Recombinant Pig Cytochrome P450 4A24 (CYP4A24)

What is Cytochrome P450 4A24 and how was it initially identified?

Cytochrome P450 4A24 (CYP4A24) is a member of the cytochrome P450 4A subfamily that was initially identified during extensive studies aimed at verifying aberrant amino acids found in CYP4A21 within a normally conserved CYP4A motif. CYP4A24 was co-amplified by PCR alongside CYP4A21 and CYP4A25 from both pig liver and kidney tissues. After detection, the sequence was characterized by restriction-enzyme analysis and subsequently cloned . Unlike some other cytochrome P450 enzymes, CYP4A24 maintains relatively stable protein expression throughout developmental stages in Göttingen Minipigs, suggesting its consistent role in various developmental phases .

How does CYP4A24 differ from other members of the porcine CYP4A subfamily?

CYP4A24 differs from other porcine CYP4A subfamily members primarily in its amino acid sequence and enzymatic activities. While it shares approximately 97% sequence identity with CYP4A25, it has more substantial differences from CYP4A21. Unlike CYP4A21, which does not catalyze omega- or (omega-1)-hydroxylation of lauric acid and instead participates in bile acid biosynthesis, CYP4A24 exhibits both omega- and (omega-1)-hydroxylase activities towards lauric acid and palmitic acid . The variable regions between CYP4A24 and CYP4A25 are primarily confined to beta-sheets 1 and 4, which potentially indicates differences in substrate specificity or regioselectivity despite their high sequence similarity .

What are the optimal methods for cloning CYP4A24 from porcine liver?

For optimal cloning of CYP4A24 from porcine liver, PCR-based approaches have proven successful. Based on the available research, the following methodology is recommended:

• Extract total RNA from fresh porcine liver tissue

• Perform reverse transcription to obtain cDNA

• Design specific primers based on conserved regions of CYP4A family while accounting for the unique regions of CYP4A24

• Amplify the CYP4A24 sequence using high-fidelity DNA polymerase

• Perform restriction-enzyme analysis to distinguish CYP4A24 from co-amplified sequences (particularly CYP4A21 and CYP4A25)

• Clone the isolated sequence into an appropriate expression vector

It's essential to note that when performing PCR amplification of CYP4A sequences from porcine liver, multiple CYP4A isoforms may be co-amplified. Therefore, careful sequence verification and restriction enzyme analysis are critical steps to ensure isolation of the specific CYP4A24 sequence .

What expression systems are most effective for producing functional recombinant CYP4A24?

For expressing functional CYP4A24, a yeast expression system is recommended as it has been successfully used to express both CYP4A24 and CYP4A25 in their active forms capable of hydroxylating lauric and palmitic acids . For optimal expression in yeast, the sequence should be codon-optimized and expressed with a suitable promoter (such as GAL1 or ADH2) .

How can researchers distinguish between expressed CYP4A24 and closely related CYP4A25 during protein purification?

Distinguishing between the closely related CYP4A24 and CYP4A25 (97% sequence identity) during protein purification presents a significant challenge. The following methodological approach is recommended:

• Use affinity tags specific to each protein by employing differentially tagged constructs (e.g., His-tag for CYP4A24 and FLAG-tag for CYP4A25)

• Employ high-resolution chromatographic techniques such as ion-exchange chromatography that can separate proteins based on subtle charge differences

• Develop isoform-specific antibodies targeting the variable regions, particularly those in beta-sheets 1 and 4 where most differences between these enzymes are located

• Utilize mass spectrometry-based approaches to identify unique peptide fragments that distinguish between these highly similar proteins

• Implement restriction enzyme analysis if working with the encoding DNA, as this approach was successfully used in the original characterization of these enzymes

For confirmation of identity, enzyme activity assays with specific substrates may provide additional verification, as subtle differences in substrate specificity or regioselectivity are expected between these isoforms .

What are the primary catalytic activities of recombinant CYP4A24?

Recombinant CYP4A24 exhibits primarily omega- and (omega-1)-hydroxylase activities towards fatty acids. The enzyme efficiently catalyzes the hydroxylation of:

• Lauric acid (C12:0) - A medium-chain fatty acid that serves as a model substrate for CYP4A enzymes

• Palmitic acid (C16:0) - A long-chain fatty acid that is a physiologically relevant substrate

The hydroxylation occurs predominantly at the terminal (omega) and penultimate (omega-1) carbon positions of these fatty acids . This enzymatic activity contributes to fatty acid metabolism in porcine liver and kidney tissues. Unlike CYP4A21, which has evolved to participate in bile acid biosynthesis and lacks the ability to hydroxylate lauric acid, CYP4A24 maintains the characteristic hydroxylase activities typical of the CYP4A subfamily .

