ENO3 Human

What is ENO3 and what is its primary function in human tissues?

ENO3, also known as beta-enolase (ENO-β), is an enzyme encoded by the ENO3 gene in humans. It belongs to the enolase family and functions as a glycolytic enzyme that catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate during glycolysis. This isoform is predominantly expressed in adult striated muscle, including skeletal and cardiac muscle tissues . During fetal muscle development, there is a transcriptional switch from expressing ENO1 (alpha-enolase) to ENO3, influenced by muscle innervation and Myo D1. Interestingly, ENO3 expression levels are higher in fast-twitch muscle fibers compared to slow-twitch fibers, suggesting specialized metabolic roles for tissues with high energy demands .

The catalytic activity of ENO3 is essential for energy production, particularly in muscle tissues that rely heavily on glycolysis. Specific activity measurements for recombinant human ENO3 indicate activity levels exceeding 5,000 pmol/min/μg, as determined by monitoring NAD absorbance changes at 340nm .

How is the structure of the ENO3 gene organized, and what regulatory elements control its expression?

The ENO3 gene spans approximately 6 kb and contains 12 exons, with the first exon being an untranslated region (non-coding). The gene's regulatory architecture is specialized for muscle-specific expression. The first intron, along with the 5'-flanking region, contains consensus sequences for muscle-specific regulatory factors including a CC(A + T-rich)6GG box, a M-CAT-box CAATCCT, and two myocyte-specific enhancer-binding factor 1 boxes .

Upstream of the first exon lies a TATA-like box and CpG-rich region containing recognition motifs for binding transcriptional regulatory factors such as Sp1, activator protein 1 and 2, CCAAT box transcription factor/nuclear factor I, and cyclic AMP . A distinctive feature of ENO3 compared to other enolase genes is that it possesses a single transcription initiation site located 26 bp downstream of the TATA-like box, whereas other enolase genes have multiple transcription initiation sites .

This unique structural organization contributes to the tissue-specific expression pattern of ENO3 and explains its predominant presence in muscle tissues.

What methodological approaches are recommended for measuring ENO3 enzymatic activity?

For accurate measurement of ENO3 enzymatic activity, researchers should consider several methodological approaches:

• Spectrophotometric assays: The standard method involves monitoring the decrease of NAD in absorbance at 340nm resulting from NADH consumption at pH 6.5 and 37°C . This approach provides a quantitative measure of ENO3's catalytic function in converting 2-phosphoglycerate to phosphoenolpyruvate.

• Optimal reaction conditions: Maintain experimental conditions at pH 6.5 and 37°C, which have been established as optimal for ENO3 activity measurement . Using appropriate buffer systems (typically Tris-HCl) with stabilizing agents like DTT (0.02%) helps maintain enzyme stability during assays.

• Substrate concentration optimization: Ensure linear enzymatic rates by optimizing substrate concentrations through preliminary kinetic studies determining Km values for 2-phosphoglycerate.

• Controls and normalization: Include proper controls to account for background NAD/NADH fluctuations and normalize activity to protein concentration, determined using methods like Bradford or BCA assays.

• Coupled enzyme assays: Consider coupling the reaction with other glycolytic enzymes to create a continuous assay system, which can improve sensitivity and reproducibility.

The specific activity of purified recombinant human ENO3 protein typically exceeds 5,000 pmol/min/μg, providing a benchmark for quality control in enzyme preparation .

What are the structural differences between ENO3 and other enolase isoforms?

ENO3 (beta-enolase) is one of three enolase isoforms in humans, alongside ENO1 (alpha-enolase) and ENO2 (gamma-enolase). Each isoform acts as a protein subunit that can form hetero- or homodimers of various combinations (αα, αβ, αγ, ββ, and γγ) . Understanding the structural differences between these isoforms provides insights into their specialized functions:

Sequence and structural comparison:

• ENO3 encodes a 433-residue protein that forms functional dimers

• The protein contains an active site that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate

• Compared to other enolase isoforms, ENO3 has a relatively conserved catalytic domain but differs in certain surface-exposed regions

Evolutionary perspective:

• Based on its comparatively small length and highly conserved intron/exon organization, ENO3 is suggested to have been the last to diverge from a common ancestral gene

• This evolutionary history may explain its specialized function in muscle tissues

Tissue distribution differences:

• ENO3 is predominantly expressed in muscle tissue

• ENO1 is ubiquitously expressed across most tissues

• ENO2 is mainly found in neuronal and neuroendocrine tissues

These structural differences underlie the functional specialization of ENO3 in muscle metabolism and explain why mutations in ENO3 primarily affect muscle function, leading to conditions such as glycogen storage disease type XIII .

