HADHB Human

What is the molecular structure and enzymatic function of HADHB in human metabolism?

HADHB (Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit) encodes the beta subunit of the mitochondrial trifunctional protein (MTP). This 51.2 kDa protein composed of 474 amino acids catalyzes the final thiolytic cleavage step in mitochondrial beta-oxidation of long-chain fatty acids. Specifically, HADHB provides the 3-ketoacyl-CoA thiolase activity, where the thiol group of Coenzyme A cleaves 3-ketoacyl CoA between C-2 and C-3, yielding an acetyl CoA molecule and an acyl CoA molecule that is two carbons shorter .

The MTP complex forms an α₂β₂ heterotetrameric structure with two HADHB (β) subunits forming a homodimer in the center, flanked by one HADHA (α) subunit on each side. Recent cryo-electron microscopy studies have elucidated this quaternary structure, revealing that proper complex assembly is essential for efficient substrate channeling through the beta-oxidation pathway .

For experimental investigation of HADHB structure:

• X-ray crystallography or cryo-EM can resolve detailed protein structure

• Protein-protein interaction studies using co-immunoprecipitation can examine HADHB-HADHA interactions

• Site-directed mutagenesis of key domains can identify critical residues for enzyme function

How does HADHB contribute to the mitochondrial fatty acid oxidation pathway?

HADHB functions within the mitochondrial trifunctional protein, which catalyzes three consecutive reactions in the long-chain fatty acid beta-oxidation pathway. This metabolic pathway represents the major energy-producing process in tissues, especially during fasting states . Within this pathway:

• HADHA (α-subunit) performs two enzymatic activities:

  • 2,3-enoyl-CoA hydratase activity

  • 3-hydroxyacyl-CoA dehydrogenase activity

• HADHB (β-subunit) performs the final step:

  • 3-ketoacyl-CoA thiolase activity

When studying HADHB's role in this pathway, researchers should consider:

• Measuring specific intermediates of beta-oxidation using LC-MS/MS

• Analyzing fatty acid utilization in cells with wild-type versus mutant HADHB

• Examining compensatory mechanisms when HADHB function is compromised

What alternative functions does HADHB perform beyond fatty acid metabolism?

Beyond its canonical role in fatty acid metabolism, HADHB demonstrates additional functions that expand its biological significance. The protein can bind RNA and decrease the stability of some mRNAs, suggesting a role in post-transcriptional regulation . This RNA-binding capacity may represent a mechanism by which metabolic status is communicated to gene expression regulation.

Methodological approaches to investigate these non-canonical functions include:

• RNA immunoprecipitation (RIP) to identify HADHB-bound transcripts

• RNA stability assays comparing wild-type and HADHB-deficient cells

• Polysome profiling to assess effects on translation

• Protein-protein interaction networks to identify non-metabolic binding partners

Researchers should design experiments that can differentiate direct effects of HADHB from indirect consequences of altered fatty acid metabolism when investigating these alternative functions.

What is the genomic organization of HADHB and how should researchers approach sequencing analysis?

The HADHB gene is located on chromosome 2p23 and contains 17 exons encoding a protein of 474 amino acids . The gene is positioned in a head-to-head orientation with HADHA, suggesting coordinated regulation of these functionally related genes.

For comprehensive genetic analysis of HADHB:

When interpreting sequencing results, researchers should consider:

• Evolutionary conservation of affected amino acids

• Structural impact using in silico prediction tools

• Population frequency in databases like gnomAD

• Functional consequences on protein stability and enzyme activity

• Potential effects on HADHA-HADHB interaction

What types of HADHB mutations have been reported and how do they affect protein function?

Various types of mutations in the HADHB gene have been identified in patients with MTP deficiency:

For functional characterization of HADHB variants:

• Express recombinant wild-type and mutant proteins to assess stability

• Perform enzyme activity assays to measure catalytic function

• Analyze complex formation with HADHA using co-immunoprecipitation

• Conduct in silico structural analysis to predict impacts on protein folding

• Assess cellular phenotypes in patient-derived cells or model systems

Structural analysis based on cryo-electron microscopy of the human MTP reveals that mutations can affect the homodimerization of HADHB subunits or disrupt the interaction with HADHA subunits, compromising the integrity of the entire complex .

How does HADHB genotype correlate with clinical phenotype in MTP deficiency?

MTP deficiency presents with a broad spectrum of clinical phenotypes correlating with specific HADHB mutations. This genotype-phenotype relationship can be stratified into several categories:

A particularly noteworthy finding is the identification of a mild phenotype in three young adults with HADHB mutations (c.694G>A p.(Ala232Thr) and c.255-1G>A) characterized by axonal neuropathy and frequent intermittent weakness episodes without myoglobinuria. These cases highlight that MTP deficiency should be considered in the differential diagnosis of patients with milder fluctuating neuromuscular symptoms, even without elevated CK or rhabdomyolysis .

Research approaches to investigate genotype-phenotype correlations include:

• Systematic collection of clinical data from patients with defined mutations

• Biochemical characterization of enzyme activity for different variants

• Development of cellular and animal models expressing specific mutations

• Longitudinal studies to assess disease progression with different genotypes

What are the optimal techniques for measuring HADHB enzyme activity in different sample types?

