Recombinant Human 3-beta-hydroxysteroid-Delta (8),Delta (7)-isomerase (EBP)
Description
Recombinant Human 3-Beta-Hydroxysteroid-Delta(8),Delta(7)-Isomerase (EBP): Overview
Recombinant Human EBP is a bioengineered form of the endoplasmic reticulum (ER) membrane protein Emopamil-Binding Protein (EBP), encoded by the EBP gene. This enzyme catalyzes the isomerization of Δ⁸-sterols (e.g., zymosterol) to Δ⁷-sterols in the cholesterol biosynthesis pathway . Its recombinant form is produced via heterologous expression systems (e.g., E. coli) and is widely used in structural, biochemical, and pharmacological studies .
Catalytic Mechanism
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Substrate Binding: Δ⁸-sterols (e.g., zymosterol) enter the ER lumen via helix H1 .
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Carbocation Formation: His76 protonates the C9α position, generating a carbocation at C8 stabilized by Trp196’s π-cation interaction .
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Isomerization: Glu80 deprotonates C7β, shifting the double bond to Δ⁷ .
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Product Release: Δ⁷-sterols exit through a membrane gate between TM1 and TM5 .
Key Residues:
Pharmacological and Non-Enzymatic Roles
EBP binds structurally diverse ligands (e.g., tamoxifen, U18666A) through its charged amine groups, mimicking the carbocation intermediate .
Ligand Interactions
Non-Enzymatic Functions:
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Hedgehog Signaling: Binds Smoothened (SMO), blocking its cholesterylation at Asn95 and inhibiting pathway activation .
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Drug Resistance: Acts as a multidrug-binding protein, conferring resistance to tamoxifen and similar compounds .
Disease Association
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Conradi-Hunermann Syndrome (CDPX2): Mutations (e.g., L18P, E103K) disrupt sterol binding or dimerization, causing skeletal and developmental defects .
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Cancer: EBP inhibition induces autophagy in tumor cells, highlighting its therapeutic potential .
| Mutation | Location | Effect |
|---|---|---|
| L18P | Helix H1 (luminal) | Blocks solvent entry; reduces activity |
| R110Q | Dimer interface | Destabilizes protein folding |
Research Tools
Product Specs
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Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C, while lyophilized forms exhibit a shelf life of 12 months at -20°C/-80°C.
Target Background
- This study expands the current phenotypic spectrum of males with hypomorphic EBP mutations and supports the hypothesis of an X-linked recessive entity independent of CDPX2. PMID: 24700572
- Mutation analysis revealed a heterozygous novel missense mutation, c.204G>T (p.W68C), in exon 2. PMID: 24915996
- Observed with non-mosaic EBP mutations in males. PMID: 24459067
- Report steroidomimetic aminomethyl spiroacetals as novel inhibitors of the enzyme Delta8,7-sterol isomerase in cholesterol biosynthesis. PMID: 24493593
- Elastin binding protein and FKBP65 modulate the kinetics of self-assembly of tropoelastin in an in vitro system. PMID: 24106871
- Results demonstrate a clear relationship between abnormal sterol profile and the EBP gene mutation. PMID: 22121851
- Postzygotic mosaicism on an ichthyosiform skin lesion in the mother of a girl with X-linked dominant chondrodysplasia punctata associated with a novel EBP mutation. PMID: 21931045
- Molecular analysis of EBP mutations was conducted. PMID: 17378690
- Emopamil binding protein (EBP)-shRNA sequences were designed and tested for their effectiveness. PMID: 17498944
- Two novel (3G-->T and 419-422delTTCT) and one known mutation were identified in the EBP gene. The significant phenotypic variability in our patients suggests that genotype-phenotype correlation is not clear-cut. PMID: 17949453
- Two unrelated Thai girls with chondrodysplasia punctata type 2 were studied. Mutation analysis via PCR-sequencing the entire coding region of emopamil binding protein (EBP) successfully identified two potentially pathogenic, novel mutations, c.616G-->T and c.382delC. PMID: 18573709
Q&A
What is the molecular structure and organization of human EBP?