What expression systems are most effective for producing recombinant human ENO3?

For researchers seeking to produce recombinant human ENO3 protein for structural or functional studies, several expression systems have proven effective, with Escherichia coli being the most commonly utilized:

• Bacterial expression in E. coli:

  • Provides high yields of functional recombinant human ENO3 protein

  • Typically includes an N-terminal His-tag to facilitate purification

  • Can achieve purity levels greater than 95% as determined by SDS-PAGE analysis

  • Conventional chromatography techniques are effective for purification

• Optimal construct design:

  • Expression of the full-length protein sequence (434 amino acids) ensures proper folding and activity

  • Addition of appropriate tags (His-tag) at the N-terminus facilitates purification without compromising enzymatic function

  • Codon optimization for E. coli can improve expression efficiency

• Purification strategy:

  • Immobilized metal affinity chromatography using the His-tag

  • Additional purification steps like ion exchange or size exclusion chromatography

  • Buffer optimization for stability (pH 8.0 with 0.02% DTT, 0.32% Tris HCl, 20% Glycerol, 0.58% Sodium chloride)

• Storage considerations:

  • Addition of stabilizing agents such as DTT (0.02%) helps maintain enzymatic activity

  • Storage in 20% glycerol prevents protein aggregation

  • Aliquoting and storing at -20°C or -80°C avoids freeze/thaw cycles that can reduce activity

• Quality control measures:

  • Verification of purity using SDS-PAGE

  • Activity testing by monitoring NAD absorbance changes at 340nm

  • Mass spectrometry confirmation of protein identity

Following these guidelines can help researchers produce high-quality ENO3 protein suitable for a variety of experimental applications.

What is ENO3's role in cancer progression and its potential as a therapeutic target?

ENO3 exhibits context-dependent roles in cancer development and progression, with emerging evidence suggesting it may function as a tumor suppressor in certain cancer types:

In hepatocellular carcinoma (HCC), ENO3 has been identified as a tumor suppressor. Research has demonstrated that ENO3 is remarkably down-regulated in human HCC tissue compared to non-cancerous tissue, and low expression correlates with poor prognosis in HCC patients . Functional studies have shown that overexpression of ENO3 suppresses proliferative, migratory, and invasive abilities of HCC cells both in vitro and in vivo, while knocking down ENO3 enhances these malignant phenotypes .

Mechanistically, ENO3 represses the epithelial-mesenchymal transition (EMT) process, which is crucial for cancer metastasis. Further investigation revealed that ENO3 suppresses the Wnt/β-catenin signaling pathway, which subsequently modulates the transcription of target genes associated with proliferation and metastasis of HCC cells .

These findings suggest that ENO3 could be a promising candidate for cancer treatment, particularly in HCC. Therapeutic strategies could include:

• Approaches to upregulate ENO3 expression in tumors where it acts as a tumor suppressor

• Targeting the Wnt/β-catenin pathway in conjunction with ENO3 modulation

• Developing biomarkers based on ENO3 expression to guide treatment decisions

The study by Frontiers in Cell and Developmental Biology highlighted that "ENO3 acted as a tumor inhibitor in HCC development and implied ENO3 as a promising candidate for HCC treatment" , underscoring its potential therapeutic significance.

How do mutations in ENO3 contribute to glycogen storage disease type XIII?

Glycogen storage disease type XIII (GSD13), also known as muscle β-enolase deficiency, is a rare inherited metabolic myopathy caused by mutations in the ENO3 gene. These mutations typically affect the enzyme's active site, disrupting its glycolytic activity and consequently impairing energy metabolism in muscle tissue .

The condition is generally characterized as an autosomal recessive disorder, but clinical observations have identified both heterozygous and homozygous mutations in affected individuals. Heterozygous mutations typically result in milder clinical presentations, while homozygous mutations tend to produce more severe symptoms, including rhabdomyolysis (the breakdown of damaged muscle tissue) .

