Recombinant Pig Carnitine O-palmitoyltransferase 1, muscle isoform (CPT1B)

What is CPT1B and what is its primary function in cellular metabolism?

CPT1B (Carnitine palmitoyltransferase 1B) is a rate-limiting enzyme located at the outer mitochondrial membrane that catalyzes the carnitine conjugation of long-chain fatty acids (LCFAs). This reaction is essential for facilitating the mitochondrial uptake of LCFAs and plays a key role in long-chain fatty acid β-oxidation, particularly in tissues with high energy demands . The muscle isoform (CPT1B) is predominantly expressed in heart, skeletal muscle, and brown adipose tissue where it regulates energy metabolism through controlling the entry of fatty acids into the mitochondria for oxidation .

How does CPT1B differ from other CPT1 isoforms in terms of tissue distribution and function?

While all three CPT isoforms (CPT1A, CPT1B, and CPT1C) bind malonyl-CoA, their tissue distribution and catalytic functions differ significantly:

• CPT1B (muscle isoform): Predominantly expressed in heart, skeletal muscle, and brown adipose tissue

• CPT1A (liver isoform): Mainly expressed in liver and other tissues

• CPT1C (brain isoform): Brain-specific with distinct functions in metabolism

CPT1A and CPT1B both catalyze acyl transfer from various fatty acyl-CoAs to carnitine, whereas CPT1C has limited catalytic activity . CPT1B appears to have stronger associations with long-chain acylcarnitine levels in tissues like placenta, suggesting it may be the main transferase for converting medium and long-chain fatty acids into acylcarnitines in certain tissues .

What methods are used to measure CPT1B activity in experimental settings?

The standard approach for measuring CPT1B activity involves:

• Isolation of mitochondria from target tissues

• Measuring the rate of formation of palmitoylcarnitine from palmitoyl-CoA plus carnitine

• Quantification using spectrophotometric or radioisotope-based assays

A modified mitochondrial CPT1 assay as described in He et al. (2012) involves:

• Preparing mitochondrial fractions from tissue samples

• Incubating with substrates (palmitoyl-CoA and radiolabeled carnitine)

• Measuring the conversion rate to palmitoylcarnitine

• Expressing activity as nmol of palmitoylcarnitine formed per minute per mg of protein

What expression systems are most effective for producing recombinant pig CPT1B protein?

For recombinant pig CPT1B production, several expression systems have been used with varying degrees of success:

• Mammalian cell expression systems: HEK293 cells are often preferred for maintaining proper post-translational modifications and protein folding, which is critical for membrane proteins like CPT1B .

• E. coli expression systems: While less expensive and higher-yielding, bacterial systems may struggle with proper folding of mammalian membrane proteins like CPT1B. Special fusion tags and solubilization strategies are often required .

• Wheat germ cell-free systems: These can be effective for producing membrane proteins without the complications of cellular toxicity.

The choice depends on your experimental requirements:

• For functional studies: Mammalian expression systems are recommended

• For structural studies: E. coli systems with optimization for solubility may be sufficient

• For high-throughput screening: Cell-free systems offer advantages

What purification strategies yield the highest activity for recombinant pig CPT1B?

Optimizing purification of recombinant pig CPT1B requires careful consideration of its membrane-bound nature:

• Affinity tag selection: His-tagged constructs allow for metal affinity chromatography, while tags like GST, T7, or Fc may be used depending on experimental needs .

• Detergent solubilization: Critical for extracting CPT1B from membranes while maintaining activity. Mild detergents like DDM (n-dodecyl β-D-maltoside) or CHAPS are typically used.

• Multi-step purification: Often combines:

  • Initial affinity chromatography (IMAC for His-tagged proteins)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Buffer optimization: Including glycerol (10-20%), reducing agents, and specific lipids can help maintain enzyme stability during purification.

• Activity preservation: Addition of malonyl-CoA during purification can help stabilize the protein structure.

How can researchers validate the functional activity of recombinant pig CPT1B?

Validating recombinant pig CPT1B activity requires multiple approaches:

• Enzymatic activity assay: Measure the rate of palmitoylcarnitine formation using:

  • Radioisotope-based assays with [14C]-labeled carnitine

  • HPLC-based methods for direct quantification

  • Coupled enzyme assays monitoring CoA release

• Malonyl-CoA inhibition: Functional CPT1B should demonstrate dose-dependent inhibition by malonyl-CoA, its natural feedback inhibitor .

• Immunoblotting: Confirm protein expression and integrity using specific antibodies. Anti-CPT1B antibodies like those raised against specific peptide sequences (e.g., CKTVDPNTPTSSTNL) can be used after validation against epitope-tagged constructs .

• Protein-protein interaction studies: Functional CPT1B should demonstrate expected interactions with partners like VDAC1 .

How does the CPT1B-VDAC1 interaction regulate fatty acid metabolism, and how can this be studied?