Human EBP is a 230-amino acid membrane protein with four transmembrane domains. The protein is encoded by the EBP gene located on chromosome Xp11.23-p11.22 . The complete amino acid sequence is:
MTTNAGPLHPYWPQHLRLDNFVPNDRPTWHILAGLFSVTGVLVVTTWLLSGRAAVVPLGTWRRLSLCWFAVCGFIHLVIEGWFVLYYEDLLGDQAFLSQLWKEYAKGDSRYILGDNFTVCMETITACLWGPLSLWVVIAFLRQHPLRFILQLVVSVGQIYGDVLYFLTEHRDGFQHGELGHPLYFWFYFVFMNALWLVLPGVLVLDAVKHLTHAQSTLDAKATKAKSKKN
The protein has been characterized as having the following structural features:
| Structural Feature | Description |
|---|---|
| Length | 230 amino acids |
| Transmembrane Domains | 4 |
| Cellular Localization | Endoplasmic reticulum membrane |
| UniProt ID | Q15125 |
| Key Functional Domains | Active site residues involved in isomerase activity |
Experimental approaches for structural determination have included X-ray crystallography and cryo-electron microscopy, which have provided insights into the catalytic mechanism of the isomerization reaction.
What is the catalytic mechanism of EBP in the isomerization reaction?
EBP catalyzes the conversion of Delta(8)-sterols to their corresponding Delta(7)-isomers by shifting the double bond from the C8-C9 to the C7-C8 position in the B-ring of the sterol nucleus . This isomerization reaction is one of the final steps in cholesterol de novo biosynthesis .
The reaction mechanism involves:
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Binding of the Delta(8)-sterol substrate to the active site
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Protonation of the Delta(8) double bond
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Formation of a carbocation intermediate
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Deprotonation at the C7 position to form the Delta(7) double bond
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Release of the Delta(7)-sterol product
Recent structural studies have revealed specific amino acid residues that participate in this acid-base catalysis, including conserved histidine and aspartic acid residues that facilitate proton transfer during the reaction .
How does EBP function differ from other sterol isomerases?
EBP belongs to a family of sterol isomerases but has distinct characteristics compared to other members. Unlike 3-beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (HSD3B2), which catalyzes both oxidative conversion of Delta(5)-ene-3-beta-hydroxy steroids and isomerization reactions, EBP specifically performs the Delta(8)-Delta(7) isomerization .
| Isomerase | Reaction Catalyzed | Substrate Specificity | Cellular Location |
|---|---|---|---|
| EBP (Delta(8)-Delta(7) isomerase) | Converts Delta(8)-sterols to Delta(7)-sterols | Specific for sterols with Delta(8) double bond | Endoplasmic reticulum |
| HSD3B2 | Oxidative conversion and isomerization of Delta(5) to Delta(4) steroids | Broader substrate range including pregnenolone and DHEA | Endoplasmic reticulum and mitochondria |
Additionally, EBP has shown high binding affinity for both enantiomers of emopamil, a calcium channel blocker, suggesting a potential secondary role beyond cholesterol biosynthesis .
What expression systems yield optimal results for recombinant human EBP production?
Successful expression of functional recombinant human EBP requires careful consideration of expression systems that can properly process membrane proteins with multiple transmembrane domains. Based on research findings, the following systems have demonstrated effectiveness:
| Expression System | Advantages | Limitations | Yield |
|---|---|---|---|
| Mammalian cell lines (HEK293, CHO) | Proper folding and post-translational modifications | Higher cost, longer production time | Moderate (0.5-2 mg/L) |
| Insect cell/baculovirus system | Good compromise between yield and proper folding | Requires specialized equipment | Good (2-5 mg/L) |
| E. coli with fusion tags | High yield, economical | May require refolding, potential for inactive protein | High but variable activity (5-10 mg/L) |
| Yeast systems (P. pastoris) | Proper folding of membrane proteins, scalable | Glycosylation patterns differ from human | Good (2-4 mg/L) |
For functional studies requiring properly folded EBP, mammalian or insect cell expression systems are recommended despite their lower yields. For structural studies requiring larger quantities, bacterial systems with subsequent refolding protocols may be more appropriate.
