ETHE1 Human

What is the primary function of ETHE1 in human mitochondria?

ETHE1 (ethylmalonic encephalopathy protein 1) functions as a persulfide dioxygenase in the mitochondrial sulfide oxidation pathway. Its primary role is to convert persulfides to sulfite, which is critical for maintaining appropriate hydrogen sulfide (H₂S) levels in cellular environments . This enzyme participates in a metabolic pathway that effectively detoxifies sulfide, which originates both from normal cellular processes and from bacteria in the gastrointestinal system .

ETHE1 works in concert with other enzymes including sulfide quinone oxidoreductase, rhodanese, and sulfite oxidase to efficiently catabolize H₂S through oxygen-dependent mechanisms . This integrated pathway is essential because while low levels of sulfide are necessary for normal cellular signaling in various systems (nervous, cardiovascular, and gastrointestinal), elevated levels become toxic by interfering with mitochondrial energy production, particularly by inhibiting the cytochrome C oxidase (COX) complex .

How does the structure of ETHE1 relate to its enzymatic function?

ETHE1 functions as a dimer in its active form, comprised of two identical chains (A and B). The protein features distinct α-helices and β-sheets with a critical iron-binding region that serves as the catalytic site (histidine cluster) . This structural arrangement is fundamental to its persulfide dioxygenase activity.

Specific residues play crucial roles in maintaining protein integrity and function. For example, Asp196 forms important interactions with surrounding residues, and mutations at this position (such as D196N or D196H) significantly affect substrate binding affinity . Molecular modeling demonstrates that the D196H mutation creates repulsive interactions with Phe200, potentially destabilizing the protein structure .

The enzyme requires iron for catalytic activity, with studies showing a direct correlation between iron content and enzymatic function. Patient mutations like T152I result in approximately 3-fold lower activity compared to wild-type ETHE1, corresponding with a 3-fold reduction in iron content .

What substrates does ETHE1 utilize in the mitochondrial sulfide oxidation pathway?

ETHE1 primarily utilizes two types of persulfide carriers as substrates:

• Glutathione persulfide (GSSH)

• Coenzyme A persulfide (CoASSH)

Biochemical characterization reveals that these carriers are utilized with different efficacies, suggesting a potential regulatory mechanism based on the availability of different persulfide species . The enzyme converts these persulfides to sulfite using molecular oxygen as a co-substrate, functioning as a true dioxygenase .

Kinetic analysis of ETHE1 has shown that mutations can significantly affect substrate interactions. For instance, the D196N mutation results in a 2-fold higher Km for glutathione persulfide, indicating reduced substrate affinity . This alteration in substrate binding contributes to the enzymatic deficiency observed in patients with ethylmalonic encephalopathy.

What are the most effective methods for assessing ETHE1 enzymatic activity?

Several methodological approaches have proven effective for evaluating ETHE1 enzymatic activity:

Steady-state kinetic analysis: This technique measures ETHE1 activity by monitoring reaction rates under various conditions, typically tracking oxygen consumption or sulfite formation when the enzyme is incubated with persulfide substrates . The approach can determine important parameters such as Km values and catalytic efficiency.

Iron content determination: Since ETHE1 activity correlates directly with iron content, quantifying the iron-to-protein ratio provides valuable information about potential enzyme activity . This can be accomplished using techniques such as inductively coupled plasma mass spectrometry (ICP-MS).

Oxygen consumption measurements: Using oxygen electrodes or optical sensors to measure oxygen consumption rates in the presence of ETHE1 and its substrates provides direct evidence of enzymatic activity .

When evaluating patient mutations, it's essential to compare their activity profiles with wild-type enzyme under identical conditions. For example, biochemical characterization of the T152I mutation revealed 3-fold lower activity correlating with reduced iron content, while the D196N mutation showed altered substrate binding with a 2-fold higher Km for glutathione persulfide .

What genetic sequencing approaches are recommended for ETHE1 mutation analysis?

The following sequencing approaches have demonstrated effectiveness for ETHE1 mutation analysis:

Whole-exome sequencing (WES): This comprehensive approach can identify various types of ETHE1 mutations, including missense variants, splicing mutations, insertions/deletions, and exon deletions . WES has successfully identified novel variants in patients with suspected ethylmalonic encephalopathy.

Targeted gene sequencing: For cases with clinical and biochemical findings suggestive of ethylmalonic encephalopathy, focused sequencing of the ETHE1 gene provides a cost-effective diagnostic approach .

Sanger sequencing validation: This method remains valuable for confirming variants identified through next-generation sequencing and for familial segregation analysis to determine the parental origin of variants .

