Recombinant Danio rerio Lipoyl synthase, mitochondrial (lias)

What is the basic function of Lipoyl Synthase (LIAS) in zebrafish mitochondria?

Zebrafish LIAS, like its human counterpart, is an iron-sulfur cluster enzyme that catalyzes the insertion of two sulfur atoms into protein-bound octanoyl substrates to form lipoyl cofactors. This reaction is essential for the function of several key metabolic enzyme complexes. LIAS contains two [4Fe-4S] clusters: a reducing cluster that serves as an electron source and an auxiliary cluster that provides the sulfur atoms for insertion reactions . The enzyme requires two equivalents of S-adenosylmethionine (SAM) to produce radical species that facilitate the consecutive sulfur insertions at the sixth and eighth carbon positions of the octanoyl substrate . This essential process supports mitochondrial metabolism, and disruption can lead to metabolic disorders.

How conserved is zebrafish LIAS compared to human LIAS?

While the search results don't provide specific sequence comparison data for zebrafish LIAS, structural and functional conservation across species is typically high for fundamental metabolic enzymes. The human LIAS protein contains characteristic iron-sulfur cluster binding motifs, including a 3-C-X2-C motif for the reducing [4Fe-4S] cluster and a C-X4-C-X5-C motif for the auxiliary cluster . These motifs are generally conserved in lipoyl synthases across species. The functional significance of this conservation is demonstrated by the fact that an R249H point mutation in human LIAS causes severe disease, including neonatal epilepsy and muscular hypotonia . Researchers working with zebrafish LIAS should consider these conserved domains when designing experimental approaches.

What are the critical structural features of recombinant LIAS that must be preserved during expression?

The most critical features of recombinant LIAS are its two [4Fe-4S] clusters, which are essential for catalytic activity. The reducing cluster provides electrons for radical generation, while the auxiliary cluster serves as the source of sulfur atoms that are inserted into the octanoyl substrate . When expressing recombinant LIAS, researchers must ensure that:

• The iron-sulfur cluster binding motifs remain intact during cloning

• Appropriate reconstitution methods are used to incorporate the [4Fe-4S] clusters

• Special handling procedures are implemented to prevent oxidative damage to the clusters

For human LIAS, researchers have successfully expressed a recombinant form lacking the mitochondrial targeting sequence (residues 28-372) . A similar approach would likely be suitable for zebrafish LIAS, adjusting for species-specific mitochondrial targeting sequences.

What expression systems are most effective for producing active recombinant zebrafish LIAS?

Based on successful approaches with human LIAS, E. coli expression systems are likely effective for zebrafish LIAS production. When expressing iron-sulfur proteins like LIAS, consider the following methodological approaches:

• Clone the zebrafish LIAS gene without its mitochondrial targeting sequence to improve solubility

• Use specialized E. coli strains that enhance iron-sulfur protein expression

• Optimize growth conditions including temperature, induction timing, and media composition

• Consider co-expression with iron-sulfur cluster assembly proteins

The human LIAS studies demonstrated successful expression and purification of recombinant LIAS (lacking the mitochondrial targeting sequence) . This provides a framework that can be adapted for zebrafish LIAS, though species-specific optimizations may be necessary.

How should iron-sulfur clusters be reconstituted in recombinant zebrafish LIAS?

Iron-sulfur cluster reconstitution is a critical step for obtaining catalytically active LIAS. Based on human LIAS studies, researchers should consider the following methodological approach:

• Purify the apoprotein under anaerobic conditions to prevent oxidative damage

• Use iron-sulfur cluster donor proteins such as ISCU or ISCA2 for physiologically relevant reconstitution

• Verify successful reconstitution through activity assays and spectroscopic methods

Research with human LIAS has shown that both ISCU and ISCA2 (both full-length and truncated versions) can successfully reconstitute LIAS activity . Specifically, ISCU cluster delivery resulted in the highest LIAS activity within 120 minutes of formation time, followed by full-length ISCA2 (95 ± 10% relative activity) . These findings provide a framework for zebrafish LIAS reconstitution approaches.