What are the most reliable methods for measuring EBP enzyme activity?
Accurate measurement of EBP activity is essential for both basic research and disease mechanism studies. Several methodological approaches have been developed:
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Radioisotope-based assays: Using radiolabeled Delta(8)-sterol substrates and measuring conversion to Delta(7) products by thin-layer chromatography or HPLC.
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LC-MS/MS methods: High-sensitivity approach detecting substrate-to-product conversion without radioisotopes.
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Spectrophotometric coupled assays: Linking the isomerase reaction to a spectrophotometrically detectable change.
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Fluorescent sterol analogs: Utilizing fluorescent Delta(8)-sterol derivatives and measuring changes in emission spectra upon isomerization.
For optimal results, the following protocol components are recommended:
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Buffer conditions: pH 7.0-7.5 with detergent concentrations above the critical micelle concentration
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Substrate concentration: 10-50 μM Delta(8)-sterol substrate
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Reaction temperature: 37°C for human EBP
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Reaction time: Linear range typically between 5-30 minutes
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Detection method: LC-MS/MS provides the best combination of sensitivity and specificity
These methodological considerations are crucial for obtaining reproducible activity measurements across different experimental setups.
What is the specific role of EBP in the cholesterol biosynthesis pathway?
EBP catalyzes one of the last steps in cholesterol de novo biosynthesis, specifically the conversion of Delta(8)-sterols to their corresponding Delta(7)-isomers . This reaction is critical for the proper synthesis of cholesterol, which serves as a precursor for steroid hormones and is an essential component of cell membranes.
The position of EBP in the cholesterol biosynthesis pathway can be visualized as follows:
| Step | Enzyme | Reaction |
|---|---|---|
| ... | Previous enzymes | Earlier steps in cholesterol synthesis |
| n-2 | Sterol reductase | Reduction of specific double bonds |
| n-1 | C-4 sterol methyloxidase | Oxidation of C-4 methyl groups |
| n | EBP (Delta(8)-Delta(7) isomerase) | Conversion of Delta(8)-sterols to Delta(7)-sterols |
| n+1 | 7-dehydrocholesterol reductase | Reduction of the Delta(7) double bond |
| n+2 | Final processing enzymes | Completion of cholesterol synthesis |
This specific positioning makes EBP a potential target for modulating cholesterol biosynthesis in research contexts. Inhibition of EBP activity leads to the accumulation of Delta(8)-sterols and disruption of downstream sterol metabolism, with potential implications for diseases characterized by cholesterol dysregulation.
What diseases are associated with EBP mutations, and what are their molecular mechanisms?
Mutations in the EBP gene are associated with several genetic disorders, most notably Chondrodysplasia Punctata 2, X-Linked Dominant (CDPX2) and Mend Syndrome . The molecular mechanisms underlying these disorders involve:
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Loss of isomerase activity: Mutations can disrupt the catalytic activity of EBP, leading to incomplete cholesterol synthesis and accumulation of abnormal sterol intermediates.
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Altered protein stability: Some mutations affect the folding and stability of the EBP protein, resulting in reduced cellular levels of functional enzyme.
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Disrupted membrane localization: Certain mutations interfere with proper targeting of EBP to the endoplasmic reticulum membrane, where it normally functions.
Research has identified the following correlation between mutation types and disease severity:
| Mutation Type | Effect on Enzyme Activity | Disease Association | Severity |
|---|---|---|---|
| Missense mutations in transmembrane domains | Partial loss of function | Milder forms of CDPX2 | Moderate |
| Nonsense mutations | Complete loss of function | Severe CDPX2, Mend Syndrome | Severe |
| Splice site mutations | Variable enzyme production | Variable presentation | Variable |
Understanding these genotype-phenotype correlations is crucial for developing targeted therapeutic approaches and for accurate genetic counseling.
How can structural studies of EBP inform drug development targeting sterol metabolism?
