Recombinant Rat Carnitine O-palmitoyltransferase 1, liver isoform (Cpt1a)

What is the functional role of CPT1A in cellular metabolism?

CPT1A is the rate-limiting enzyme in the carnitine palmitoyltransferase system, responsible for facilitating the transfer of long-chain fatty acids from the cytosol into the mitochondrial matrix for β-oxidation. It catalyzes the conversion of long-chain acyl-CoA to acylcarnitine, which can then be transported across the inner mitochondrial membrane by carnitine-acylcarnitine translocase. This process is essential for energy production from fatty acids, particularly during fasting or high energy demand states .

Methodological approach: To study CPT1A's role in metabolism, researchers typically employ enzyme activity assays using spectrophotometric detection of CoA released during the enzymatic reaction with DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)). This provides a reliable proxy for enzyme activity based on the direct relationship between CPT1A catalytic activity and increasing CoA concentration .

How is CPT1A structurally organized, particularly its transmembrane domains?

CPT1A is anchored to the outer mitochondrial membrane through two transmembrane domains, with both the N-terminus and the catalytic C-terminus facing the cytosol. Transmembrane domain 2 (TM2) has been particularly studied for its role in oligomerization through GXXXG(A) motifs, which affects the enzyme's sensitivity to malonyl-CoA inhibition .

Methodological approach: To investigate the structure-function relationship of CPT1A's transmembrane domains, researchers can use complementary genetic assays that facilitate measurement of helix-helix interactions in the Escherichia coli inner membrane, combined with multiple quantitative biophysical methods .

What experimental systems are most suitable for studying rat CPT1A?

Several experimental systems have proven effective for studying rat CPT1A:

• In vitro expression systems: E. coli for expressing catalytic domains (Leu572~Lys773) with N-terminal His and GST tags .

• Yeast expression: Pichia pastoris for full-length enzyme expression with appropriate post-translational modifications .

• Mammalian cell systems: Expi293 cells transfected with CPT1A plasmid for reliable and robust source of catalytically active human CPT1A .

• Animal models: Cardiac-specific CPT1A knockout mice and AAV9-mediated cardiac-specific CPT1A overexpression for in vivo studies .

• Primary cell cultures: Isolated hepatocytes for glucose production studies .

Methodological recommendation: For optimal protein expression, direct transfection of mammalian cells (such as Expi293) with a CPT1A plasmid has been demonstrated to provide reliable enzyme activity for high-throughput screening applications .

What methods are most effective for detecting and quantifying CPT1A?

Researchers employ several complementary approaches to detect and quantify CPT1A:

• Immunodetection: ELISA using specific anti-CPT1A monoclonal antibodies with sensitivity reaching 9.375 pg/ml .

• Protein analysis: Western blotting for protein expression levels in tissue or cell samples .

• Transcriptional analysis: RT-qPCR for mRNA expression evaluation, which shows variable correlation with protein levels depending on the tissue context .

• Activity assays: Spectrophotometric detection of CoA using DTNB or radioisotope-based assays using 3H-carnitine (though the latter has safety limitations) .

Methodological insight: The ELISA approach allows detection of CPT1A in the transfected cell pellet with approximately 13-fold higher sensitivity compared to control cell pellets, while minimal CPT1A is detected in the supernatant, confirming its tight association with mitochondrial membranes .

How does oligomerization of transmembrane domains affect CPT1A function and malonyl-CoA sensitivity?

The oligomerization state of CPT1A, particularly mediated by transmembrane domain 2 (TM2), significantly impacts the enzyme's sensitivity to malonyl-CoA inhibition. Research has shown that TM2 can form oligomers (up to hexamers) through close-packing of GXXXG(A) motifs .

Experimental evidence demonstrates that disruption of these GXXXG(A) motifs reduces the oligomeric state to trimers or lower, which correlates with increased sensitivity to malonyl-CoA inhibition. Specifically, the IC50 decreases from 30.3 ± 5.0 to 3.0 ± 0.6 μM when these motifs are disrupted .

Methodological approach: To study this phenomenon, researchers can employ mutations designed to disrupt close-packing of the GXXXG(A) motifs in TM2 peptides and analyze changes in the oligomeric state using complementary genetic assays and biophysical methods. These changes can then be correlated with functional effects on malonyl-CoA sensitivity in the full-length enzyme .

What are the methodological approaches for expressing recombinant rat CPT1A with optimal enzymatic activity?

Expressing functionally active recombinant rat CPT1A requires consideration of several technical factors:

• Expression system selection:

  • E. coli: Suitable for producing the catalytic domain (e.g., Leu572~Lys773) with N-terminal His and GST tags .

  • Yeast (Pichia pastoris): Effective for expressing full-length enzyme with post-translational modifications .

  • Mammalian cells (Expi293): Provides human-like post-translational modifications and proper folding .