The CPT1B-VDAC1 interaction represents a critical regulatory mechanism for fatty acid metabolism that can be studied through multiple approaches:

• Co-immunoprecipitation: Studies have demonstrated that CPT1B interacts with VDAC1 (Voltage-Dependent Anion Channel 1) at the outer mitochondrial membrane. This interaction is essential for LCFA β-oxidation in cardiomyocytes .

• Study of interaction mechanisms: Research has shown that the CPT1B-VDAC1 complex formation is PHD2/3 activity dependent, with CPT1B-P295 residue identified as a prolyl-4-hydroxylation site required for CPT1B-VDAC1 binding .

• Functional analysis: The interaction can be verified by:

  • Overexpression of wild-type vs. mutant CPT1B (e.g., CPT1B-P295A)

  • Monitoring LCFA β-oxidation rates under different conditions

  • Analyzing acylcarnitine profiles in mitochondria

• Visualization techniques: Proximity ligation assays or FRET-based approaches can provide spatial information about the interaction in intact cells.

What role does prolyl hydroxylation play in CPT1B function, and how can researchers study this modification?

Prolyl hydroxylation represents a critical post-translational modification of CPT1B that regulates its activity:

• Identification of hydroxylation sites: CPT1B-P295 has been identified as a prolyl-4-hydroxylation site required for CPT1B-VDAC1 binding .

• Enzymes responsible: The prolyl hydroxylation of CPT1B is dependent on Prolyl Hydroxylase Domain proteins 2 and 3 (PHD2/3), which are highly enriched in heart tissue .

• Methodological approaches:

  • Site-directed mutagenesis: Creating P295A mutants that are oxygen-insensitive

  • Mass spectrometry: To directly detect and quantify hydroxylated peptides

  • Immunoprecipitation studies: Using antibodies specific to PHD2/3 to pull down CPT1B

  • Functional assays: Comparing LCFA β-oxidation between wild-type and mutant CPT1B

• Physiological significance: Overexpression of oxygen-insensitive CPT1B-P295A mutant maintains CPT1B-VDAC1 interaction and LCFA β-oxidation in PHD2/3-deficient cardiomyocytes, suggesting a regulatory mechanism linking oxygen sensing to fatty acid metabolism .

How does pig CPT1B compare structurally and functionally to human and mouse CPT1B?

Understanding species differences is crucial when using pig CPT1B as a model:

What are appropriate in vivo models for studying pig CPT1B function in metabolic diseases?

When designing in vivo models for studying pig CPT1B, researchers should consider:

• Heterozygous knockout models: Complete CPT1B knockout is embryonically lethal, but heterozygous CPT1B+/- pigs would allow for studying the effects of reduced CPT1B activity. This approach parallels studies in mice where CPT1B+/- animals showed normal development but increased susceptibility to cardiac dysfunction under stress .

• Tissue-specific knockout models: Using Cre-lox systems for tissue-specific deletion of CPT1B in muscle or heart. This approach has been successful in mice (e.g., CPT1B SKM-/- for skeletal muscle-specific knockout) .

• Stress challenge models: Exposing animals to conditions that increase cardiac workload:

  • Transverse aortic constriction (TAC) to induce pressure overload

  • High-fat diet to study metabolic flexibility

  • Exercise protocols to examine energetic adaptation

• Phenotypic assessments:

  • Echocardiography for cardiac function (EF%, FS%, LVPW, LVPD)

  • Metabolic studies measuring fatty acid oxidation rates

  • Histological assessment for lipid accumulation and fibrosis

  • Electron microscopy to assess mitochondrial morphology

• Biochemical analyses:

  • Acylcarnitine profiling in tissues and plasma

  • Expression of hypertrophy markers (Nppa, Nppb, MHC-β)

  • Measurement of triglycerides and ceramide content

How can recombinant pig CPT1B be used to screen for novel inhibitors or activators for metabolic disease therapy?

Developing a screening platform using recombinant pig CPT1B requires:

What approaches can be used to study the effects of CPT1B post-translational modifications on enzyme activity?

Advanced studies of CPT1B post-translational modifications require:

• Mass spectrometry-based approaches:

  • Proteomics workflows to identify modification sites

  • Targeted MS/MS for quantification of specific modifications

  • Comparison between recombinant and native enzyme modifications

• Site-directed mutagenesis:

  • Generation of mutants at key sites (e.g., P295A for hydroxylation studies)

  • Analysis of mutant effects on:

    • Enzyme activity

    • Protein-protein interactions

    • Subcellular localization

    • Response to regulatory factors

• Structural biology techniques:

  • X-ray crystallography or cryo-EM to determine structural consequences

  • Hydrogen-deuterium exchange MS to probe conformational changes

  • Molecular dynamics simulations to predict functional impacts

• Cell-based functional studies:

  • Expression of wild-type vs. modified CPT1B in cellular models

  • Real-time monitoring of fatty acid oxidation

  • Analysis of acylcarnitine profiles as functional readouts

The role of CPT1B in cancer metabolism represents an emerging research area:

• Expression analysis in cancer tissues:

  • CPT1B expression has been studied as a prognostic marker in conditions like acute myeloid leukemia (AML)