Recent structural elucidation of EBP has revealed important insights for drug development . The protein's structure shows specific binding pockets that can be targeted for modulating cholesterol biosynthesis. Key considerations for structure-based drug design include:
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Active site targeting: Compounds designed to interact with catalytic residues can inhibit the isomerase activity with high specificity.
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Allosteric modulation: Several potential allosteric sites have been identified that could enable fine-tuning of enzyme activity rather than complete inhibition.
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Ligand-binding properties: EBP's high-affinity binding to emopamil and other phenylalkylamine calcium antagonists provides a structural basis for developing dual-action compounds that might target both sterol metabolism and calcium signaling .
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Membrane-protein interface: The protein-lipid interactions at the membrane interface offer unique opportunities for developing compounds that disrupt proper enzyme positioning.
Structural information has already led to the development of several experimental compounds that show promise in modulating EBP activity in vitro, with potential applications in disorders of cholesterol metabolism.
What are the latest methodological advances in studying EBP-substrate interactions?
Recent technological advances have significantly enhanced our ability to study EBP-substrate interactions:
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Cryo-electron microscopy: High-resolution structures of EBP in complex with substrates and inhibitors have revealed detailed binding mechanisms and conformational changes during catalysis.
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Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique allows for the mapping of protein dynamics and ligand-induced conformational changes without requiring protein crystallization.
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Surface plasmon resonance (SPR) and microscale thermophoresis (MST): These methods enable real-time measurement of binding kinetics between EBP and various ligands or substrates.
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Computational approaches: Molecular dynamics simulations have provided insights into the dynamics of substrate entry, binding, and product release from the active site.
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CRISPR-based screening: Genome-wide screens have identified potential interacting partners and regulators of EBP activity in cellular contexts.
These methodological advances have collectively enhanced our understanding of how EBP functions at the molecular level and have opened new avenues for therapeutic intervention in conditions associated with abnormal sterol metabolism.
What are emerging research areas connecting EBP to broader cellular functions?
Beyond its established role in cholesterol biosynthesis, emerging research suggests EBP may have additional functions that warrant further investigation:
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Autophagy regulation: Preliminary evidence suggests connections between EBP activity and cellular autophagy pathways, potentially linking sterol metabolism to broader cellular quality control mechanisms .
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Oligodendrocyte formation: EBP appears to play a role in oligodendrocyte development, suggesting implications for myelin formation and potentially for demyelinating disorders .
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Calcium signaling: Given EBP's binding affinity for calcium channel modulators like emopamil, potential cross-talk between sterol metabolism and calcium signaling pathways represents an intriguing area for further research .
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Sterol-dependent membrane organization: The specific sterols produced through EBP activity may have distinct effects on membrane organization and function beyond serving as precursors for cholesterol.
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Interaction with other metabolic pathways: How EBP activity and the resulting sterol intermediates influence or are influenced by other metabolic pathways remains an active area of investigation.
These research directions highlight the potential for EBP-focused studies to contribute insights beyond cholesterol metabolism, potentially informing our understanding of cellular homeostasis more broadly.
What considerations are important for designing EBP inhibitors with therapeutic potential?
Developing effective and specific EBP inhibitors presents several challenges and opportunities that researchers should consider:
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Selectivity among sterol isomerases: Ensuring compounds specifically target EBP rather than other isomerases like HSD3B2 requires careful design based on structural differences .
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Membrane penetration: As an endoplasmic reticulum membrane protein, effective inhibitors must navigate cellular membranes to reach their target.
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Therapeutic window: Complete inhibition of EBP would disrupt cholesterol biosynthesis, potentially causing adverse effects. Partial inhibition may be preferable for therapeutic applications.
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Structure-activity relationship studies: Systematic modification of lead compounds is essential for optimizing potency, selectivity, and pharmacokinetic properties.
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Cell-based validation assays: Beyond enzyme inhibition assays, compounds should be validated in cellular models that can assess effects on the complete sterol biosynthesis pathway.
Promising approaches include developing reversible inhibitors that allow for titrated modulation of enzyme activity, and potentially exploring the dual-targeting of EBP and related enzymes in the cholesterol biosynthesis pathway for synergistic effects.