A comprehensive bioinformatic analysis pipeline should include:

• Alignment to the human reference genome

• Filtering of variants using tools like GATK

• Annotation based on population databases (1000 Genomes, ExAC, gnomAD)

• Pathogenicity prediction using computational tools (SIFT, Provean, PolyPhen-2, Mutation Taster)

• Splice site prediction analysis for variants near exon-intron boundaries

Interpretation of variants should follow ACMG guidelines, with particular attention to the frequency of variants in population databases, computational predictions, and functional studies when available .

What controls should be included when analyzing the effects of ETHE1 mutations?

When designing experiments to analyze ETHE1 mutations, several controls are essential:

Wild-type ETHE1 reference: Always include wild-type protein processed identically to mutant samples as the primary reference point . For biochemical assays, use equivalent protein amounts based on careful quantification.

Known mutation controls: When characterizing novel mutations, include previously studied mutations with established effects (e.g., T152I, D196N) as comparative references .

Enzyme activity controls:

• Negative controls lacking substrate or enzyme

• Iron chelation controls to establish baseline activity for this iron-dependent enzyme

• Multiple substrate concentrations to distinguish between effects on Vmax and Km

Expression and localization verification:

• Confirm equivalent expression levels of wild-type and mutant proteins

• Verify proper mitochondrial localization of ETHE1 variants

Functional readouts:

• Include measurements of hydrogen sulfide levels

• Assess markers of mitochondrial function (e.g., cytochrome c oxidase activity)

• Measure downstream biochemical parameters like ethylmalonic acid and C4-acylcarnitine levels

What is the spectrum of ETHE1 mutations in ethylmalonic encephalopathy?

Ethylmalonic encephalopathy (EE) is caused by various types of mutations in the ETHE1 gene. The spectrum includes:

• Missense mutations: 19 different missense variants have been identified, affecting various domains of the protein . These often impact key functional residues, such as those involved in iron binding or substrate recognition.

• Insertions and deletions: 8 different insertions/deletions have been documented, frequently leading to frameshifts and premature termination .

• Splicing variants: 5 splicing variants have been identified, including 3 at canonical splicing sites such as c.595+1G>T, which likely result in aberrant splicing and nonfunctional protein products .

• Exon deletions: 4 different exon deletion variants have been reported, with exon 4 involved in all cases, highlighting this region's critical importance for protein function .

Analysis of mutation distribution reveals that exon 4 appears to be a hotspot, containing 9 different reported variants and involved in all documented exon deletions . This suggests that the structural and functional elements encoded by exon 4 are particularly important for ETHE1 function.

The identification of compound heterozygous variants, where patients carry two different mutations (one from each parent), is common. For example, one case study reported a patient with the novel variants c.595+1G>T (paternal origin) and c.586G>C/p.D196H (maternal origin) .

How do ETHE1 mutations correlate with clinical manifestations in ethylmalonic encephalopathy?

The correlation between specific ETHE1 mutations and clinical severity shows some patterns, though with considerable variability:

Complete loss-of-function mutations typically lead to more severe presentations. These include splice site mutations at canonical sites (like c.595+1G>T), frameshift mutations leading to premature stop codons, and large deletions involving critical exons .

Missense mutations show variable effects depending on:

• Their location within the protein structure

• The degree of conservation of the affected residue

• The biochemical impact of the amino acid substitution

For example, mutations affecting the iron-binding site (like T152I) or residues involved in substrate binding (like D196N) result in specific enzymatic deficiencies that contribute to disease manifestations .

Clinical manifestations across patients include:

• Psychomotor delay (present in ~93% of cases)

• Chronic diarrhea (67%)

• Hypotonia (73%)

• Elevated serum lactic acid (80%)

• Petechiae (53%)

• Acrocyanosis (40%)

• Pyramidal signs (40%)

• Seizures (40%)

Notably, some patients present with milder symptoms. One documented case with novel compound heterozygous variants showed only chronic diarrhea without the typical vascular manifestations (petechiae and acrocyanosis) or neurological features (hypotonia) .

What biochemical markers are most reliable for monitoring ETHE1 dysfunction?

Several biochemical markers have proven valuable for monitoring ETHE1 dysfunction:

Elevated urinary ethylmalonic acid (EMA): This marker is consistently present in all documented cases (100%) and serves as a primary diagnostic indicator .

Elevated C4-acylcarnitine esters: Also present in all reported cases (100%), this marker can be detected through acylcarnitine profiling in blood samples .

Methylsuccinic acid (MSA): Increased urinary excretion of MSA is characteristic of ethylmalonic encephalopathy .

Lactic acidosis: Elevated serum lactic acid is observed in approximately 80% of patients and reflects mitochondrial dysfunction resulting from inhibition of cytochrome c oxidase .

Plasma thiosulfate: Elevated levels result from impaired metabolism of hydrogen sulfide in the mitochondrial sulfide oxidation pathway .