• Optimization strategies:

  • Direct transfection of mammalian cells with CPT1A plasmid

  • Use of appropriate cell lysis procedures that maintain enzyme activity

  • Appropriate buffer composition: PBS (pH 7.4) containing 0.01% SKL, 5% Trehalose .

• Activity validation:

  • Spectrophotometric DTNB-based detection of free thiols

  • Testing with known inhibitors (etomoxir, perhexiline, chlorpromazine) .

For high-throughput applications, the direct cell lysis of human CPT1A-transformed Expi293 cells without the need for purification of recombinant proteins has been demonstrated to be highly effective .

How does CPT1A-mediated fatty acid oxidation contribute to cancer cell resistance to immune surveillance?

CPT1A-mediated fatty acid oxidation (FAO) has been identified as a critical mechanism for cancer cell resistance to cytolytic immune cells :

Methodological approach: To study this phenomenon, researchers can use shRNA to knock down CPT1A in human cancer cell lines and assess changes in FAO rates using FAO diffusion assays and Seahorse assays, then evaluate susceptibility to immune-mediated cytolysis in co-culture systems with various immune effector cells .

What is the role of CPT1A in cardiac stress responses and cardioprotection?

Research has demonstrated that CPT1A upregulation appears to be an adaptive rather than maladaptive response to cardiac stress :

• Clinical evidence:

  • Increased CPT1A protein levels observed in hearts of HFrEF (Heart Failure with reduced Ejection Fraction) patients

  • Similar findings in two different NICM (Non-Ischemic Cardiomyopathy) patient cohorts at two different institutions .

• Experimental findings:

  • Cardiac-specific CPT1A knockout mice (csCPT1a ko) show exacerbated response to pressure overload (TAC)

  • AAV9-mediated cardiac CPT1A overexpression (54% increase) attenuates adverse cardiac remodeling

  • CPT1A functions beyond fat metabolism to inhibit gene programs associated with cardiac remodeling, including profibrotic, hypertrophic, and cell death responses .

• Regulatory mechanisms:

  • miR370 regulates CPT1A expression in preclinical models

  • CPT1A mediates cardiac natriuretic peptide production .

Methodological approach: To investigate CPT1A's role in cardiac function, researchers can employ cardiac-specific CPT1A knockout mice (using Cre-loxP system) and AAV9-mediated gene delivery for cardiac-specific CPT1A overexpression, followed by TAC (Transverse Aortic Constriction) for pressure overload studies and assessment of cardiac remodeling parameters .

How does CPT1A influence ferroptosis resistance in cancer cells?

Recent research has uncovered CPT1A's critical role in ferroptosis resistance, particularly in cancer stem cells :

• Mechanistic insights:

  • CPT1A restrains ubiquitination and degradation of c-Myc

  • c-Myc transcriptionally activates CPT1A expression, creating a positive feedback loop

  • This loop enhances cellular antioxidant capacity by activating the NRF2/GPX4 system

  • CPT1A reduces phospholipid polyunsaturated fatty acids through ACSL4 downregulation .

• Implications for cancer therapy:

  • CPT1A inhibition enhances ferroptosis in cancer stem cells

  • Targeting CPT1A improves immune checkpoint blockade efficacy

  • L-carnitine derived from tumor-associated macrophages contributes to ferroptosis resistance .

Methodological approach: To study this phenomenon, researchers have employed approaches including metabolomics, transcriptomics, and lung epithelial-specific Cpt1a-knockout mouse models, combined with clinical analysis .

What technical considerations are important for designing high-throughput CPT1A activity assays?

When developing high-throughput assays for CPT1A activity, several technical aspects should be considered :

• Detection method selection:

  • Radioisotope-based assays using 3H-carnitine offer high sensitivity but have safety limitations and limited access to scintillation equipment

  • Spectrophotometric detection of CoA using DTNB provides a reliable alternative that is more suitable for high-throughput applications .

• Source of enzyme:

  • Direct cell lysis of human CPT1A-transformed Expi293 cells eliminates the need for purification of recombinant proteins

  • This approach provides highly reproducible and dose-responsive quantification for CPT1A activity .

• Validation approach:

  • Use of previously reported CPT1A inhibitors (etomoxir, perhexiline, and chlorpromazine) as controls

  • Establishing dose-response curves to confirm assay sensitivity and reproducibility .

• Assay optimization:

  • The spectrophotometric DTNB-based detection of free thiols provides a proxy for enzyme activity based on the direct relationship between CPT1A catalytic activity and increasing CoA concentration

  • This method has been validated to provide highly reproducible and dose-responsive quantification for CPT1A activity .

This optimized approach allows for cost-effective, safe, and scalable identification of CPT1A inhibitors, which may lead to potential treatments for type 2 diabetes and cancer .