At the molecular level, ENO3 mutations lead to:

• Reduced catalytic efficiency: Mutations in the active site impair the enzyme's ability to convert 2-phosphoglycerate to phosphoenolpyruvate

• Protein instability: Some mutations may affect protein folding or stability, leading to reduced enzyme levels

• Impaired dimerization: Since ENO3 functions as a dimer, mutations affecting protein-protein interfaces can disrupt functional enzyme formation

The pathophysiological consequences include:

• Reduced ATP production in muscle cells, particularly during anaerobic exercise

• Impaired ability to meet energy demands during intense physical activity

• Accumulation of glycolytic intermediates upstream of the enzymatic block

• Potential compensatory upregulation of alternative metabolic pathways

Advances in genetic testing, such as exome sequencing and targeted gene panels, have improved access to diagnosis for muscle β-enolase deficiency . These technologies allow for more precise identification of the specific ENO3 mutations, which can inform prognosis and potential treatment approaches.

What methodological approaches are most effective for investigating ENO3's role in the Wnt/β-catenin signaling pathway?

Research has identified that ENO3 suppresses the Wnt/β-catenin signaling pathway in hepatocellular carcinoma, which subsequently impacts the transcription of target genes associated with proliferation and metastasis . To rigorously investigate this regulatory relationship, researchers should employ multiple complementary methodological approaches:

• Gene expression modulation techniques:

  • Overexpression studies using transfection of ENO3 expression vectors

  • Knockdown experiments using siRNA or shRNA targeting ENO3

  • CRISPR-Cas9 genome editing to create ENO3 knockout or knock-in cell lines

  • Inducible expression systems to study time-dependent effects

• Protein-protein interaction analysis:

  • Co-immunoprecipitation (Co-IP) to detect physical interactions between ENO3 and Wnt/β-catenin pathway components

  • Proximity ligation assays to visualize protein interactions in situ

  • Mass spectrometry-based interactome analysis to identify all interaction partners

  • In vitro binding assays with purified proteins to confirm direct interactions

• Wnt/β-catenin pathway activity assessment:

  • TOPFlash/FOPFlash luciferase reporter assays to measure β-catenin-dependent transcriptional activity

  • Western blot analysis of active (non-phosphorylated) β-catenin levels

  • Immunofluorescence to track β-catenin nuclear translocation

  • Chromatin immunoprecipitation (ChIP) to assess β-catenin binding to target gene promoters

• Target gene expression analysis:

  • qRT-PCR and RNA-seq to measure expression of Wnt/β-catenin target genes

  • ChIP-seq to identify β-catenin binding sites at target gene promoters

  • Promoter-reporter assays to assess transcriptional regulation

• Functional validation studies:

  • Cell proliferation, migration, and invasion assays following ENO3 modulation

  • Rescue experiments combining ENO3 overexpression with Wnt pathway activators

  • In vivo xenograft models to confirm observations in a physiological context

These methodologies, when used in combination, provide a comprehensive framework for understanding how ENO3 regulates Wnt/β-catenin signaling. The study published in Frontiers in Cell and Developmental Biology demonstrated that "ENO3 suppressed the Wnt/β-catenin signal, which subsequently modulated the transcription of its target genes associated with the proliferation and metastasis capacity of HCC cells" , highlighting the importance of this regulatory mechanism.

What are the emerging techniques for detecting and quantifying ENO3 protein-protein interactions?

Understanding ENO3's protein interaction network is crucial for elucidating its multifunctional roles in both normal physiology and disease states. Several advanced techniques have emerged as valuable tools for investigating ENO3 protein-protein interactions:

• Proximity-dependent labeling methods:

  • BioID and TurboID approaches, where ENO3 is fused to a biotin ligase that biotinylates nearby proteins

  • APEX2 proximity labeling, utilizing an engineered ascorbate peroxidase to tag proximal proteins

  • These methods capture transient or weak interactions that might be missed by traditional co-immunoprecipitation

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize co-localization at nanometer resolution

  • Förster resonance energy transfer (FRET) to detect direct protein interactions in living cells

  • Fluorescence lifetime imaging microscopy (FLIM) to measure protein proximity with high sensitivity

  • Live-cell imaging to track dynamic interactions in real-time

• Mass spectrometry-based approaches:

  • Affinity purification coupled with mass spectrometry (AP-MS) using tagged ENO3

  • Cross-linking mass spectrometry (XL-MS) to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to detect conformational changes upon binding

  • Thermal proteome profiling to detect interaction-induced changes in thermal stability

• Protein complementation assays:

  • Split fluorescent protein systems (e.g., split GFP) where fragments reunite when fused proteins interact

  • NanoBiT system using complementary fragments of NanoLuc luciferase

  • Mammalian membrane two-hybrid (MaMTH) system for studying interactions involving membrane proteins

• Computational prediction and validation:

  • Machine learning approaches to predict potential interaction partners

  • Molecular docking simulations to model interaction interfaces

  • Network analysis to place ENO3 interactions in broader biological contexts

When studying ENO3's role in cancer, these techniques can reveal how it interacts with components of the Wnt/β-catenin pathway, as demonstrated in hepatocellular carcinoma research . For investigating its role in muscle metabolism, these methods can identify interactions with other glycolytic enzymes and regulatory proteins in muscle tissue .

These emerging technologies, especially when used in combination, provide researchers with powerful tools to map ENO3's interactome with unprecedented detail and contextual relevance.

How do post-translational modifications affect ENO3 function in different physiological contexts?

Post-translational modifications (PTMs) can significantly alter ENO3's enzymatic activity, subcellular localization, protein-protein interactions, and stability. Understanding these modifications is crucial for comprehending ENO3's diverse roles in both normal physiology and disease states:

• Phosphorylation:

  • May regulate enzymatic activity in response to cellular energy status

  • Could affect interaction with other glycolytic enzymes or signaling proteins

  • Potentially mediates ENO3's non-glycolytic functions, such as its role in suppressing the Wnt/β-catenin pathway in cancer cells

  • Research methods: Phospho-specific antibodies, mass spectrometry-based phosphoproteomics, site-directed mutagenesis

• Acetylation:

  • The research shows that TFG-TEC, an oncoprotein, activates ENO3 expression by increasing acetylation of histone H3 at the ENO3 promoter

  • Protein acetylation might also directly affect ENO3 function

  • Research methods: Acetylation-specific antibodies, mass spectrometry, HDAC inhibitor treatments

• Glycosylation:

  • May affect protein stability and localization

  • Could be particularly relevant in cancer contexts where aberrant glycosylation is common

  • Research methods: Glycoprotein staining, lectin affinity, mass spectrometry

• Oxidative modifications:

  • Cysteine oxidation could affect enzymatic activity, especially important during oxidative stress in muscle tissue

  • May be relevant in muscle pathologies including glycogen storage disease type XIII

  • Research methods: Redox proteomics, site-directed mutagenesis of cysteine residues

Methodological approaches for studying ENO3 PTMs:

• Unbiased PTM profiling:

  • Mass spectrometry-based proteomics to identify all modifications

  • Enrichment strategies for specific PTMs (e.g., phosphopeptide enrichment)

  • Comparison of PTM patterns between normal muscle tissue and disease states

• Functional assessment of PTMs:

  • Site-directed mutagenesis to create non-modifiable versions (e.g., S→A for phosphorylation sites)

  • In vitro enzymatic assays comparing wild-type and mutant proteins

  • Cellular studies examining localization, interactions, and function

Understanding how these modifications regulate ENO3 could provide insights into its role in glycogen storage disease type XIII and its tumor suppressor function in hepatocellular carcinoma , potentially revealing new therapeutic targets.

What are the best practices for purifying active ENO3 protein for structural and functional studies?

Obtaining pure, active ENO3 protein is essential for structural and functional studies. Based on established protocols for recombinant human ENO3 protein production, the following best practices are recommended:

• Expression system selection:

  • Escherichia coli has been successfully used for expressing functional human ENO3

  • The protein can include an N-terminal His-tag to facilitate purification

  • The full-length protein (434 amino acids) should be expressed to ensure proper folding and activity

• Construct design considerations:

  • Include the complete coding sequence (amino acids 1-434)

  • Add purification tags that minimally interfere with protein function (His-tag at N-terminus is effective)

  • Consider codon optimization for the expression system

  • Include cleavage sites for tag removal if necessary for downstream applications

• Purification strategy:

  • Conventional chromatography has proven effective for purifying ENO3 to >95% purity

  • Two-step purification typically yields the best results:

    • Initial purification using immobilized metal affinity chromatography (IMAC) via the His-tag

    • Secondary purification using size exclusion chromatography to ensure homogeneity

• Buffer optimization:

  • Optimal buffer conditions include pH 8.00

  • Include stabilizing components: 0.02% DTT, 0.32% Tris HCl, 20% Glycerol, 0.58% Sodium chloride