Accurate measurement of HADHB enzyme activity is crucial for both research and diagnostic applications. Multiple methodological approaches can be employed:

For research applications, the specific protocol should be selected based on:

Which antibodies and immunodetection methods are most effective for HADHB research?

Multiple validated antibodies and detection methods are available for HADHB research:

Methodological considerations for different applications:

• Western Blot:

  • Use fresh lysates with protease inhibitors

  • Include mitochondrial markers for normalization

  • Verify specificity with appropriate controls

  • Expected band size: 51 kDa

• Immunohistochemistry:

  • Optimize antigen retrieval for formalin-fixed tissues

  • Use mitochondrial co-staining to confirm localization

  • Include tissue from known MTP-deficient patients as controls

• Flow Cytometry:

  • Permeabilize cells appropriately to access mitochondrial proteins

  • Use mitochondrial membrane potential dyes for co-localization

  • Compare with isotype control antibodies

• Immunoprecipitation:

  • Use approximately 1mg cell lysate per 10μl antibody-conjugated beads

  • Include appropriate negative controls

  • Confirm specificity by mass spectrometry

How can researchers develop cellular and animal models for studying HADHB function?

Developing appropriate models is essential for studying HADHB function, disease mechanisms, and potential therapeutic approaches:

Methodological considerations for model development:

• Validate models by confirming HADHB expression levels and enzyme activity

• Characterize metabolic profiles using targeted metabolomics

• Assess mitochondrial function using respirometry, membrane potential, and ROS production

• Challenge models with metabolic stressors to reveal phenotypes (fasting, exercise, temperature)

• Use dietary interventions to modulate phenotype severity

For phenotypic characterization, researchers should examine:

• Fatty acid oxidation capacity using labeled substrate oxidation assays

• Accumulation of intermediate metabolites by mass spectrometry

• Tissue-specific manifestations, particularly in muscle and nerve

• Compensatory mechanisms that may mask phenotypes

• Stress-induced decompensation that mimics clinical triggers

How can contradictory findings about HADHB mutations and clinical manifestations be reconciled?

Researchers face several challenges when reconciling contradictory findings regarding HADHB mutations and their clinical manifestations:

To systematically address these challenges:

• Establish international patient registries with standardized data collection

• Develop consensus guidelines for phenotypic classification

• Implement multi-omics approaches (genomics, transcriptomics, proteomics, metabolomics)

• Create in vitro systems to test variant combinations and environmental conditions

• Design longitudinal studies to track phenotypic evolution over time

The case of three patients with similar mild phenotypes despite different HADHB mutations illustrates the complexity of genotype-phenotype correlations. These patients presented with axonal neuropathy and intermittent weakness episodes without myoglobinuria, challenging the traditional association of MTP deficiency with severe manifestations or rhabdomyolysis .

What emerging techniques can advance our understanding of HADHB's role in health and disease?

Several cutting-edge methodological approaches can advance HADHB research:

Implementation strategies:

• Combine multiple approaches to triangulate findings

• Establish interdisciplinary collaborations between metabolic specialists, neurologists, and basic scientists

• Develop computational models to integrate diverse datasets

• Apply systems biology approaches to understand network-level effects of HADHB dysfunction

• Translate fundamental findings into potential therapeutic strategies

What therapeutic approaches are being developed for HADHB deficiency, and how should their efficacy be evaluated?

Current and emerging therapeutic strategies for HADHB deficiency require rigorous evaluation methodologies:

For clinical trial design, researchers should consider:

• Appropriate control groups and randomization

• Selection of clinically meaningful endpoints

• Biomarkers that reflect disease activity and treatment response

• Long-term follow-up to assess durability of effects

• Patient-reported outcomes to capture quality of life impacts

• Stratification based on genotype and baseline disease severity

What are the highest priority research questions regarding HADHB that remain unanswered?

Several critical knowledge gaps persist in HADHB research that warrant focused investigation:

Methodological considerations for addressing these questions:

• Develop collaborative networks to increase patient numbers

• Standardize phenotypic assessments across centers

• Implement multiple complementary approaches in parallel

• Create biorepositories of patient samples for future analyses

• Utilize advanced computational methods to integrate diverse datasets

How can multi-omics approaches be integrated to advance HADHB research?

Integrating multi-omics approaches provides a comprehensive view of HADHB biology:

Methodological framework for integration:

• Collect matched samples for multiple omics analyses

• Apply standardized protocols for sample preparation and analysis

• Develop computational pipelines for data integration

• Use network analysis to identify key nodes and pathways

• Validate findings in multiple model systems

• Translate integrated insights into testable therapeutic hypotheses

What standardized protocols should be developed for international collaboration in HADHB research?

To facilitate meaningful international collaboration, standardized protocols are essential:

For successful implementation:

• Establish an international HADHB/MTP deficiency consortium

• Secure funding for infrastructure to support collaborative efforts

• Develop data sharing agreements that respect privacy regulations

• Create repositories for reference materials and biological samples

• Organize regular workshops for protocol harmonization and training

• Implement quality control programs to ensure consistent results across sites

Collaborative research studies are particularly needed to determine benefit from early treatment identified through newborn screening, as individual studies have had small sample sizes and inconclusive results regarding complications such as neuropathy and retinopathy .