Recombinant Mouse Carnitine O-palmitoyltransferase 1, brain isoform (Cpt1c)

What is the tissue distribution of CPT1C expression?

CPT1C is predominantly expressed in the brain and testis, distinguishing it from other CPT1 family members . Within the brain, CPT1C protein has been detected through coimmunoprecipitation assays in the cortex, cerebellum, and hippocampus . More recent research has revealed that CPT1C is also expressed in human mesenchymal stem cells (hMSCs) and certain cancer cells . This tissue-specific expression pattern suggests specialized functions in these cell types that differ from the conventional role of CPT1 family members in fatty acid metabolism.

What cellular compartments contain CPT1C protein?

CPT1C is primarily localized to the endoplasmic reticulum (ER) in neurons, where it interacts with AMPA receptors to regulate their trafficking . CPT1C also plays a role in regulating late endosome/lysosome (LE/Lys) anterograde transport in neurons through its interaction with protrudin . In human mesenchymal stem cells, CPT1C has been shown to enhance autophagy and promote lipid droplet synthesis, suggesting localization to autophagosomes and regions involved in lipid metabolism .

What are recommended methods for detecting CPT1C expression in tissue samples?

Based on the research data, several methods have proven effective for detecting CPT1C:

• Immunoblotting: Using antibodies produced against specific peptide sequences (e.g., CKTVDPNTPTSSTNL) with affinity purification from rabbit serum . The specificity should be verified using epitope-tagged constructs.

• Coimmunoprecipitation assays: These have been successfully used to detect CPT1C interactions with GluA1 in brain tissues (cortex, cerebellum, and hippocampus) . This approach requires:

  • Brain tissue homogenization

  • Immunoprecipitation with anti-GluA1 antibodies

  • Western blotting with anti-CPT1C antibodies

  • Appropriate negative controls (e.g., neutral IgG immunoprecipitation)

• RT-PCR and qPCR: For mRNA detection in tissues where protein levels might be low.

For experimental validation, it's essential to include CPT1C knockout tissues as negative controls to confirm antibody specificity .

How can researchers effectively express and purify recombinant CPT1C protein?

Based on protocols for related proteins like CPT1A, the following approach is recommended:

• Expression system: Use bacterial expression systems (E. coli) with N-terminal His-tags for initial purification studies . For more complex studies requiring post-translational modifications, consider mammalian expression systems.

• Purification protocol:

  • Express full-length mature protein (similar to positions 2-773 in CPT1A)

  • Purify using nickel affinity chromatography for His-tagged proteins

  • Consider buffer composition containing Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • Lyophilization can be used for long-term storage

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol for long-term storage at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

• Quality control: Verify purity (>90%) using SDS-PAGE and confirm identity by mass spectrometry or western blotting.

What experimental approaches are optimal for studying CPT1C's malonyl-CoA binding properties?

To study the malonyl-CoA binding properties of CPT1C, researchers should consider:

• Radioligand binding assays: Using radiolabeled [14C]malonyl-CoA to determine binding affinity (Kd).

• Surface plasmon resonance (SPR): For real-time binding kinetics analysis.

• Isothermal titration calorimetry (ITC): To measure the thermodynamic parameters of binding.

• Mutagenesis studies: Target residues known to be important for malonyl-CoA binding in other CPT1 isoforms to confirm their importance in CPT1C.

When designing these experiments, consider that CPT1C has high-affinity malonyl-CoA binding but lacks detectable catalytic activity with conventional acyl-CoA substrates . This suggests the need to explore binding with unconventional lipid substrates that might be specific to brain tissue.

How does CPT1C regulate AMPAR trafficking in neurons?

CPT1C enhances AMPAR trafficking through the following mechanism:

• CPT1C interacts directly with GluA1-containing AMPARs, including GluA1/GluA2 heteromers, which are dominant in hippocampal pyramidal neurons .

• This interaction affects the palmitoylation state of GluA1, as indicated by research showing "CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1" .

• The palmitoylation modification influences AMPAR surface expression and trafficking.

• In CPT1C knockout animals, the number of AMPARs at hippocampal neuron synapses is reduced, and these animals show reduced AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs) .

• CPT1C-mediated regulation of AMPARs appears to be a common event throughout the CNS, as CPT1C interaction with GluA1 has been demonstrated in cortex, cerebellum, and hippocampus .

The methodological approach to study this involves coimmunoprecipitation assays from different brain regions, electrophysiological recordings, and palmitoylation assays.

What is the role of CPT1C in cellular response to glucose deprivation?