How does the substrate specificity of CYP4A24 compare to other cytochrome P450 enzymes?

CYP4A24 displays a substrate specificity profile that is characteristic of the CYP4A subfamily but with distinctive features:

The substrate specificity of CYP4A24 primarily involves medium to long-chain fatty acids, with a preference for terminal and subterminal hydroxylation. The variable regions between CYP4A24 and its close homolog CYP4A25, which are confined to beta-sheets 1 and 4, may confer subtle differences in substrate specificity or regioselectivity between these highly similar enzymes .

What factors influence the catalytic efficiency of recombinant CYP4A24?

Several critical factors influence the catalytic efficiency of recombinant CYP4A24:

• Expression System Selection: The choice of expression system significantly impacts proper folding and post-translational modifications. Yeast systems have proven effective for producing catalytically active CYP4A24 .

• Redox Partner Coupling: Efficient electron transfer from NADPH through cytochrome P450 reductase to CYP4A24 is essential for optimal catalytic activity. The source and ratio of redox partners (reductase:CYP) significantly influences hydroxylation rates.

• Membrane Environment: As a membrane-bound enzyme, CYP4A24 requires an appropriate lipid environment for structural stability and optimal substrate access.

• pH and Ionic Conditions: Maintaining optimal pH (typically 7.2-7.6) and appropriate ionic strength is crucial for maximal enzyme activity.

• Substrate Concentration: Substrate availability within the appropriate concentration range avoiding inhibition is necessary for accurate assessment of catalytic efficiency.

• Temperature: Maintaining physiologically relevant temperature (37°C for mammalian enzymes) ensures proper enzyme conformation and activity.

Researchers should carefully control these parameters when designing experiments to assess CYP4A24 catalytic activity, particularly when making comparisons between different experimental conditions or enzyme variants .

How does CYP4A24 expression change during porcine development?

CYP4A24 exhibits a unique expression pattern during porcine development that differs from many other cytochrome P450 enzymes. While many CYP enzymes show gradual increases in protein abundance during development, CYP4A24 demonstrates relatively stable protein expression over time in Göttingen Minipigs . This stable expression pattern contrasts with enzymes like CYP51A1, which shows high expression during fetal stages followed by a decrease during the first month of life and a subsequent increase toward adulthood .

The consistent expression of CYP4A24 throughout developmental stages suggests that this enzyme plays a fundamental role in basic metabolic processes that remain essential throughout the organism's life cycle, rather than serving developmental stage-specific functions.

Are there sex-related differences in CYP4A24 protein abundance in adult pigs?

Based on the available research data, CYP4A24 does not appear to show significant sex-related differences in protein abundance in adult Göttingen Minipigs. This is notable because several other porcine CYP enzymes do exhibit sex-related differences in expression. For instance, CYP1A1, CYP1A2, CYP2A19, CYP2E1_2, CYP3A22, CYP4V2_2a, and CYP4V2_2b all show higher expression in female compared to male Göttingen Minipigs .

The absence of significant sex-related differences in CYP4A24 expression suggests that its metabolic functions may be equally important in both sexes and are not substantially influenced by sex hormones. This information is particularly valuable for researchers designing studies involving CYP4A24, as they may not need to control for sex as a significant variable when focusing specifically on this enzyme .

What methodologies are most reliable for quantifying CYP4A24 protein expression in tissue samples?

For reliable quantification of CYP4A24 protein expression in tissue samples, mass spectrometry-based approaches have proven most effective, particularly for distinguishing between highly similar CYP isoforms. The recommended methodological approach includes:

• Sample Preparation:

  • Careful extraction of microsomes from liver or kidney tissue

  • Protein denaturation, reduction, and alkylation

  • Tryptic digestion to generate peptide fragments

• Mass Spectrometry Analysis:

  • Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

  • Targeted analysis using multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM)

  • Selection of unique peptides that specifically distinguish CYP4A24 from closely related isoforms

• Quantification Approach:

  • Use of stable isotope-labeled peptide standards

  • Normalization against housekeeping proteins

  • Validation with QC samples of known concentration

This mass spectrometry-based approach has been successfully applied in developmental studies of Göttingen Minipigs and allows for accurate differentiation between CYP4A24 and other highly similar CYP enzymes, such as CYP4A25 (97% sequence identity) .

