Recombinant Mouse Trans-2,3-enoyl-CoA reductase-like (Tecrl)

What is the molecular structure of mouse Tecrl protein and how does it differ from Tecr?

Trans-2,3-enoyl-CoA reductase-like (Tecrl) is a protein related to but distinct from Trans-2,3-enoyl-CoA reductase (Tecr). While Tecr is well-characterized as an enzyme involved in fatty acid elongation with 308 amino acids in mouse models, Tecrl has a similar structure but different functional properties. Tecrl has gained research attention due to its associations with cardiac arrhythmias rather than primarily metabolic functions. The structural differences between these proteins contribute to their distinct physiological roles, with Tecrl being particularly important in cardiac tissue function .

What expression systems are optimal for producing recombinant mouse Tecrl protein?

For recombinant production of mouse Tecr protein, E. coli expression systems have proven effective, as demonstrated in commercial production protocols. The full-length mouse Trans-2,3-enoyl-CoA reductase (1-308 amino acids) can be expressed with an N-terminal His tag for purification purposes. This approach allows for high yield and purity (>90% as determined by SDS-PAGE) . For Tecrl specifically, similar prokaryotic expression systems may be employed, though mammalian expression systems might better preserve post-translational modifications that could be critical for functional studies, particularly when investigating cardiac-related phenotypes .

What are the recommended storage and handling protocols for recombinant Tecrl protein?

Recombinant Tecrl protein requires careful handling to maintain structural integrity and enzymatic activity. Based on established protocols for similar proteins like Tecr, the following storage conditions are recommended:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• For short-term storage (up to one week), maintain working aliquots at 4°C

• For reconstitution, use deionized sterile water to achieve 0.1-1.0 mg/mL concentration

• Add glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage

• Store in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

These conditions help preserve protein stability and activity for experimental applications.

What are the known mutations in Tecrl and their association with cardiac arrhythmias?

Tecrl mutations have been identified as causative factors in life-threatening inherited arrhythmias with overlapping features of Long QT Syndrome (LQTS) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Significant mutations include:

• p.Arg196Gln mutation: Identified in unrelated French Canadian patients with similar cardiac electrical phenotypes characterized by normal QT interval at baseline but adrenergic-induced QT prolongation and ventricular arrhythmias .

• c.331+1G>A splice site mutation: Found in a consanguineous Sudanese family with CPVT. This mutation affects the splice donor site of intron 3, resulting in complete exon 3 skipping (45bp deletion) as confirmed by PCR analysis .

These mutations represent critical research targets for understanding arrhythmogenic mechanisms in otherwise unexplained cardiac events .

How do Tecrl mutations affect cardiac electrophysiology at the cellular level?

Functional studies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with Tecrl mutations have revealed significant electrophysiological abnormalities:

• Action potential (AP) prolongation: Cardiomyocytes derived from patients homozygous for Tecrl mutations (TECRL-Hom-hiPSC-CMs) displayed marked prolongation of action potential duration at 20% repolarization (APD20) compared to control cells .

• Repolarization abnormalities: There was a clear trend for increased action potential duration at 50% (APD50) and 90% (APD90) repolarization in mutant cells, suggesting fundamental alterations in ionic currents responsible for repolarization .

• Calcium handling: Analysis of intracellular calcium dynamics in hiPSC-CMs from affected individuals showed abnormalities consistent with arrhythmogenic mechanisms, providing a potential link between the genetic defect and the clinical phenotype .

These cellular findings provide a mechanistic explanation for the mixed LQTS/CPVT phenotype observed clinically in patients with Tecrl mutations .

What experimental models are most suitable for investigating Tecrl function?

Several experimental models have proven valuable for investigating Tecrl function and pathology:

• Human iPSC-derived cardiomyocytes (hiPSC-CMs): Cardiomyocytes derived from patients with homozygous and heterozygous Tecrl mutations provide a physiologically relevant model for studying the functional consequences of these mutations. This model allows for detailed electrophysiological characterization and calcium imaging studies .

• Gene knockdown models: Lentiviral vectors encoding Tecrl-specific shRNAs have been used to create knockdown models in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study action potential properties in the absence of Tecrl function .

• Heterologous expression systems: For basic functional studies of wild-type and mutant Tecrl, expression in standardized cell lines allows for controlled biochemical and functional analysis.

These complementary approaches enable researchers to investigate different aspects of Tecrl biology from molecular interactions to cellular phenotypes .

What genetic analysis techniques are most effective for identifying novel Tecrl mutations?

Whole-exome sequencing (WES) has proven particularly effective for identifying Tecrl mutations in patients with unexplained arrhythmias. The workflow for genetic analysis typically includes:

• Patient selection: Identify individuals with clinical features of both LQTS and CPVT who test negative for mutations in common disease-associated genes.

• WES: Perform whole-exome sequencing with appropriate coverage (typically >100x) to identify rare variants.

• Filtering strategies: Apply bioinformatic filtering to prioritize variants based on:

  • Allele frequency (<0.01% in population databases such as gnomAD, ExAC)

  • Conservation scores (phastCons, GERP++)

  • Predicted functional impact (SIFT, PolyPhen2)

  • Segregation with disease in families

• Validation: Confirm candidate variants using Sanger sequencing and assess segregation in available family members .

This approach has successfully identified causative Tecrl mutations in cases where conventional genetic testing for arrhythmia genes was negative .

How can researchers assess the functional impact of Tecrl mutations?

To evaluate the functional consequences of Tecrl mutations, researchers employ multiple complementary approaches:

• mRNA splicing analysis: For mutations affecting splice sites (e.g., c.331+1G>A), RT-PCR using primers spanning the affected region can determine alterations in splicing. For example, the c.331+1G>A mutation results in a 45bp deletion corresponding to exon 3 skipping, which can be visualized as a shorter PCR product (126bp vs. 171bp for wild-type) .

