Recombinant Horse Carnitine O-palmitoyltransferase 1, liver isoform (CPT1A)

What is CPT1A and what is its primary function in cellular metabolism?

Specifically, CPT1A facilitates the transport of activated long-chain fatty acids across the mitochondrial membrane by converting them to acylcarnitines. This process is essential for maintaining energy supply via fatty acid oxidation, particularly during fasting conditions when glucose availability is limited .

How is the structure of CPT1A organized at the molecular level?

CPT1A exists in a complex molecular arrangement in the mitochondrial outer membrane. Research using blue native electrophoresis followed by immunoblotting and mass spectrometry has revealed that CPT1A forms large molecular mass complexes with other proteins .

The protein contains conserved functional motifs and amino acid residues that are essential for its enzymatic activity. Bioinformatics analysis of CPT1A from various species has confirmed these conserved elements. For example, in the large yellow croaker (Larimichthys crocea), the complete cDNA sequence of cpt1a has an open reading frame of 2319 bp encoding a protein of 772 amino acids with conserved functional motifs .

Recent studies suggest that while earlier models proposed CPT1A exists as a trimer that under native conditions could form a dimer of trimers (hexamer) creating a channel for acylcarnitine translocation, the enzyme actually forms hetero-oligomeric complexes with metabolically relevant enzymes and channel proteins such as ACSL (long chain acyl-CoA synthetase) and VDAC (voltage-dependent anion channel) .

How do CPT1A expression patterns differ across tissues and conditions?

CPT1A shows tissue-specific expression patterns. In studies of various species including fish, the highest mRNA expression of cpt1a is observed in the liver . This aligns with its role as the liver isoform of CPT1.

Expression levels change significantly in response to physiological conditions. During fasting, cpt1a mRNA expression dramatically increases to support enhanced fatty acid oxidation for energy production, while CPT1 enzymatic activity can remain stable . This suggests complex post-transcriptional regulatory mechanisms.

In disease states such as heart failure, CPT1A protein levels are significantly altered. Research has shown increased CPT1a protein in hearts of Heart Failure with reduced Ejection Fraction (HFrEF) patients across different non-ischemic cardiomyopathy (NICM) cohorts .

How does oxidative stress affect CPT1A function and what are the mechanisms involved?

Metabolomics research has identified CPT1A as a novel target for oxidative inactivation. Studies using integrative liquid chromatography-mass spectrometry (LC-MS) and observing rapid metabolomic changes in response to hydrogen peroxide (H₂O₂)-induced oxidative stress in HeLa cells revealed characteristic metabolite profiles that uniquely indicated CPT1A inactivation .

The enzymatic activity of CPT1A significantly declines when exposed to H₂O₂ in several human cell types. This inactivation appears to be a direct effect of H₂O₂ in vitro and occurs substantially when cells are cultured with reagents that generate reactive oxygen species (ROS) .

This suggests that CPT1A inhibition might be a general phenomenon under various stress conditions associated with ROS generation, providing insight into mechanisms for oxidative dysfunction in mitochondrial metabolism. The generality of this finding suggests CPT1A could be an important target in conditions characterized by oxidative stress .

What protein complexes does CPT1A form in the mitochondrial outer membrane?

Research using blue native electrophoresis, immunoprecipitation, and immunocapture techniques has revealed that CPT1A forms heteromeric protein complexes in the mitochondrial outer membrane (MOM). These complexes contain not only CPT1A but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC) .

Experimental data from multiple techniques have confirmed strong protein-protein interactions between these three components:

These findings challenge the earlier model of CPT1A functioning solely as a homo-oligomer and suggest that CPT1A, ACSL, and VDAC form a metabolic complex that transfers activated fatty acids through the mitochondrial outer membrane .

What is the role of CPT1A in cancer progression and response to therapy?

The role of CPT1A in cancer appears to be context-dependent, with studies showing varying patterns across different cancer types. In colorectal cancer (CRC), CPT1A expression is decreased compared to paired healthy tissue, and lower expression correlates with decreased patient survival, suggesting that CPT1A may suppress tumor progression in this context .

Functional studies using CRISPR to knockout CPT1A in CRC cell lines that express CPT1A, and overexpressing CPT1A in cell lines with low expression, demonstrated that increased CPT1A expression decreased cell survival and DNA repair in response to radiation in culture. In xenograft models, CPT1A decreased tumor growth basally, and radiotherapy could further decrease tumor growth in CPT1A-expressing tumors .

Interestingly, CPT1A may also mediate response to therapy. In both breast cancer and colorectal cancer models, CPT1A expression affects sensitivity to treatments. For example, in human breast cancer MDA-MB-231 cells, IFN-γ treatment up-regulates CPT1A expression and phosphorylation of AMPKα, which is associated with elevated fatty acid oxidation activity .

How do genetic variations in CPT1A affect its function and associated clinical phenotypes?

Genetic variations in CPT1A can lead to significant changes in enzyme function and associated clinical presentations. The most well-characterized variant is the CPT1A Arctic Variant, which is particularly prevalent in circumpolar populations .