CPT1C plays a crucial protective role during glucose deprivation through several mechanisms:

• Enhancement of autophagy: CPT1C overexpression in human mesenchymal stem cells (hMSCs) increases autophagic flux during glucose deficiency, which is essential for cell survival under stress conditions .

• Promotion of lipid droplet synthesis: CPT1C increases the number of lipid droplets, which serve as energy reservoirs .

• ATP maintenance: The enhanced autophagy and increased lipid droplets lead to higher intracellular ATP levels during glucose deprivation .

• Nutrient sensing mechanism: CPT1C senses intracellular malonyl-CoA levels, which decrease during glucose deprivation due to reduced fatty acid synthesis .

• Regulation of endosome/lysosome transport: Under energy stress (glucose depletion or AMPK activation), malonyl-CoA synthesis is inhibited, causing unbound CPT1C to prevent protrudin-mediated transfer of kinesin-1 to late endosomes/lysosomes, temporarily arresting axon growth to conserve energy .

The protective effect of CPT1C during glucose deprivation is abolished when either autophagy or lipolysis is inhibited, confirming that both processes are essential for CPT1C-mediated cell survival .

How does CPT1C coordinate nutrient sensing with cellular transport mechanisms?

CPT1C functions as a sophisticated nutrient sensor that coordinates cellular transport with metabolic status:

• Malonyl-CoA sensing: CPT1C binds malonyl-CoA, which serves as an indicator of nutrient sufficiency as its levels reflect the metabolic state of fatty acids and glucose .

• Regulation of LE/Lys transport:

  • Under sufficient nutrient supply: High malonyl-CoA levels cause CPT1C to promote plus-end transport of late endosomes/lysosomes through enhancement of protrudin function .

  • Under energy stress: Low malonyl-CoA levels result in unbound CPT1C preventing protrudin-mediated transfer of kinesin-1 to LE/Lys, reducing anterograde transport .

• Impact on neuronal development: This mechanism affects axon growth, which is promoted under nutrient sufficiency and temporarily arrested during energy stress .

• Coordination with amino acid sensing: While malonyl-CoA levels depend on fatty acid and glucose metabolism (but not amino acids), the positioning of LE/Lys is also regulated independently by amino acid levels through PI3P production .

What phenotypes are observed in CPT1C knockout mouse models?

CPT1C knockout (KO) mice exhibit several notable phenotypes:

• Metabolic abnormalities:

  • Increased susceptibility to obesity when fed a high-fat diet, suggesting CPT1C is protective against the effects of excessive fat consumption .

  • Altered glucose homeostasis and potential changes in insulin sensitivity, though specific details would require further investigation of the glucose tolerance tests mentioned in the research .

• Neuronal defects:

  • Reduced number of AMPARs at hippocampal neuron synapses .

  • Decreased AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs) in hippocampal pyramidal cells .

  • Poor dendrite arborization in neurons, through a mechanism independent of protrudin .

• Cellular abnormalities:

  • Altered late endosome/lysosome positioning and transport .

  • Potentially compromised response to nutrient stress, particularly glucose deprivation.

These phenotypes highlight CPT1C's important role in both metabolic regulation and neuronal development, suggesting potential therapeutic implications for conditions involving neuronal dysfunction and metabolic disorders.

How might CPT1C function in cancer cells compared to neurons?

The expression of CPT1C in both neurons and cancer cells suggests potentially different functional roles:

• In neurons:

  • Functions primarily as a nutrient sensor through malonyl-CoA binding .

  • Regulates AMPAR trafficking through palmitoylation mechanisms .

  • Controls endosome/lysosome transport in response to energy availability .

  • Promotes proper dendrite and axon development .

• In cancer cells:

  • May provide metabolic adaptability during nutrient stress, similar to its role in protecting hMSCs during glucose deprivation .

  • Could potentially enhance tumor cell survival under hypoxic or nutrient-poor conditions.

  • Might contribute to altered lipid metabolism, which is a hallmark of many cancer types.

  • The specific role of CPT1C in tumor cells was noted as "different from that described in tumor cells" , suggesting unique adaptations of its function in the cancer context.

The methodological approach to investigate these differences would involve comparative studies using neuron and cancer cell models, focusing on metabolic adaptations, response to nutrient stress, and potential moonlighting functions of CPT1C in different cellular contexts.

What potential therapeutic applications might target CPT1C function?

Based on the research findings, potential therapeutic applications targeting CPT1C might include:

• Neuroprotective strategies:

  • Enhancing CPT1C function could potentially protect neurons during metabolic stress or nutrient deprivation, relevant for stroke or neurodegenerative conditions.

  • Targeting the CPT1C-AMPAR interaction could modulate synaptic plasticity in conditions with aberrant glutamatergic signaling.