• Protein expression and localization: Immunofluorescence staining and Western blot analysis can determine if mutations affect protein stability, expression levels, or subcellular localization.

• Electrophysiological characterization: Patch-clamp recordings of action potentials in hiPSC-CMs from patients or cells with engineered Tecrl mutations provide direct evidence of functional alterations. Key parameters to assess include action potential duration at 20%, 50%, and 90% repolarization .

• Calcium imaging: Analysis of intracellular calcium dynamics using fluorescent indicators can reveal abnormalities in calcium handling that may contribute to arrhythmogenesis .

These methods collectively provide a comprehensive assessment of how Tecrl mutations impact cellular physiology and potentially lead to arrhythmias .

What are the optimal techniques for purifying recombinant Tecrl protein for in vitro studies?

For researchers requiring purified Tecrl protein for biochemical and structural studies, the following purification protocol is recommended:

• Expression system: Express full-length mouse Tecrl protein with an N-terminal His tag in E. coli.

• Purification method:

  • Use affinity chromatography with Ni-NTA resin for initial capture

  • Apply gradient elution with increasing imidazole concentration

  • Further purify using size exclusion chromatography if higher purity is required

• Quality control:

  • Verify purity by SDS-PAGE (aim for >90% purity)

  • Confirm identity by Western blot and/or mass spectrometry

  • Assess activity using appropriate enzymatic assays

• Storage:

  • Lyophilize purified protein for long-term stability

  • Store at -20°C/-80°C

  • Reconstitute in Tris/PBS-based buffer with 6% Trehalose (pH 8.0)

  • Add glycerol (5-50%) for cryoprotection

This approach yields high-quality protein suitable for structural studies, antibody production, and functional assays .

How do Tecrl mutations manifest clinically and what is the significance for patient management?

Tecrl mutations manifest with a unique clinical phenotype that combines features of both Long QT Syndrome (LQTS) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), creating diagnostic and management challenges:

• Clinical presentation:

  • Normal QT interval at baseline but adrenergic-induced QT prolongation

  • Stress-induced atrial and ventricular tachycardia

  • High risk of sudden cardiac death

  • Cardiac arrest as a presenting symptom in many cases

• Diagnostic considerations:

  • Consider Tecrl testing in patients with features of both LQTS and CPVT

  • Particularly when conventional genetic testing is negative

  • Family history may be significant in consanguineous families

• Management implications:

  • Patients require careful monitoring and aggressive anti-arrhythmic therapy

  • Beta-blockers and ICD placement may be warranted

  • Exercise restriction and avoidance of adrenergic triggers

  • Family screening for asymptomatic carriers

The recognition of Tecrl as a causative gene has important implications for genetic testing panels, which should now include this gene to improve diagnostic yield in inherited arrhythmia cases .

What are the emerging therapeutic approaches targeting Tecrl-associated arrhythmias?

Research into Tecrl-associated arrhythmias has identified several potential therapeutic approaches:

• Conventional therapies:

  • Beta-blockers remain first-line therapy due to the adrenergic-sensitive nature of arrhythmias

  • Implantable cardioverter-defibrillators (ICDs) for high-risk patients

  • Sodium channel blockers may have a role based on electrophysiological characteristics

• Targeted approaches under investigation:

  • Calcium channel modulators targeting the calcium handling abnormalities

  • Gene therapy approaches to restore normal Tecrl function

  • Pharmacological chaperones to rescue misfolded Tecrl protein

• Precision medicine strategies:

  • hiPSC-CM models from patients can be used for personalized drug screening

  • Patient-specific therapy based on cellular phenotype and response to various compounds

The overlap between LQTS and CPVT phenotypes suggests that combination therapy addressing both repolarization abnormalities and calcium handling may be necessary for optimal management .

What are the critical knowledge gaps in Tecrl research that require further investigation?

Despite significant advances, several critical knowledge gaps remain in Tecrl research:

• Structure-function relationship:

  • Detailed structural characterization of wild-type and mutant Tecrl protein

  • Molecular mechanisms by which Tecrl mutations affect protein function

  • Protein-protein interactions critical for normal Tecrl function

• Tissue-specific effects:

  • Why cardiac tissue is particularly affected despite broader expression

  • Whether Tecrl has roles in other tissues that are currently unrecognized

  • Cell-type specific functions within the heart (atrial vs. ventricular)

• Genotype-phenotype correlations:

  • Why some mutations cause predominantly LQTS-like features while others present more like CPVT

  • Modifying factors (genetic or environmental) that influence phenotypic expression

  • Long-term natural history of Tecrl-associated disease

Addressing these knowledge gaps will require multidisciplinary approaches combining structural biology, genetics, electrophysiology, and clinical research.

How can high-throughput screening approaches facilitate Tecrl research?

High-throughput screening approaches offer significant potential for advancing Tecrl research:

• Compound screening:

  • hiPSC-CM models can be used to screen drug libraries for compounds that normalize the electrophysiological phenotype

  • Calcium imaging-based screening to identify modulators of calcium handling

  • Target-based screening to identify direct Tecrl modulators

• Genetic modifier screening:

  • CRISPR-based screens to identify genes that modify Tecrl-associated phenotypes

  • RNA interference screens to identify pathway components

  • Overexpression libraries to identify potential compensatory mechanisms

• Patient-derived cell repositories:

  • Development of biobanks with cells from multiple patients with different Tecrl mutations

  • Creation of isogenic cell lines with engineered mutations for controlled studies

  • Differentiation protocols optimized for Tecrl functional studies

These approaches could accelerate therapeutic discovery while also providing mechanistic insights into Tecrl biology.