
How is CH25H regulated?
CH25H is induced by interferons and Toll-like receptor signaling, and modulated post-transcriptionally by miR-7705.
What diseases are associated with cholesterol 25-hydroxylase?
It has been linked to neurodegeneration, diabetic kidney disease, atherosclerosis, steatosis, and colorectal cancer.
Can CRISPR be used to study CH25H?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are widely used to dissect CH25H function.
What are the substrates of cholesterol 25-hydroxylase?
The substrates are cholesterol, molecular oxygen, and a hydrogen donor (AH2).
Where is CH25H located in the cell?
CH25H is an endoplasmic reticulum membrane protein.
Does 25-HC have antiviral properties?
Yes, 25-HC can inhibit viral entry, as shown for porcine deltacoronavirus.
How can I measure cholesterol 25-hydroxylase activity?
Enzymatic activity is typically measured by quantifying 25-HC levels using LC-MS/MS.
Conclusion
Cholesterol 25-hydroxylase activity (GO:0001567) is a critical enzymatic step that produces the pleiotropic oxysterol 25-HC, influencing immunity, metabolism, and disease. Its regulation by interferons and its broad downstream effects make it a compelling target for therapeutic intervention. CRISPR-based models and advanced screening technologies are essential to fully understand its roles and to translate these findings into clinical applications.
References
- 1. Toral-Rios D et al.. 2024. Cholesterol 25-hydroxylase mediates neuroinflammation and neurodegeneration in a mouse model of tauopathy.. J Exp Med 221(4) PMID: 38442267
- 2. Zhang L et al.. 2024. Cholesterol 25-Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4.. Adv Sci (Weinh) 11(29):e2309642 PMID: 38816950
- 3. Xiao J et al.. 2024. 25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages.. Immunity 57(5):1087-1104.e7 PMID: 38640930
- 4. Zhou D et al.. 2025. Cholesterol 25-Hydroxylase Enhances Myeloid-Derived Suppressor Cell (MDSC) Immunosuppression via the Stimulator of Interferon Genes (STING)-Tank-Binding Kinase 1 (TBK1)-Receptor-Interacting Protein Kinase 3 (RIPK3) Pathway in Colorectal Cancer.. MedComm (2020) 6(10):e70411 PMID: 41020041
- 5. Dong Z et al.. 2022. Hepatic Reduction in Cholesterol 25-Hydroxylase Aggravates Diet-induced Steatosis.. Cell Mol Gastroenterol Hepatol 13(4):1161-1179 PMID: 34990887
- 6. Wang L et al.. 2025. Interferon-induced miR-7705 modulates the anti-virus activity of cholesterol 25-hydroxylase.. J Virol 99(9):e0119825 PMID: 40928246
- 7. Ke W et al.. 2021. Cholesterol 25-hydroxylase suppresses porcine deltacoronavirus infection by inhibiting viral entry.. Virus Res 295:198306 PMID: 33476696
- 8. Canfrán-Duque A et al.. 2023. Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling.. Circulation 147(5):388-408 PMID: 36416142