What purification challenges are specific to zebrafish LIAS, and how can they be addressed?

While the search results don't address zebrafish LIAS purification specifically, iron-sulfur proteins share common purification challenges:

• Oxygen sensitivity: All steps should be performed under anaerobic conditions

• Cluster stability: Buffer composition (pH, salt concentration, reducing agents) must be optimized

• Protein solubility: Detergents or solubilizing agents may be needed without disrupting structure

Researchers should adapt purification protocols used for human LIAS, which has been successfully purified in a recombinant form . Preliminary small-scale purifications with different buffer conditions can help identify optimal conditions for zebrafish LIAS stability and activity.

What methods are most reliable for measuring zebrafish LIAS enzymatic activity?

LC-MS activity assays provide the most direct and quantitative measurement of LIAS activity. For zebrafish LIAS, researchers can adapt the approach used for human LIAS:

• Incubate reconstituted LIAS with octanoyl-containing substrate, SAM, and necessary cofactors

• Detect and quantify the lipoylated product formation using LC-MS

• Include appropriate controls to verify that product formation is dependent on active, reconstituted LIAS

Studies with human LIAS have successfully used LC-MS to detect double sulfur insertion into octanoyl-containing substrates, with product peaks identified and quantified . This methodology should be adaptable for zebrafish LIAS with appropriate substrate considerations.

How can researchers distinguish between partial and complete reconstitution of both [4Fe-4S] clusters?

Distinguishing between partial and complete reconstitution requires multiple analytical approaches:

• UV-visible spectroscopy: Characteristic absorbance features of [4Fe-4S] clusters

• EPR spectroscopy: Analysis of cluster electronic states

• Activity assays: Full activity requires both clusters to be intact

• Iron and sulfur quantification: Stoichiometric analysis to confirm cluster composition

When analyzing reconstitution success, researchers should compare activity levels achieved with different iron-sulfur cluster donor proteins. For human LIAS, ISCU was found to be the most effective donor, followed by full-length ISCA2 . These comparative approaches can help determine optimal reconstitution conditions for zebrafish LIAS.

What controls are essential when assessing recombinant zebrafish LIAS activity?

Essential controls for zebrafish LIAS activity assays include:

• Negative control: Reaction without reconstituted LIAS to confirm substrate stability

• Apoprotein control: Non-reconstituted LIAS to verify the necessity of iron-sulfur clusters

• SAM-free control: To demonstrate SAM requirement for radical generation

• Time-course analysis: To establish linear reaction rates and enzyme stability

These controls help distinguish authentic LIAS activity from non-enzymatic reactions. Human LIAS studies incorporated control experiments that verified product formation was only facilitated by active, reconstituted LIAS , providing a template for zebrafish LIAS experimental design.

Why is zebrafish a valuable model organism for studying LIAS function and related metabolic disorders?

Zebrafish offers numerous advantages for LIAS research:

• Genetic manipulability: Ease of creating transgenic and knockout lines

• Optical transparency: Enables in vivo visualization of metabolic processes

• Rapid development: Accelerates experimental timelines

• Similarities to human metabolism: Provides translational relevance

Zebrafish demonstrates striking similarities to humans in redox signaling, antioxidant enzymes, and lipid metabolism pathways . These similarities make zebrafish particularly valuable for studying the role of LIAS in oxidative stress responses and metabolic regulation. Additionally, zebrafish embryos can be generated in large numbers and respond robustly to oxidative stress, making them ideal for high-throughput studies of LIAS function .

How can transgenic zebrafish models be used to study LIAS function in oxidative stress responses?