What are the human orthologs of porcine CYP4A24 and how do they differ functionally?

The primary human ortholog of porcine CYP4A24 is CYP4A11, which shares significant sequence similarity and functional characteristics:

The functional differences between porcine CYP4A24 and human CYP4A11 are primarily in substrate preference and regioselectivity of hydroxylation. While both enzymes metabolize fatty acids, human CYP4A11 plays a significant role in the production of 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid, which has important functions in vascular regulation. This difference has implications for using porcine models to study human drug metabolism and toxicity .

How suitable is CYP4A24 as a model for human CYP4A-mediated drug metabolism?

The suitability of CYP4A24 as a model for human CYP4A-mediated drug metabolism depends on the specific research question and compounds being studied:

Strengths as a model:

Limitations as a model:

• Differences in regioselectivity (omega vs. omega-1 hydroxylation)

• Potential differences in substrate specificity beyond fatty acids

• Different regulation patterns during development

• Potential differences in drug-drug interactions

What experimental designs best translate findings from recombinant CYP4A24 studies to human applications?

To effectively translate findings from recombinant CYP4A24 studies to human applications, researchers should implement the following experimental design strategies:

• Parallel Testing Platform:

  • Conduct side-by-side comparisons of recombinant porcine CYP4A24 and human CYP4A11 using identical experimental conditions

  • Include multiple substrate concentrations to generate complete kinetic profiles

  • Analyze both primary metabolites and secondary transformation products

• Integrated Multi-System Approach:

  • Combine recombinant enzyme studies with liver microsome assays from both species

  • Use hepatocytes or liver slices to account for cellular uptake and efflux processes

  • Employ PBPK (Physiologically Based Pharmacokinetic) modeling to scale from in vitro to in vivo

• Correlation Analysis Framework:

  • Establish quantitative structure-activity relationships (QSAR) for both enzymes

  • Develop mathematical models to predict human metabolism based on porcine data

  • Validate with a diverse set of probe substrates with known human metabolic profiles

• Validation Strategy:

  • Include positive and negative control compounds with well-characterized metabolism

  • Verify findings with in vivo studies in Göttingen Minipigs when feasible

  • Cross-reference with existing human clinical data when available

This comprehensive approach accounts for species differences while maximizing the translational value of research conducted with recombinant CYP4A24, particularly in drug development and toxicology applications .

What are the most effective methods for studying CYP4A24 enzyme kinetics?

For studying CYP4A24 enzyme kinetics, a comprehensive methodological approach incorporating multiple analytical techniques is recommended:

• Substrate Selection and Preparation:

  • Primary substrates: Lauric acid and palmitic acid

  • Prepare substrate solutions in appropriate solvents (typically DMSO) with final solvent concentration <1% in reaction mixtures

  • Use radiolabeled substrates for increased sensitivity where appropriate

• Reaction Conditions Optimization:

  • Buffer: 0.5 M potassium phosphate (K₃PO₄) buffer at pH 7.4

  • Temperature: 37°C (physiological)

  • Include NADPH-regenerating system (NADP+, glucose-6-phosphate, glucose-6-phosphate dehydrogenase)

  • Optimize protein concentration through linearity studies

• Kinetic Parameter Determination:

  • Measure initial reaction velocities at multiple substrate concentrations (typically 8-10 concentrations ranging from 0.1 × Km to 10 × Km)

  • Plot data using appropriate enzyme kinetic models (Michaelis-Menten, Hill, or substrate inhibition equations)

  • Determine key parameters (Km, Vmax, kcat, kcat/Km) using non-linear regression

• Analytical Methods:

  • HPLC or UPLC with appropriate detection (UV, fluorescence, or mass spectrometry)

  • Gas chromatography-mass spectrometry (GC-MS) for volatile metabolites

  • LC-MS/MS for comprehensive metabolite profiling and quantification

• Data Analysis Approach:

  • Use appropriate software (GraphPad Prism, SigmaPlot, or R) for kinetic modeling

  • Apply statistical methods to assess goodness of fit and parameter reliability

  • Compare kinetic parameters between experimental conditions using appropriate statistical tests

This methodology has been successfully applied in studies involving related CYP enzymes and provides a robust framework for characterizing CYP4A24 enzyme kinetics .