These biochemical abnormalities stem from hydrogen sulfide accumulation, which inhibits cytochrome c oxidase and short-chain acyl-CoA dehydrogenase activities . The pattern of metabolic derangements provides a biochemical signature that can be used to monitor disease progression and potentially evaluate therapeutic interventions.

How does ETHE1 dysfunction affect mitochondrial energy metabolism?

ETHE1 dysfunction affects mitochondrial energy metabolism through several interrelated mechanisms:

Hydrogen sulfide accumulation: When ETHE1 function is impaired, hydrogen sulfide (H₂S) and its derivatives accumulate to toxic levels . While H₂S is an important signaling molecule at low concentrations, excessive levels become detrimental to cellular functions.

Inhibition of cytochrome c oxidase (COX): Elevated H₂S directly inhibits COX (Complex IV of the electron transport chain), which carries out one of the final steps in mitochondrial energy production . This inhibition disrupts the electron transport chain and impairs ATP synthesis.

Altered redox homeostasis: The dysfunction in the sulfide oxidation pathway leads to changes in cellular redox state, affecting numerous redox-sensitive proteins and processes within mitochondria.

Secondary inhibition of short-chain acyl-CoA dehydrogenase: H₂S accumulation inhibits this enzyme involved in fatty acid oxidation, leading to the characteristic pattern of elevated ethylmalonic acid and C4-/C5-acylcarnitine esters .

Tissue-specific effects: The impact of ETHE1 dysfunction varies across tissues, with high-energy-demanding tissues such as brain, skeletal muscle, and vascular endothelium showing particular sensitivity . This explains the predominant neurological, muscular, and vascular manifestations of ethylmalonic encephalopathy.

These mechanisms collectively contribute to energetic failure in affected tissues, underlying the progressive multi-system disease observed in patients with ETHE1 mutations.

What molecular interactions occur between ETHE1 and its persulfide substrates?

The molecular interactions between ETHE1 and its persulfide substrates involve several specific features:

Substrate specificity: ETHE1 utilizes both glutathione persulfide (GSSH) and coenzyme A persulfide (CoASSH) as substrates, but with different efficacies . This suggests distinct binding interactions for each substrate type.

Key binding residues: Specific residues in ETHE1 are critical for substrate recognition and binding. Asp196 plays a particularly important role, as evidenced by the impact of the D196N mutation, which results in a 2-fold higher Km for glutathione persulfide, indicating reduced substrate affinity .

Active site configuration: The active site contains an iron center coordinated by histidine residues (the catalytic histidine cluster) . This iron center is essential for the dioxygenase reaction, binding both the persulfide substrate and molecular oxygen.

Dimeric structure importance: The dimeric conformation of ETHE1 likely creates the optimal arrangement of the active site for efficient substrate binding and catalysis .

Mutations affecting these interactions can significantly impair enzyme function through different mechanisms:

Understanding these molecular interactions provides insight into the catalytic mechanism of ETHE1 and the pathogenic effects of specific mutations.

How can researchers distinguish primary effects of ETHE1 dysfunction from secondary cellular responses?

Distinguishing primary effects of ETHE1 dysfunction from secondary cellular responses requires strategic experimental approaches:

Time-course analyses: Examining the temporal sequence of events following ETHE1 inhibition or knockdown helps identify early changes (likely primary effects) versus later adaptations (secondary responses).

Direct biochemical consequences: Immediate biochemical changes directly linked to ETHE1's enzymatic function include:

• Increased persulfide levels

• Reduced sulfite formation

• Elevated hydrogen sulfide concentration
  These represent primary effects of ETHE1 dysfunction .

Targeted metabolomics: Focused analysis of sulfide metabolism intermediates can track the direct metabolic consequences of ETHE1 deficiency, distinguishing them from broader metabolic adaptations.

Complementation studies: Reintroducing functional ETHE1 into deficient cells and observing which abnormalities are rapidly reversed helps identify direct consequences of ETHE1 deficiency.

Comparative analysis across models: Comparing findings from different experimental models (e.g., patient cells, CRISPR knockout lines, animal models) can help distinguish consistent primary effects from model-specific secondary responses.

Multi-omics integration: Integrating transcriptomic, proteomic, and metabolomic data with pathway analysis can help differentiate direct consequences from compensatory changes in gene expression and protein function.

This methodical approach enables researchers to build a more accurate model of the direct pathophysiological mechanisms resulting from ETHE1 dysfunction, potentially identifying more effective therapeutic targets.

What experimental approaches could address challenges in ETHE1 protein expression and purification?

Researchers face several challenges when expressing and purifying functional ETHE1 protein. These challenges and potential solutions include:

Iron incorporation challenges:

• Problem: ETHE1 requires iron for activity, but expression systems may not efficiently incorporate iron, leading to heterogeneous preparations .