The Arctic Variant of CPT1A is inherited in an autosomal recessive manner and results in a fatty acid oxidation disorder where the body has difficulty breaking down fatty acids from both food and body fat . This variant is remarkably common in some populations:

Clinical manifestations of CPT1A deficiency typically appear between 8-18 months of age and include metabolic crisis characterized by hypoglycemia (hypoketotic), seizures due to hypoglycemia, and in rare cases, death, especially when associated with concomitant infectious disease . Initial signs of metabolic crisis include sleepiness, irritability, and poor appetite.

Management of patients with CPT1A variants focuses on avoiding fasting states and ensuring adequate glucose availability during illness .

What are the optimal methods for expressing and purifying recombinant CPT1A for enzymatic studies?

Based on successful recombinant CPT1A expression studies, the following approach has proven effective:

• Expression System Selection: E. coli or baculovirus-infected insect cells are commonly used expression systems for recombinant CPT1A. For the full-length membrane protein, insect cells often provide better folding capabilities.

• Construct Design: The complete cDNA sequence should include the full open reading frame. In one study with large yellow croaker CPT1A, this was 2319 bp encoding a protein of 772 amino acids .

• Purification Strategy: Affinity chromatography using tagged recombinant proteins has been successful. Immobilized CPT1A-specific antibodies can also be used for immunocapture approaches .

• Enzymatic Activity Verification: Recombinant CPT1A protein (rCPT1A) should be tested for catalytic activity using standardized assays. In one study, the rCPT1A showed Michaelis constant (Km) of approximately 1.38 mM and maximal reaction rates (Vmax) for carnitine of approximately 12.66 nmols/min/mg protein .

• Storage Conditions: Based on antibody storage recommendations, which can be relevant for stabilizing the protein structure, buffer conditions of PBS with 0.02% sodium azide and 50% glycerol pH 7.3 at -20°C can provide stability for extended periods .

How can CPT1A activity be accurately measured in laboratory settings?

Accurate measurement of CPT1A activity is crucial for understanding its function in normal and pathological conditions. Based on current methodologies, the following approaches are recommended:

What experimental approaches can be used to study CPT1A-protein interactions?

Several complementary techniques have been successfully employed to characterize CPT1A interactions with other proteins:

• Blue Native Electrophoresis: This technique preserves protein-protein interactions and has been used to identify large molecular mass complexes containing CPT1A, ACSL, and VDAC. Following separation, proteins can be detected by immunoblotting or identified by mass spectrometry .

• Immunoprecipitation: Using antibodies against CPT1A or its potential interacting partners, protein complexes can be isolated and analyzed. This approach has revealed strong interactions between CPT1A, ACSL, and VDAC .

• Immunocapture: Immobilized CPT1A-specific antibodies can be used to capture not only CPT1A but also its interacting partners. This approach has strengthened findings from blue native electrophoresis and immunoprecipitation studies .

• Mass Spectrometry: Following isolation of CPT1A complexes by the above methods, mass spectrometry can be used for protein identification and characterization of post-translational modifications that might regulate these interactions.

• Proximity Ligation Assays: This technique can detect protein-protein interactions in situ in fixed cells or tissues, providing spatial information about where these interactions occur within the cell.

• Co-immunoprecipitation (Co-IP): As evidenced by published applications with CPT1A antibodies, Co-IP has been successfully used to study CPT1A protein interactions .

• Crosslinking Experiments: Chemical crosslinkers can stabilize transient protein-protein interactions, facilitating their isolation and identification.

How can researchers effectively modulate CPT1A activity in cellular and animal models?

Modulating CPT1A activity is crucial for understanding its role in normal physiology and disease states. Based on current research, several approaches have proven effective:

• Genetic Approaches:

  • CRISPR/Cas9 Knockout: Complete deletion of CPT1A has been successfully used in cancer cell lines to study its role in tumor cell response to various stresses and therapies .

  • Conditional Knockout: Cardiac-specific CPT1A knockout mice (csCPT1a ko) have been generated to study the role of CPT1A in heart function under pathological stress .

  • Overexpression: AAV9-mediated cardiac-specific CPT1A overexpression has been used to investigate the protective effect of CPT1A in heart failure models .

  • RNA Interference: siRNA targeting CPT1A (SI-CPT1A) has been used to knockdown CPT1A expression and study its effects on cell metabolism and function .

• Pharmacological Approaches:

  • CPT1 Inhibitors: Compounds like etomoxir can be used to inhibit CPT1A activity.

  • Fatty Acid Oxidation Modulators: Compounds that alter substrate availability or co-factor levels can indirectly affect CPT1A function.

• Metabolic Modulation:

  • Fasting Protocols: Fasting dramatically increases cpt1a mRNA expression and can be used to study physiological regulation of CPT1A .

  • Reactive Oxygen Species (ROS) Induction: Treatment with H₂O₂ or other ROS-generating compounds can be used to study oxidative inactivation of CPT1A .