• Stem cell-based therapies:

  • Manipulating CPT1C expression in mesenchymal stem cells could enhance their survival in avascular or nutrient-poor environments during regenerative medicine applications .

  • This approach might improve hMSC efficacy in tissue engineering and cell-based therapies.

• Metabolic disorder treatments:

  • Since CPT1C KO mice are more susceptible to diet-induced obesity , CPT1C activators might have potential as anti-obesity therapeutics.

  • Targeting brain-specific metabolic regulation through CPT1C might offer fewer peripheral side effects compared to systemic metabolic interventions.

• Cancer therapy:

  • Inhibiting CPT1C function could potentially reduce cancer cell survival in nutrient-restricted tumor microenvironments.

  • Combinatorial approaches targeting CPT1C alongside conventional therapies might prevent adaptive resistance mechanisms in tumors.

Methodological considerations for therapeutic development would include high-throughput screening for modulators of CPT1C-malonyl-CoA binding, structure-based drug design targeting its unique properties, and cell-type specific delivery strategies.

How might post-translational modifications regulate CPT1C function?

This advanced research question addresses the potential regulatory mechanisms affecting CPT1C:

• Potential phosphorylation regulation:

  • AMPK activation influences CPT1C function during energy stress , suggesting possible direct phosphorylation.

  • Kinases involved in metabolic regulation (PKA, AKT, mTOR) might phosphorylate CPT1C to coordinate its function with cellular signaling pathways.

• Palmitoylation dynamics:

  • CPT1C influences GluA1 palmitoylation , but CPT1C itself might also undergo palmitoylation as a regulatory mechanism.

  • Reciprocal regulation between CPT1C and palmitoylation machinery warrants investigation.

• Ubiquitination and protein stability:

  • Turnover rates and degradation pathways for CPT1C remain poorly characterized.

  • Ubiquitination might regulate CPT1C abundance under different metabolic conditions.

• Conformational changes:

  • Malonyl-CoA binding likely induces conformational changes in CPT1C that alter its interactions with partners like protrudin .

  • Structural studies would help elucidate these mechanisms.

Methodological approaches should include phosphoproteomic analysis, site-directed mutagenesis of potential modification sites, and structural studies comparing different binding states.

What is the evolutionary significance of CPT1C functioning as a pseudoenzyme?

This question explores the evolutionary adaptation of CPT1C:

• Functional repurposing:

  • CPT1C contains conserved residues for carnitine acyltransferase activity and malonyl-CoA binding but lacks catalytic activity with conventional substrates .

  • This suggests evolutionary repurposing from metabolic enzyme to specialized sensor/regulator.

• Brain-specific metabolic regulation:

  • The brain has unique metabolic requirements and regulations compared to other tissues.

  • CPT1C may have evolved to provide brain-specific regulation of metabolism and cellular processes without disrupting systemic fatty acid oxidation.

• Neuronal specialization:

  • CPT1C's role in regulating endosome/lysosome transport and AMPAR trafficking suggests adaptation to neuron-specific functions.

  • The pseudoenzyme may serve as a metabolic checkpoint for energy-intensive processes like axon growth.

• Substrate specificity evolution:

  • CPT1C might be specialized for "the metabolism of a distinct class of fatty acids involved in brain function" , suggesting potential catalytic activity with yet-unidentified neuronal lipids.

Research approaches should include comparative genomics across species, reconstruction of ancestral CPT1 sequences, and screening for noncanonical substrates in brain tissue.

How does CPT1C integrate with other cellular nutrient sensing pathways?

This complex question examines CPT1C's position in the broader cellular metabolic network:

• Coordination with mTOR signaling:

  • Late endosome/lysosome positioning regulated by CPT1C affects mTORC1 activation .

  • This suggests CPT1C functions upstream of mTOR in nutrient sensing.

• Interaction with AMPK pathway:

  • AMPK activation, which occurs during energy stress, influences CPT1C-mediated regulation .

  • The reciprocal relationship between AMPK and CPT1C requires further elucidation.

• Connection to autophagy regulation:

  • CPT1C enhances autophagic flux during glucose deprivation .

  • How this connects with canonical autophagy regulators (ULK1, Beclin-1) remains to be fully characterized.

• Integration with lipid metabolism sensors:

  • As a malonyl-CoA sensor, CPT1C likely coordinates with other lipid metabolism regulators (SREBP, PPARs).

  • The specific signaling cascades connecting these pathways are not fully mapped.

Methodological approaches should include interaction proteomics, pathway analysis in CPT1C KO models under various nutrient conditions, and computational modeling of integrated nutrient sensing networks.