ECH1 Human

What is ECH1 and what is its primary function in human cells?

ECH1 (delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase) is a member of the hydratase/isomerase superfamily that plays an important role in the auxiliary step of the fatty acid beta-oxidation pathway . The protein shows high sequence similarity to enoyl-CoA hydratases across multiple species, particularly within conserved domains characteristic of these enzymes. Its primary function is to isomerize 3-trans,5-cis-dienoyl-CoA to 2-trans,4-trans-dienoyl-CoA, a critical step in the metabolism of unsaturated fatty acids with odd-numbered double bonds .

For researchers investigating ECH1 function, enzyme activity assays measuring isomerization rates provide direct functional evidence. These assays typically employ spectrophotometric measurements or HPLC analysis of substrate-product conversion. Complementary approaches include metabolomic profiling of cells with modulated ECH1 expression to identify changes in fatty acid intermediates, providing insight into the specific metabolic pathways affected by ECH1 activity.

What cellular compartments does ECH1 localize to?

ECH1 contains a C-terminal peroxisomal targeting sequence and primarily localizes to peroxisomes . Interestingly, studies of the rat ortholog indicate that the protein can also localize to the matrix of both peroxisomes and mitochondria, suggesting potential dual compartmentalization depending on cellular conditions . This dual localization may reflect the importance of ECH1 in fatty acid metabolism occurring in both organelles.

To methodologically investigate ECH1 localization, researchers should employ a multi-faceted approach including:

• Subcellular fractionation followed by Western blotting

• Immunofluorescence microscopy with organelle-specific markers

• Expression of fluorescently-tagged ECH1 constructs

• Proximity labeling approaches such as BioID or APEX

For quantitative assessment, high-content imaging systems with automated image analysis can measure colocalization coefficients with known peroxisomal and mitochondrial markers, providing statistical robustness to localization studies.

How should ECH1 protein be stored and handled for research applications?

Recombinant ECH1 protein requires specific storage and handling conditions to maintain structural integrity and enzymatic activity. Based on manufacturer specifications, the optimal formulation includes 20 mM Tris-HCl buffer (pH 8.0), 10% glycerol, 1 mM DTT, and 50 mM NaCl . This formulation provides pH stability, prevents protein denaturation, maintains reducing conditions, and provides ionic strength.

Researchers should aliquot the protein upon receipt to minimize freeze-thaw cycles and validate protein integrity by SDS-PAGE before experimental use . For enzymatic assays, activity controls should be included to ensure the protein maintains its functional properties throughout experimental procedures.

What expression systems are used to produce recombinant ECH1?

Recombinant human ECH1 protein for research applications is typically produced in E. coli expression systems . This bacterial expression platform offers advantages for producing non-glycosylated human proteins like ECH1, including high yield, cost-effectiveness, and established purification protocols. The recombinant protein is typically tagged with a polyhistidine sequence (His-tag) at the N-terminus to facilitate purification by metal affinity chromatography .

For researchers producing their own ECH1 protein, optimization of expression conditions is critical. This includes selection of appropriate E. coli strains (BL21(DE3) derivatives are common), induction parameters (IPTG concentration, temperature, duration), and purification strategies. Following purification, validation of protein identity and purity should be performed using techniques such as SDS-PAGE (targeting >90% purity), Western blotting with anti-ECH1 antibodies, and mass spectrometry .

What are the optimal experimental conditions for studying ECH1 enzymatic activity in vitro?

For rigorous biochemical characterization of ECH1 activity, researchers should establish well-controlled reaction conditions that mimic physiological environments while enabling precise measurements. Based on biochemical principles and the protein's characteristics, the following parameters are recommended:

Activity assays should include appropriate controls such as heat-inactivated enzyme preparations and reaction mixtures lacking substrate. For kinetic analysis, researchers should collect time-course data with multiple substrate concentrations to determine Km, Vmax, and kcat values. These parameters provide quantitative measures of enzyme efficiency and can be compared across experimental conditions or between wild-type and mutant proteins.

How can researchers analyze potential interactions between ECH1 and other proteins in fatty acid metabolism?