  • Cytokine Treatment: IFN-γ treatment has been shown to up-regulate CPT1A expression and phosphorylation of AMPKα in human breast cancer cells .

Example of experimental outcomes from CPT1A modulation:

How is CPT1A involved in metabolic disorders and what are the therapeutic implications?

CPT1A deficiency is a well-characterized metabolic disorder with significant clinical implications. This fatty acid oxidation disorder results in the body's inability to break down certain fats, leading to potential metabolic crises .

The most common manifestation of CPT1A deficiency is the Arctic Variant, which is particularly prevalent in circumpolar populations. In the Yu'pik population in Alaska, 50% are homozygous for this variant, and the total incidence in Alaskan newborns is 7% per year .

Clinical features of CPT1A deficiency include:

• Onset of symptoms typically between 8-18 months of age

• Initial signs of metabolic crisis: sleepiness, irritability, poor appetite

• Metabolic crisis characterized by hypoketotic hypoglycemia, seizures, and rarely death, especially during infectious illness

Management strategies focus on preventing fasting states:

• Regular feeding schedules for healthy children

• During illness, ensuring glucose-containing fluids (Pedialyte, juice, sports drinks)

• For NPO (nothing by mouth) patients, dextrose-containing IV fluids (D5-NS or D5-1/2NS)

Newborn screening programs have incorporated testing for CPT1A deficiency, allowing early intervention and management. The Alaska Newborn Screen (processed in Oregon) added CPT1A screening in fall 2003 .

Research into potential therapeutic approaches for CPT1A-related disorders continues, with a focus on:

• Nutritional management strategies

• Novel enzyme replacement approaches

• Gene therapy possibilities for severe forms of the disorder

What is the significance of CPT1A in cardiac function and heart failure?

Recent research has revealed a critical and previously unrecognized role for CPT1A in cardiac function and the response to pathological stress. Multiple independent studies have shown that CPT1A protein is increased in the hearts of patients with Heart Failure with reduced Ejection Fraction (HFrEF) .

This increase in CPT1A appears to be an adaptive rather than maladaptive response. Evidence supporting this includes:

• Increased CPT1A protein in hearts of HFrEF patients, replicated in two different non-ischemic cardiomyopathy (NICM) patient cohorts at two different institutions .

• CPT1A upregulation is adaptive in the pathologically stressed heart:

  • Cardiac-specific CPT1A knockout mice (csCPT1a ko) subjected to transverse aortic constriction (TAC, a model of pressure overload) showed worse outcomes compared to wild-type controls .

  • AAV9-mediated cardiac-specific CPT1A overexpression attenuated adverse cardiac remodeling in response to pressure overload .

• Beyond its role in fat metabolism, CPT1A functions in adult myocardium to inhibit gene programs associated with adverse cardiac remodeling, including profibrotic, hypertrophic, and cell death responses .

• There is evidence for directional regulation of CPT1A expression by miR370 in preclinical animal models, which correlates to changes in CPT1A and miR370 in human heart failure .

These findings suggest that therapeutic approaches aimed at increasing or preserving CPT1A function in the heart could potentially benefit patients with heart failure or at risk for adverse cardiac remodeling following pathological stress.

How can recombinant CPT1A be utilized in drug discovery and development?

Recombinant CPT1A can serve as a valuable tool in drug discovery and development pipelines, particularly for conditions where modulating fatty acid oxidation might have therapeutic benefits. Based on current research, several applications are possible:

• High-throughput Screening Platforms:

  • Purified recombinant CPT1A can be used in enzymatic assays to screen compound libraries for novel inhibitors or activators.

  • Such screens can identify compounds with greater specificity and fewer off-target effects than existing CPT1A modulators.

• Structure-based Drug Design:

  • With recombinant protein, structural studies (X-ray crystallography, cryo-EM) can reveal the molecular architecture of CPT1A.

  • This structural information can guide rational design of drugs targeting specific functional domains of the enzyme.

• Target Validation:

  • In cancer research, where CPT1A has shown context-dependent roles, recombinant CPT1A can help validate it as a therapeutic target.

  • For example, in colorectal cancer, where CPT1A expression correlates with better patient survival and increased radiation sensitivity , recombinant CPT1A could help identify compounds that increase CPT1A activity specifically in tumor cells.

• Biomarker Development:

  • Recombinant CPT1A can be used to develop and standardize assays measuring CPT1A activity in patient samples.

  • Such assays could identify patients most likely to benefit from therapies targeting fatty acid metabolism.

• Therapeutic Protein Development:

  • For conditions with CPT1A deficiency, optimized recombinant CPT1A could potentially be developed as an enzyme replacement therapy.

  • While challenging due to the membrane-bound nature of CPT1A, innovative delivery systems might overcome these limitations.

• Counter-screening:

  • For drugs under development for other targets, testing against recombinant CPT1A can help identify potential metabolic side effects related to fatty acid oxidation.