  • DTT helps maintain cysteine residues in reduced state

  • Glycerol prevents protein aggregation and improves stability

• Quality control measures:

  • Verify purity using SDS-PAGE (target >95%)

  • Confirm identity using western blotting or mass spectrometry

  • Assess specific activity (target >5,000 pmol/min/μg) by measuring NAD absorbance changes at 340nm

  • Check oligomeric state using native PAGE or size exclusion chromatography (ENO3 functions as a dimer)

• Storage conditions:

  • Aliquot and store at -20°C or -80°C to avoid freeze/thaw cycles

  • For short-term storage (1-2 weeks), keep at 4°C

  • Handle as an active protein that may elicit biological responses

Following these practices should yield purified ENO3 protein suitable for both structural studies (e.g., crystallography, cryo-EM) and functional analyses (enzymatic assays, protein-protein interaction studies).

How can researchers effectively use CRISPR-Cas9 gene editing to study ENO3 function?

CRISPR-Cas9 gene editing offers powerful approaches for investigating ENO3 function in various experimental models. Here's a comprehensive methodological guide for researchers:

By systematically applying these CRISPR-Cas9 approaches, researchers can gain comprehensive insights into ENO3's multifaceted roles in normal physiology and disease contexts, as demonstrated in studies showing its tumor suppressor function in hepatocellular carcinoma and its critical role in muscle metabolism .

What analytical techniques are most sensitive for detecting changes in ENO3 expression in patient samples?

Detecting changes in ENO3 expression in patient samples requires sensitive and specific analytical techniques. For researchers investigating ENO3 in clinical contexts, such as hepatocellular carcinoma or glycogen storage disease type XIII , the following methods are recommended:

• Nucleic acid-based methods:

  a) Quantitative PCR (qPCR):

  • Highly sensitive for detecting transcript levels

  • Requires small amounts of starting material (valuable for limited patient samples)

  • Can be performed on fresh, frozen, or FFPE tissue samples

  • Best practices: Use validated reference genes for normalization; design primers spanning exon-exon junctions

  b) Digital PCR:

  • Provides absolute quantification without standard curves

  • Higher precision and reproducibility than qPCR

  • Less affected by PCR inhibitors that may be present in clinical samples

  • Particularly valuable for detecting subtle expression changes

• Protein detection methods:

  a) Immunohistochemistry (IHC):

  • Allows visualization of ENO3 protein in tissue context

  • Can reveal spatial distribution within muscle fibers or tumor tissue

  • Permits comparison between affected and unaffected regions within the same sample

  • Crucial for confirming the downregulation of ENO3 in hepatocellular carcinoma tissues

  b) Western blotting:

  • Semi-quantitative assessment of protein levels

  • Can detect full-length protein and potential variants

  • Useful for comparing expression levels between patient and control samples

  • Enhanced sensitivity with chemiluminescent or fluorescent detection systems

• Advanced technologies:

  a) Mass spectrometry-based proteomics:

  • Can provide absolute quantification of ENO3 protein

  • Allows detection of post-translational modifications

  • Approaches include selected reaction monitoring (SRM) or multiple reaction monitoring (MRM)

  • Particularly valuable for research on post-translational modifications affecting enzyme activity

  b) Single-cell RNA sequencing:

  • Reveals expression patterns in individual cells

  • Can identify cell populations with altered ENO3 expression

  • Particularly valuable for heterogeneous samples like tumors

  • Helps understand cell-type specific expression patterns

The choice of technique should be guided by the specific research question, sample availability, and required sensitivity. For many clinical studies, a combination of approaches (e.g., qPCR for screening followed by IHC for validation) may provide the most comprehensive assessment of ENO3 expression changes, as was done in studies examining ENO3's role as a tumor suppressor in hepatocellular carcinoma .

What are the most effective experimental models for studying ENO3 in muscle development and disease?

