ECHS1 Human, Active

What is ECHS1 and what are its primary functions in human metabolism?

ECHS1 (enoyl-CoA hydratase, short chain 1) is a multifunctional mitochondrial matrix enzyme that catalyzes the second step of the β-oxidation spiral of fatty acids, specifically the hydration of chain-shortened α,β-unsaturated enoyl-CoA thioesters to produce β-hydroxyacyl-CoA . Beyond fatty acid metabolism, ECHS1 plays a crucial role in amino acid catabolism, particularly in valine metabolism, where it converts methacrylyl-CoA to (S)-3-hydroxyisobutyryl-CoA and acryloyl-CoA to 3-hydroxypropionyl-CoA . This dual functionality makes ECHS1 essential for both energy production and amino acid processing.

How is ECHS1 activity measured in experimental settings?

ECHS1 enzyme activity can be determined through spectrophotometry by measuring the absorbance of unsaturated substrate (crotonyl-CoA) over time. The specific activity is defined as the amount of enzyme that hydrolyzes 1.0 μmole of crotonoyl-CoA to hydroxybutyryl-CoA per minute at pH 7.5 at 25°C . In patient-derived cells, ECHS1 enzyme activity in mitochondrial fractions can be measured and normalized to control activity to evaluate the functional impact of mutations . For research requiring recombinant ECHS1, commercially available preparations typically have specific activity >150 units/mg .

What are the most common ECHS1 mutations identified in patients?

Multiple mutations have been identified in ECHS1, with most being missense mutations. Among the reported variants, c.476A>G (p.Gln159Arg) has been found in multiple unrelated ECHS1-deficient patients from diverse ethnic backgrounds, suggesting it may be a potential hotspot . Recent studies have identified novel mutations, including c.463G>A (p.Gly155Ser), which was detected in three patients from unrelated families, potentially indicating a founder effect . Compound heterozygosity is common in ECHS1 deficiency, with patients typically carrying two different mutations . The mutation spectrum continues to expand, with at least 34 different mutations identified across 42 reported patients .

What is the clinical and biochemical spectrum of ECHS1 deficiency?

ECHS1 deficiency presents with a heterogeneous phenotype, with disease onset typically in the first year of life and clinical course ranging from neonatal death to survival into adulthood . The most prominent clinical features include:

Biochemically, patients typically show elevated serum lactate and brain MRI reveals white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism . Urinary excretion of 2-methyl-2,3-dihydroxybutyrate is significantly increased, indicating impaired valine oxidation . Unlike HIBCH deficiency (a related disorder), ECHS1 deficiency does not typically show elevated 3-hydroxyisobutyryl-carnitine levels, providing a potential biomarker distinction between these conditions .

How does ECHS1 deficiency differ from other mitochondrial disorders?

Unlike typical fatty acid oxidation disorders that present with liver failure, hypoketotic glycaemia, and rhabdomyolysis, ECHS1 deficiency manifests primarily as Leigh syndrome or Leigh-like encephalopathy, which is more commonly associated with oxidative phosphorylation (OXPHOS) defects . This unique presentation reflects the dual role of ECHS1 in both fatty acid and amino acid metabolism.

The specific biochemical markers that distinguish ECHS1 deficiency include:

• Normal acylcarnitine profiles in serum (unlike many fatty acid oxidation disorders)

• Increased butyrylcarnitine revealed by in vitro palmitate loading of patient fibroblasts

• Elevated urinary 2-methyl-2,3-dihydroxybutyrate

• Normal 3-hydroxyisobutyryl-carnitine (distinguishing it from HIBCH deficiency)

What functional assays can be used to validate novel ECHS1 variants?

To validate novel ECHS1 variants, researchers can employ multiple complementary approaches:

• Protein expression analysis: Immunoblotting to detect ECHS1 protein levels in patient-derived fibroblasts or other relevant cells .

• Enzyme activity measurement: Spectrophotometric assay measuring the hydration of crotonyl-CoA in patient samples compared to controls .

• Palmitate loading assay: Exposing fibroblasts to palmitate and measuring acylcarnitine profiles, specifically looking for increased butyrylcarnitine, which unmasks the functional defect in mitochondrial β-oxidation of short-chain fatty acids .

• Rescue experiments: Transfection of patient cells with wild-type ECHS1 to demonstrate restoration of enzyme activity, confirming the causative nature of identified variants .

• 3D protein modeling: Constructing models of human ECHS1 to predict the impact of specific variants on protein structure and function .

What cell and animal models are available for studying ECHS1 function and deficiency?

Several experimental models can be employed for ECHS1 research:

• Patient-derived fibroblasts: Primary cell lines from patients provide a valuable resource for studying the functional consequences of ECHS1 mutations .

• Myoblast cultures: Patient-derived myoblasts can be used to study ECHS1 function in muscle tissue, which is often affected in patients .

• Cancer cell lines: Various cancer cell lines with manipulated ECHS1 expression (knockdown/overexpression) have been used to study its role in cancer metabolism, particularly in colorectal cancer (HCT116, SW480), hepatocellular carcinoma, and other cancer types .

• Genetic models: While not explicitly mentioned in the provided materials, CRISPR-Cas9 technology could be employed to generate cellular models with specific ECHS1 mutations.

How do toxic metabolites accumulate in ECHS1 deficiency and what mechanisms explain their neurotoxicity?

ECHS1 deficiency leads to accumulation of methacrylyl-CoA and acryloyl-CoA, two highly reactive intermediates in the valine catabolic pathway . These toxic metabolites:

• Spontaneously react with sulfhydryl groups of cysteine and cysteamine, depleting critical cellular antioxidants .

• Disrupt the function of pyruvate dehydrogenase complexes and electron transport chains, contributing to energy metabolism defects .