How can site-directed mutagenesis be applied to study structure-function relationships in CYP4A24?

Site-directed mutagenesis provides a powerful approach for investigating structure-function relationships in CYP4A24, particularly focusing on regions that differ from closely related enzymes:

• Target Selection Strategy:

  • Focus on the variable regions between CYP4A24 and CYP4A25 in beta-sheets 1 and 4, which likely contribute to differences in substrate specificity or regioselectivity

  • Target conserved motifs in the CYP4A family to assess their role in CYP4A24 function

  • Identify residues in the substrate binding pocket and substrate access channels

• Mutagenesis Methodology:

  • PCR-based mutagenesis using complementary primers containing the desired mutation

  • Use of commercially available kits (QuikChange, Q5 Site-Directed Mutagenesis) for efficient mutation introduction

  • Sequential mutations for addressing complex structure-function questions

• Expression and Analysis of Mutants:

  • Express wild-type and mutant proteins in parallel under identical conditions

  • Verify proper folding using CO-difference spectra

  • Conduct comprehensive kinetic analyses with multiple substrates

• Systematic Mutation Analysis Framework:

  • Alanine scanning: Replace individual residues with alanine to assess their contribution

  • Conservative vs. non-conservative substitutions: Compare effects of similar vs. dissimilar amino acid replacements

  • Reciprocal mutations: Swap residues between CYP4A24 and related enzymes (e.g., CYP4A25) to confirm their role in functional differences

• Integrated Analysis Approach:

  • Correlate functional changes with structural predictions using homology modeling

  • Use molecular dynamics simulations to assess effects on protein dynamics

  • Develop structure-function relationships across multiple mutations and substrates

This comprehensive mutagenesis approach has proven valuable for understanding cytochrome P450 enzymes and can be specifically tailored to elucidate the unique structural features of CYP4A24 that determine its catalytic properties .

What analytical techniques best detect and quantify CYP4A24-generated metabolites?

For optimal detection and quantification of CYP4A24-generated metabolites, a multi-platform analytical approach is recommended:

• Liquid Chromatography-Mass Spectrometry (LC-MS/MS):

  • Primary technique for comprehensive metabolite profiling

  • Multiple Reaction Monitoring (MRM) for targeted quantification of known metabolites

  • High-resolution MS for untargeted metabolomics and novel metabolite identification

  • Ion mobility spectrometry for separation of isomeric metabolites

• Gas Chromatography-Mass Spectrometry (GC-MS):

  • Excellent for volatile and thermally stable metabolites

  • Often requires derivatization for hydroxy-fatty acids

  • Provides complementary separation to LC-MS for comprehensive coverage

• Sample Preparation Optimization:

  • Liquid-liquid extraction for fatty acid metabolites

  • Solid-phase extraction for selective enrichment

  • Derivatization strategies to enhance sensitivity (e.g., pentafluorobenzyl esters for negative chemical ionization GC-MS)

• Specialized Detection Techniques:

  • Radiolabeled substrate metabolism with radiometric detection

  • UV detection at appropriate wavelengths (typically 205-220 nm for fatty acid metabolites)

  • Fluorescence detection after appropriate derivatization

• Data Analysis and Metabolite Identification:

  • Use of authentic standards for definitive identification

  • Structural elucidation based on fragmentation patterns

  • Software tools for automated metabolite identification

  • Quantification using internal standards (preferably stable isotope-labeled)

This comprehensive analytical approach enables reliable detection and quantification of both expected metabolites (omega and omega-1 hydroxylated fatty acids) and potentially novel metabolites from CYP4A24 enzymatic activity .

What are common challenges in expressing active recombinant CYP4A24 and how can they be addressed?

Researchers frequently encounter several challenges when expressing active recombinant CYP4A24. Below are the most common issues and recommended solutions:

• Low Expression Yield:

  • Problem: Insufficient protein production limiting experimental capabilities

  • Solution: Optimize codon usage for the expression host; use stronger promoters; adjust induction conditions (temperature, inducer concentration, duration); consider fusion tags that enhance expression

• Misfolding and Aggregation:

  • Problem: Formation of inclusion bodies or misfolded protein

  • Solution: Express at lower temperatures (16-25°C); co-express with molecular chaperones; use specialized E. coli strains designed for membrane protein expression; add detergents or lipids during purification

• Low Catalytic Activity:

  • Problem: Expressed protein shows reduced or no enzymatic activity

  • Solution: Ensure proper heme incorporation by supplementing with δ-aminolevulinic acid; optimize redox partner ratio and source; verify correct N-terminal modification; incorporate membrane mimetics (nanodiscs, liposomes)

• Instability During Purification:

  • Problem: Loss of activity during extraction and purification

  • Solution: Add glycerol (20%) and protease inhibitors to all buffers; avoid freeze-thaw cycles; maintain constant temperature during purification; use mild detergents at minimal concentrations

• Distinguishing from Similar Isoforms:

  • Problem: Difficulty separating CYP4A24 from CYP4A25 (97% identical)

  • Solution: Use highly specific affinity tags; develop isoform-specific antibodies; employ high-resolution chromatographic techniques; verify by mass spectrometry

Implementing these solutions has shown success in expressing functional cytochrome P450 enzymes in heterologous systems and can be specifically adapted for CYP4A24 expression challenges .

How can researchers troubleshoot inconsistent enzyme activity results with recombinant CYP4A24?

When faced with inconsistent enzyme activity results for recombinant CYP4A24, researchers should implement a systematic troubleshooting approach:

• Enzyme Quality Assessment:

  • Verify protein concentration with multiple methods (BCA, Bradford)

  • Confirm proper folding using CO-difference spectroscopy (characteristic 450 nm peak)

  • Check for degradation using SDS-PAGE and western blotting

  • Assess heme incorporation ratio (A417/A280 ratio)

• Reaction Conditions Verification:

  • Confirm NADPH system functionality with control reactions

  • Verify buffer pH and composition consistency

  • Control temperature precisely during incubations

  • Confirm absence of inhibitory contaminants in reagents

• Substrate Preparation Quality Control:

  • Verify substrate purity using analytical methods

  • Prepare fresh substrate solutions to avoid degradation

  • Control solvent concentration across experiments

  • Use internal standards to normalize extraction efficiency

• Analytical Method Robustness:

  • Implement system suitability tests before analytical runs

  • Include quality control samples at multiple concentrations

  • Use stable isotope-labeled internal standards

  • Validate method linearity, precision, and accuracy

• Experimental Design Considerations:

  • Perform time-course studies to ensure linearity

  • Include positive controls (e.g., CYP4A25 or liver microsomes)

  • Use statistical replicates (minimum triplicates)

  • Implement factorial design to identify interactive factors

By systematically addressing these potential sources of variability, researchers can significantly improve the consistency and reliability of enzyme activity results with recombinant CYP4A24 .

What methodological advances have improved the study of membrane-bound enzymes like CYP4A24?

Recent methodological advances have significantly enhanced the study of membrane-bound enzymes like CYP4A24:

• Nanodiscs and Membrane Mimetics:

  • Phospholipid bilayer nanodiscs provide a native-like membrane environment

  • Allow precise control of lipid composition and oligomeric state

  • Enable structural studies of membrane-bound state

  • Improve enzyme stability and activity compared to detergent solubilization

• Advanced Expression Systems:

  • Engineered yeast strains with modified membrane composition

  • Mammalian cell lines with reduced endogenous P450 activity

  • Cell-free expression systems allowing direct incorporation into liposomes

  • Specialized E. coli strains optimized for membrane protein expression

• Biophysical Characterization Techniques:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics

  • Single-molecule FRET for conformational changes

  • Cryo-electron microscopy for structural determination

  • Solid-state NMR for membrane-embedded protein structures

• Computational Approaches:

  • Molecular dynamics simulations in explicit membrane environments

  • Hybrid quantum mechanics/molecular mechanics for reaction mechanisms

  • Machine learning approaches for predicting substrate interactions

  • Systems biology modeling of enzyme networks

• Mass Spectrometry Innovations:

  • Protein MS for absolute quantification (AQUA, QconCAT approaches)

  • Intact protein analysis for post-translational modifications

  • Cross-linking MS for protein-protein interactions

  • Ion mobility separation for conformational analysis

These methodological advances provide researchers with powerful tools to overcome traditional challenges associated with studying membrane-bound enzymes like CYP4A24, enabling more detailed structural, functional, and quantitative analyses .

What are the most promising applications of recombinant CYP4A24 in pharmaceutical research?