Investigation of ECH1's protein-protein interactions requires a multi-method approach to capture both stable and transient interactions within metabolic pathways. Given ECH1's role in fatty acid metabolism and its dual localization, interaction studies should focus on both peroxisomal and mitochondrial protein networks.

Methodologically, researchers should begin with affinity purification approaches using tagged ECH1 as bait, followed by mass spectrometry (AP-MS) to identify potential interacting partners. For validation and detailed characterization, techniques including:

• Bimolecular fluorescence complementation (BiFC) for visualizing interactions in living cells

• Förster resonance energy transfer (FRET) for measuring interaction distances

• Co-immunoprecipitation with endogenous proteins for confirming physiological relevance

• Proximity-dependent labeling methods (BioID, APEX2) for capturing transient interactions

When analyzing interaction data, researchers should apply stringent statistical filtering (typically fold change >2, p<0.01) and validate key interactions using at least two independent methods. Functional validation through co-expression, co-depletion, or enzymatic coupling assays provides evidence for the biological significance of identified interactions.

What approaches are most effective for studying the effects of ECH1 mutations or deficiency?

To investigate the functional consequences of ECH1 mutations or deficiency, researchers should employ complementary genetic and biochemical approaches. CRISPR-Cas9 gene editing provides a precise method for creating cellular models with ECH1 knockout or specific mutations. Lentiviral shRNA systems offer an alternative approach for temporary knockdown when complete gene deletion is not desired.

For phenotypic characterization, a comprehensive analysis should include:

For clinical relevance, patient-derived cell lines or induced pluripotent stem cells (iPSCs) carrying ECH1 mutations can be differentiated into relevant cell types to study tissue-specific effects. Rescue experiments with wild-type ECH1 and various mutant constructs help distinguish between loss-of-function and gain-of-function effects.

How can researchers integrate ECH1 functional studies with broader metabolic pathways?

Understanding ECH1's role within the context of integrated metabolism requires systems biology approaches that capture pathway interactions and regulatory mechanisms. Metabolic flux analysis using stable isotope-labeled substrates (13C-labeled fatty acids) enables quantitative measurement of carbon flow through ECH1-dependent and alternative pathways.

Researchers should combine targeted experimental manipulations with computational modeling:

• Experimental approaches:

  • Metabolic tracing using labeled substrates and mass spectrometry

  • Pathway inhibition studies using specific inhibitors of related enzymes

  • Time-course analyses of metabolite changes after ECH1 modulation

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

• Computational approaches:

  • Constraint-based metabolic modeling (e.g., flux balance analysis)

  • Kinetic modeling of fatty acid oxidation pathways

  • Network analysis of metabolic interactions

  • Machine learning for pattern detection in complex datasets

These integrated approaches help identify compensatory mechanisms, regulatory feedback loops, and pathway crosstalk that may not be apparent from isolated studies of ECH1 function.

What methodological considerations are important when studying ECH1 in different tissue contexts?

ECH1 expression and function may vary significantly across tissue types, reflecting tissue-specific metabolic requirements. The Human Protein Atlas and other tissue expression databases provide insight into expression patterns across tissues , but functional studies require tissue-specific methodological adaptations.

For tissue-specific investigations, researchers should consider:

• Expression analysis:

  • Single-cell RNA sequencing to resolve cell type-specific expression

  • Immunohistochemistry with validated antibodies for protein localization

  • Tissue microarrays for comparative expression profiling

• Functional studies:

  • Primary cell isolation from relevant tissues

  • Organoid cultures maintaining tissue architecture

  • Tissue-specific conditional knockout animal models

  • Metabolism studies under tissue-relevant substrate conditions

• Pathological relevance:

  • Expression analysis in disease-affected tissues

  • Correlation with tissue-specific biomarkers

  • Patient-derived samples for validation studies

When interpreting results, researchers should account for tissue-specific factors including metabolic specialization, environmental conditions, and developmental stage. These considerations ensure that findings accurately reflect the biological role of ECH1 in specific physiological contexts.