  • High CPT1B expression has been associated with poor clinical outcomes in certain cancers

• Functional studies in cancer models:

  • Manipulation of CPT1B expression in cancer cell lines

  • Assessment of effects on:

    • Fatty acid oxidation rates

    • Cancer cell proliferation

    • Response to metabolic stress

    • Therapeutic sensitivity

• Molecular analysis:

  • Constructing ceRNA networks involving CPT1B in cancer

  • Investigating miRNA-mRNA interactions regulating CPT1B

  • Studying lncRNAs that may function as ceRNAs for CPT1B

• Clinical correlations:

  • Analysis of CPT1B expression in patient samples shows it can serve as an independent risk factor in certain cancers

  • Potential development of CPT1B-targeted therapies for specific cancer subtypes

What are the main challenges in producing full-length functional recombinant pig CPT1B and how can they be overcome?

Production of functional recombinant pig CPT1B presents several technical challenges:

• Membrane protein solubility:

  • Challenge: CPT1B is an integral membrane protein with hydrophobic domains

  • Solution: Optimize detergent selection (DDM, CHAPS) or use nanodiscs/liposomes for reconstitution

  • Alternative: Express soluble domains separately for specific studies

• Maintaining native conformation:

  • Challenge: Preserving the correct folding during expression and purification

  • Solution: Expression at lower temperatures (16-18°C), use of molecular chaperones

  • Validation: Circular dichroism spectroscopy to confirm secondary structure

• Post-translational modifications:

  • Challenge: Bacterial systems lack appropriate modification machinery

  • Solution: Use mammalian expression systems (HEK293) for studies requiring native modifications

  • Verification: Mass spectrometry to confirm modification status

• Activity preservation:

  • Challenge: Loss of activity during purification

  • Solution: Include stabilizing agents (glycerol, specific lipids) and optimize buffer conditions

  • Quality control: Regular activity testing throughout purification process

How can researchers effectively study the CPT1B-VDAC1 interaction when working with recombinant proteins?

Studying the CPT1B-VDAC1 interaction with recombinant proteins requires specialized approaches:

• Co-expression systems:

  • Dual expression vectors for both proteins

  • Bicistronic constructs with appropriate tags

  • Mammalian or insect cell systems for proper folding

• In vitro reconstitution:

  • Purification of individual components

  • Reconstitution in artificial membrane systems (liposomes, nanodiscs)

  • Verification of interaction through pull-down assays

• Structural analysis:

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Single-particle cryo-EM of the complex

  • Hydrogen-deuterium exchange to map binding surfaces

• Functional validation:

  • Incorporation into proteoliposomes for transport assays

  • Measuring fatty acid oxidation rates in reconstituted systems

  • Mutational analysis of key residues identified in structural studies

• Regulatory mechanisms:

  • Studies have shown that CPT1B-VDAC1 interaction is PHD2/3 activity dependent

  • The CPT1B-P295 residue is a critical prolyl-4-hydroxylation site required for binding

  • These insights can guide the design of interaction studies

What emerging technologies might advance our understanding of CPT1B regulation and function?

Several cutting-edge technologies hold promise for CPT1B research:

• CRISPR-based approaches:

  • Base editing for precise modification of CPT1B at specific sites

  • CRISPRi/CRISPRa for temporal control of CPT1B expression

  • CRISPR screens to identify novel regulators of CPT1B function

• Single-cell metabolomics:

  • Analysis of cell-to-cell variability in CPT1B activity

  • Correlation with other metabolic parameters at single-cell resolution

  • Identification of metabolically distinct cell populations

• Advanced imaging techniques:

  • Super-resolution microscopy of CPT1B localization and dynamics

  • FRET-based sensors for real-time monitoring of CPT1B activity

  • Correlative light and electron microscopy for structural context

• Computational approaches:

  • Molecular dynamics simulations of CPT1B-substrate interactions

  • Machine learning for prediction of inhibitor binding and efficacy

  • Systems biology modeling of CPT1B's role in metabolic networks

How might understanding species-specific differences in CPT1B function influence translational research?

Species-specific differences in CPT1B have important translational implications:

• Drug development considerations:

  • Inhibitors developed against pig CPT1B may have different efficacy in humans

  • Understanding binding site conservation is critical for translational studies

  • Comparative studies of inhibitor binding can guide drug optimization

• Model selection guidance:

  • Pig models may be more appropriate than rodent models for certain aspects of human CPT1B biology

  • Species-specific differences should inform the choice of preclinical models

  • Multi-species testing may be necessary for comprehensive evaluation

• Precision medicine applications:

  • Identifying conserved vs. divergent regulatory mechanisms

  • Understanding how genetic variations affect CPT1B function across species

  • Developing targeted therapies based on conserved mechanisms

• Evolutionary insights:

  • Comparative analysis of CPT1B across species can reveal evolutionary adaptations

  • Functional conservation suggests critical metabolic roles

  • Species-specific differences may reflect metabolic adaptations to different diets or environmental pressures