• May induce secondary deficiencies in multiple mitochondrial enzymes, similar to what is observed in β-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency .

• Likely contribute to the brain pathology observed in both ECHS1 and HIBCH deficiencies, reflecting the particular vulnerability of neural tissue to disruptions in energy metabolism .

The neurotoxicity appears to be exacerbated by the brain's high energy demands and the tendency of these reactive species to target mitochondrial proteins essential for neuronal function.

What is the relationship between ECHS1 deficiency and secondary OXPHOS dysfunction?

One of the most intriguing aspects of ECHS1 deficiency is its association with secondary oxidative phosphorylation (OXPHOS) defects, despite ECHS1 not being directly involved in the respiratory chain . Several mechanisms may explain this relationship:

• Direct inhibition: Accumulating toxic metabolites (methacrylyl-CoA and acryloyl-CoA) may directly inhibit OXPHOS complexes .

• Disruption of complex biogenesis: Recent evidence suggests ECHS1 deficiency may interfere with the biogenesis and/or stability of OXPHOS protein complexes, rather than simply inhibiting their function .

• Altered mitochondrial membrane potential: Research in cancer cells has shown that ECHS1 knockdown can affect mitochondrial membrane potential, which is crucial for OXPHOS function .

• Redox imbalance: ECHS1 deficiency may promote reactive oxygen species (ROS) production, which can damage OXPHOS components .

• Acetylation regulation: Decreased formation of acetyl-CoA due to impaired β-oxidation may hamper posttranslational acetylation of mitochondrial proteins, a mechanism emerging as a critical regulator of mitochondrial function .

What is the role of ECHS1 in cancer metabolism and drug resistance?

Recent research has revealed surprising roles for ECHS1 in cancer biology:

• Altered expression in cancer: ECHS1 shows aberrant expression in various cancers, with both up- and down-regulation reported depending on cancer type . It was first discovered to be down-regulated in hepatocellular carcinoma but has since been shown to have varying expression patterns across different cancers .

• Regulation of cancer cell proliferation and metastasis: ECHS1 has been shown to regulate cancer cell proliferation and metastatic potential through multiple mechanisms:

  • Remodeling fatty acid metabolism

  • Regulating the PI3K/Akt/mTOR signaling pathway

  • Affecting ceramide glycosylation and sphingolipid metabolism

• Drug resistance mechanisms: ECHS1 has been implicated in resistance to:

  • Doxorubicin in hepatocellular carcinoma

  • Oxaliplatin/5-fluorouracil in colorectal cancer

  • The mechanistic link appears to involve sphingolipid metabolic dysregulation, particularly alterations in ceramide metabolism that increase glycosphingolipid synthesis through promotion of UDP-glucose ceramide glycosyltransferase (UGCG)

• Therapeutic targeting: Small molecule inhibitors targeting ECHS1 have shown promising anti-cancer effects in preclinical studies:

  • 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) enhances pro-apoptotic effects in breast cancer by suppressing ECHS1 expression

  • Remoldin reverses fatty acid metabolism reprogramming in colorectal and breast cancer by downregulating ECHS1

  • Eliglustat, a specific inhibitor of UGCG, can reverse the drug resistance phenotype caused by ECHS1 overexpression

What therapeutic strategies are being explored for ECHS1 deficiency?

Current therapeutic approaches for ECHS1 deficiency are primarily focused on gene therapy:

• AAV9 Gene Replacement Therapy: The Cure Mito Foundation is working on an ECHS1 AAV9 gene replacement therapy that aims to deliver a functional ECHS1 gene using AAV9 to restore mitochondrial function . This approach involves:

  • Loading a healthy copy of the patient's defective gene into a virus (AAV9) that has had its own DNA removed

  • Injecting trillions of these viruses into the patient's spinal fluid

  • The viruses then binding to cells in the patient's spinal cord and brain, delivering the healthy genes to the cell's nucleus

• Metabolic treatments: Although not explicitly detailed in the search results, metabolic interventions that bypass or reduce the accumulation of toxic metabolites may be potential therapeutic avenues, similar to approaches used for other metabolic disorders.

• UGCG inhibition: In cancer research, targeting UGCG with inhibitors like Eliglustat has shown promise in reversing phenotypes associated with ECHS1 dysregulation . While primarily studied in cancer contexts, this approach might offer insights for treating certain aspects of ECHS1 deficiency.

What are the key methodological considerations for designing experiments to study ECHS1-related mechanisms?

When designing experiments to study ECHS1-related mechanisms, researchers should consider:

• Cell type selection: Different tissues show varying sensitivity to ECHS1 deficiency, with neural and cardiac tissues being particularly vulnerable. Choosing relevant cell types (neurons, cardiomyocytes, or patient-derived fibroblasts/myoblasts) is crucial .

• Metabolic state considerations: ECHS1 functions in both fed and fasting states, so experimental conditions should account for metabolic variations:

  • Consider nutrient availability in culture media

  • Evaluate effects under different substrate conditions (glucose vs. fatty acids)

  • Account for potential compensation by other metabolic pathways

• Functional readouts: Beyond measuring ECHS1 enzyme activity, consider broader functional consequences:

  • Mitochondrial membrane potential

  • Reactive oxygen species production

  • ATP generation

  • Cell viability and apoptosis markers

• Pathway interactions: Design experiments that capture the interplay between:

  • Fatty acid oxidation

  • Valine metabolism

  • OXPHOS function

  • Sphingolipid metabolism (particularly in cancer studies)

• Validation approaches: Use multiple complementary methods to confirm findings:

  • Genetic approaches (knockdown/overexpression/rescue)

  • Biochemical assays (enzyme activity, metabolite profiling)

  • Imaging techniques (to assess mitochondrial morphology and function)

  • In vivo confirmation when possible