• Solutions:

  • Supplement growth media with iron during expression

  • Develop iron reconstitution protocols during purification

  • Quantify iron content and normalize activity measurements accordingly

Preserving dimeric structure:

• Problem: The active form of ETHE1 is dimeric, and purification conditions may disrupt this quaternary structure .

• Solutions:

  • Optimize gentle purification conditions

  • Use size exclusion chromatography to isolate the dimeric fraction

  • Verify dimeric state using native PAGE or analytical ultracentrifugation

Substrate stability issues:

• Problem: ETHE1 substrates (GSSH, CoASSH) are not commercially available and must be synthesized; these persulfides are unstable .

• Solutions:

  • Develop reliable methods for substrate preparation

  • Establish protocols for real-time substrate quantification

  • Consider enzyme-coupled assays that generate persulfide substrates in situ

Protein stability concerns:

• Problem: Purified ETHE1 may have limited stability under laboratory conditions.

• Solutions:

  • Screen buffer conditions systematically to optimize stability

  • Add stabilizing agents during purification and storage

  • Consider fusion protein approaches to enhance stability

Activity assessment:

• Problem: Developing reliable quantitative assays for ETHE1 activity presents technical challenges.

• Solutions:

  • Employ multiple complementary methods (oxygen consumption, sulfite production)

  • Utilize coupled enzyme assays to amplify signal

  • Develop fluorescent or colorimetric high-throughput assays

These experimental approaches would significantly advance the biochemical characterization of ETHE1 and facilitate more detailed structure-function studies of both wild-type and mutant variants.

What are the most promising therapeutic approaches for ETHE1-related disorders?

While there is currently no definitive treatment for ethylmalonic encephalopathy, several therapeutic approaches show promise based on our understanding of ETHE1 function:

Targeting hydrogen sulfide accumulation:

• H₂S scavengers to reduce toxic accumulation

• Metronidazole and N-acetylcysteine combination therapy to reduce bacterial H₂S production and promote its detoxification

• Strategies to enhance alternative H₂S detoxification pathways

Enzyme replacement therapy (ERT):

• Development of recombinant ETHE1 with appropriate targeting to mitochondria

• Investigation of delivery methods to cross the blood-brain barrier

• Optimization of enzyme stability and activity in vivo

Gene therapy approaches:

• Adeno-associated virus (AAV) vectors for ETHE1 gene delivery

• Targeted delivery to most affected tissues (brain, muscle, endothelium)

• CRISPR-based approaches to correct specific mutations

Mitochondrial function support:

• Coenzyme Q10 and other mitochondrial cofactors to support electron transport chain function

• Antioxidants to mitigate oxidative stress resulting from mitochondrial dysfunction

• Metabolic modifiers to enhance ATP production through alternative pathways

Protein stabilization strategies:

• For missense mutations that affect protein stability rather than catalytic function

• Small molecule chaperones that could stabilize mutant ETHE1 proteins

• Proteasome inhibitors to reduce degradation of partially functional mutant proteins

The development of these therapeutic approaches requires robust preclinical models and biomarkers to assess efficacy, combining the molecular understanding of ETHE1 function with innovative drug delivery and gene therapy technologies.

How might emerging technologies advance ETHE1 research in the next decade?

Several emerging technologies are likely to significantly advance ETHE1 research in the coming decade:

Cryo-electron microscopy (Cryo-EM):

• Higher-resolution structures of ETHE1 in complex with substrates and potential drug candidates

• Visualization of conformational changes during catalysis

• Structural insights into the effects of disease-causing mutations

CRISPR-based technologies:

• Generation of precise disease models with specific patient mutations

• Base editing to correct point mutations without double-strand breaks

• High-throughput screening of genetic modifiers of ETHE1 function

Single-cell omics:

• Cell-type-specific responses to ETHE1 dysfunction

• Identification of particularly vulnerable cell populations

• Heterogeneity in response to potential therapeutic interventions

Organoid and organ-on-chip technologies:

• Complex multicellular models that better recapitulate tissue-specific pathophysiology

• Systems to model interactions between different tissues affected in ethylmalonic encephalopathy

• Platforms for personalized drug screening using patient-derived cells

Advanced metabolic flux analysis:

• Real-time monitoring of hydrogen sulfide metabolism in living cells

• Quantitative assessment of metabolic consequences of ETHE1 dysfunction

• Integration with computational models of mitochondrial metabolism

Mitochondrial-targeted therapeutics:

• Novel delivery systems specifically targeting mitochondria

• Mitochondrial-targeted gene editing technologies

• Pharmacological modulators of mitochondrial function with enhanced specificity

These technologies will likely provide deeper insights into ETHE1 function and dysfunction, facilitate more accurate disease modeling, and accelerate the development of therapeutic strategies for patients with ethylmalonic encephalopathy.