Recombinant CYP4A24 holds significant potential for several pharmaceutical research applications:

• Comparative Drug Metabolism Studies:

  • Use as a model system for understanding species differences in drug metabolism

  • Comparative analysis with human CYP4A11 to predict cross-species metabolic variations

  • Development of in vitro-to-in vivo extrapolation (IVIVE) models specific for fatty acid-metabolizing CYPs

• Drug Candidate Screening:

  • Early identification of compounds metabolized by CYP4A enzymes

  • Assessment of potential drug-drug interactions involving fatty acid metabolism

  • Evaluation of species-specific toxicity related to CYP4A-mediated metabolism

• Biomarker Development:

  • Identification of specific CYP4A24 metabolites as potential biomarkers of porcine liver function

  • Correlation of metabolite profiles with disease states in porcine models

  • Translational research on fatty acid metabolism biomarkers across species

• Physiologically Based Pharmacokinetic (PBPK) Modeling:

  • Integration of CYP4A24 kinetic data into PBPK models for the Göttingen Minipig

  • Improved prediction of drug disposition in preclinical porcine studies

  • Development of more accurate scaling factors for extrapolation to humans

• Novel Drug Target Identification:

  • Understanding the role of CYP4A-mediated metabolism in disease states

  • Identification of potential therapeutic targets in fatty acid metabolism pathways

  • Development of inhibitors or modulators of CYP4A activity for therapeutic applications

These applications leverage the unique properties of CYP4A24 and its relationship to human CYP4A enzymes to enhance pharmaceutical research and development processes.

How might genetic polymorphisms in CYP4A24 affect its function in different pig breeds?

Genetic polymorphisms in CYP4A24 across different pig breeds likely have significant implications for enzyme function and metabolic capabilities:

• Potential Functional Consequences:

  • Altered substrate specificity or regioselectivity

  • Modified catalytic efficiency (changed Km or Vmax values)

  • Differences in protein stability or membrane integration

  • Variable expression levels or tissue distribution

  • Altered regulatory responses to environmental or dietary factors

• Breed-Specific Considerations:

  • Conventional breeds vs. minipigs (e.g., Göttingen Minipig)

  • Regional/geographic variations in pig populations

  • Effects of selective breeding for specific traits

  • Wild boar vs. domesticated pig comparisons

• Research Methodology for Polymorphism Studies:

  • Systematic sequencing of CYP4A24 across diverse pig populations

  • Functional characterization of variant alleles using recombinant expression

  • Correlation of genotypes with metabolic phenotypes

  • Population genetic analysis to identify selection pressures

• Implications for Translational Research:

  • Selection of appropriate pig models for specific research questions

  • Consideration of genetic background in interpreting metabolic data

  • Potential for personalized/precision medicine approaches in veterinary applications

  • Improved understanding of evolutionary adaptations in xenobiotic metabolism

The study of CYP4A24 polymorphisms across pig breeds may reveal important insights into the evolutionary adaptation of metabolic enzymes and could inform more precise selection of porcine models for specific research applications .

What interdisciplinary approaches could advance understanding of CYP4A24 in biological systems?

Advancing our understanding of CYP4A24 in biological systems requires innovative interdisciplinary approaches that integrate multiple scientific disciplines:

• Systems Biology Integration:

  • Network analysis of CYP4A24 interactions with other metabolic enzymes

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Mathematical modeling of fatty acid metabolism pathways

  • Identification of regulatory networks controlling CYP4A24 expression

• Structural Biology and Computational Chemistry:

  • Cryo-EM structures of CYP4A24 in membrane environments

  • Molecular dynamics simulations of substrate binding and product release

  • Quantum mechanical modeling of reaction mechanisms

  • Machine learning approaches for predicting substrate specificity

• Developmental Biology and Epigenetics:

  • Analysis of developmental regulation of CYP4A24 expression

  • Epigenetic modifications affecting CYP4A24 gene expression

  • Impact of maternal environment on offspring CYP4A24 activity

  • Comparative analysis across developmental stages

• Environmental Toxicology and Nutrition Science:

  • Effects of dietary components on CYP4A24 activity

  • Impact of environmental contaminants on fatty acid metabolism

  • Nutritional programming of CYP4A expression

  • Xenobiotic-nutrient interactions mediated by CYP4A24

• Translational Medicine and Comparative Physiology:

  • Parallel studies in multiple species to identify conserved mechanisms

  • Development of porcine disease models relevant to human conditions

  • Biomarker discovery spanning from porcine models to human applications

  • One Health approaches linking animal and human health through metabolic pathways

By integrating these interdisciplinary approaches, researchers can develop a comprehensive understanding of CYP4A24's role in biological systems, from molecular mechanisms to physiological functions and disease implications .