To study ENO3 function in muscle development and disease, researchers can utilize various experimental models, each with specific advantages for addressing different research questions:

• Cellular Models:

  a) Primary human myoblasts/myotubes:

  • Isolated from muscle biopsies

  • Allow study of ENO3 during myogenic differentiation

  • Can be derived from patients with ENO3-related disorders like glycogen storage disease type XIII

  • Most physiologically relevant for human disease studies

  b) Established muscle cell lines:

  • C2C12 (mouse) or L6 (rat) myoblasts that can differentiate into myotubes

  • Allow genetic manipulation (overexpression, knockdown, CRISPR editing)

  • Useful for high-throughput screening

  • Well-characterized model for studying muscle differentiation

  c) iPSC-derived myogenic cells:

  • Generated from patient fibroblasts or blood cells

  • Maintain genetic background of donors

  • Can model developmental aspects

  • Valuable for personalized medicine approaches

• Animal Models:

  a) Transgenic mice:

  • Eno3 knockout or knockin models

  • Tissue-specific or inducible expression systems

  • Allow whole-body phenotyping

  • Can model glycogen storage disease type XIII

  • Valuable for studying fiber-type specific expression patterns

  b) Disease-specific models:

  • Mouse models of muscle diseases

  • Cancer xenograft models to study ENO3's tumor suppressor function

  • Exercise and disuse models to study regulation under different physiological conditions

• Ex Vivo Systems:

  a) Isolated muscle fibers:

  • Maintain three-dimensional architecture

  • Allow functional studies

  • Can be electrically stimulated

  • Useful for studying fiber-type differences in ENO3 expression and function

  b) Tissue explants:

  • Preserve tissue architecture and cell-cell interactions

  • Allow short-term culture for intervention studies

  • Bridge the gap between in vitro and in vivo models

• Human Samples:

  a) Patient biopsies:

  • Most directly relevant to human disease

  • Can be analyzed for ENO3 expression patterns and mutations

  • Limited availability and heterogeneity may present challenges

  • Critical for validating findings from model systems

When selecting experimental models, researchers should consider the specific research question, whether it focuses on ENO3's enzymatic function, its role in muscle development, its tumor suppressor activity in cancer , or its involvement in genetic disorders like glycogen storage disease type XIII .

What computational approaches can predict the impact of ENO3 mutations on protein structure and function?

Computational approaches offer valuable tools for predicting how mutations in ENO3 might affect protein structure and function, which is particularly relevant for understanding glycogen storage disease type XIII and potential cancer-related alterations. The following methodological framework outlines key computational strategies:

• Sequence-Based Analysis:

  a) Conservation analysis:

  • Multiple sequence alignment across species and enolase isoforms

  • Identification of highly conserved residues likely crucial for function

  • Tools: Clustal Omega, MUSCLE, ConSurf

  b) Mutation effect prediction:

  • Algorithms estimating functional impact based on evolutionary conservation and physicochemical properties

  • Tools: SIFT, PolyPhen-2, PROVEAN, CADD, MutationTaster

  • Valuable for prioritizing mutations identified in patients with glycogen storage disease type XIII

• Structural Analysis:

  a) Structure modeling:

  • Homology modeling based on crystal structures of human enolases

  • Ab initio modeling for regions lacking templates

  • Tools: SWISS-MODEL, I-TASSER, AlphaFold2

  • Critical for understanding how mutations affect the catalytic site of ENO3

  b) Molecular dynamics simulations:

  • Analysis of mutation effects on protein stability and flexibility

  • Identification of conformational changes affecting enzyme function

  • Assessment of substrate binding and catalytic activity changes

  • Tools: GROMACS, AMBER, NAMD

• Functional Site Analysis:

  a) Active site assessment:

  • Prediction of changes in substrate binding pocket geometry

  • Effects on catalytic residues positioning

  • Tools: CASTp, COACH, FTMap

  • Essential for understanding how mutations disrupt ENO3's glycolytic function

  b) Protein-protein interaction interface analysis:

  • Impact of mutations on dimerization or interaction with other proteins

  • Particularly relevant for understanding how ENO3 interacts with Wnt/β-catenin pathway components

  • Tools: HADDOCK, ClusPro, InterEvDock

• Integrated Approaches:

  a) Machine learning methods:

  • Neural networks trained on protein structures and mutation effects

  • Integration of multiple data sources for improved prediction accuracy

  • Tools: DeepDDG, mCSM, DynaMut

  b) Network analysis:

  • Integration of ENO3 in protein-protein interaction networks

  • Prediction of pathway perturbations caused by mutations

  • Valuable for understanding ENO3's role in signaling pathways like Wnt/β-catenin

These computational approaches, especially when used in combination, can provide valuable insights into the molecular mechanisms by which mutations affect ENO3 function. The predictions can guide experimental design and help prioritize variants for functional validation, ultimately contributing to better understanding of ENO3-related disorders and potential therapeutic strategies.