Transgenic zebrafish expressing fluorescent oxidative stress sensors provide powerful tools for studying LIAS function:

• H₂O₂ reporter zebrafish: Transgenic lines expressing the HyPer probe can visualize H₂O₂ gradients in vivo

• Oxidized lipid imaging: Transgenic fish expressing GFP-tagged antibodies to modified lipids enable visualization of oxidative damage

• LIAS mutant or knockdown models: Can be combined with oxidative stress reporters to assess metabolic consequences

The HyPer probe, consisting of circularly permuted YFP inserted in the H₂O₂-sensing regulatory domain of OxyR, shows spectral shifts in response to oxidation/reduction, allowing real-time monitoring of oxidative stress . This approach could be valuable for assessing how LIAS dysfunction affects cellular redox status.

What methodological approaches can be used to study LIAS function in zebrafish embryo development?

To study LIAS function during zebrafish development, researchers can employ:

• Morpholino knockdown or CRISPR/Cas9 knockout of LIAS

• Controlled exposure periods using the fish embryo acute toxicity test (FET) framework

• Histological evaluation of tissues dependent on lipoic acid cofactors

• Temporal analysis of LIAS expression and activity during development

Zebrafish embryos can be maintained under controlled conditions in 24-well plates with regular media changes, following protocols similar to the OECD TG 236 fish embryo acute toxicity test . This standardized approach enables consistent assessment of developmental phenotypes resulting from LIAS manipulation.

How does lipid metabolism in zebrafish relate to LIAS function, and what analytical techniques are applicable?

Zebrafish lipid metabolism shares important similarities with humans, making it relevant for LIAS studies:

• Lipoprotein profiles: When fed high-cholesterol diets, zebrafish develop lipoprotein profiles similar to humans

• Lipid transport: Zebrafish express homologs of key human apolipoproteins

• Analytical techniques: Advanced lipidomics methods used in zebrafish studies can be applied to LIAS research

MS/MS ALL™ infusion methods allow automated collection of TOF MS and TOF MS/MS data of all lipids, using step-wise sampling of precursor ions . This approach enables comprehensive lipidomic profiling that can reveal how LIAS dysfunction affects lipid metabolism. In vitamin E-deficient zebrafish brains, this technique identified alterations in docosahexaenoic acid (DHA)-containing phospholipids and increased hydroxy-DHA-phosphatidylcholines, indicating lipid peroxidation . Similar approaches could reveal metabolic consequences of LIAS deficiency.

What are the most common issues when expressing recombinant zebrafish LIAS, and how can they be resolved?

Common expression issues and solutions include:

• Poor solubility: Optimize expression temperature (typically lower temperatures improve solubility), adjust induction conditions, or use solubility-enhancing fusion tags

• Inactive protein: Ensure anaerobic conditions during purification, verify iron-sulfur cluster incorporation

• Low yield: Optimize codon usage for E. coli, test different media formulations, or scale up culture volume

When working with iron-sulfur proteins like LIAS, expression challenges often relate to proper cluster assembly. Co-expression with iron-sulfur cluster assembly proteins may improve yield of active protein, as demonstrated in studies with human iron-sulfur proteins .

How can researchers address inconsistent activity in reconstituted zebrafish LIAS preparations?

Inconsistent activity may result from several factors:

• Incomplete cluster reconstitution: Optimize reconstitution conditions including iron source, sulfide concentration, and incubation time

• Cluster degradation: Ensure strict anaerobic conditions and include appropriate reducing agents

• Substrate limitations: Verify substrate purity and stability

• Enzyme instability: Optimize buffer conditions to maintain protein stability

Comparative testing of different iron-sulfur cluster donor proteins (ISCU, ISCA2) can help identify optimal reconstitution approaches, as demonstrated in human LIAS studies . Additionally, activity assays should be designed to verify that product formation is specifically dependent on reconstituted LIAS .

What methodological precautions are necessary when analyzing zebrafish tissues for LIAS activity?

When analyzing zebrafish tissues for LIAS activity:

• Rapid sample processing: Minimize time between tissue collection and analysis to prevent degradation

• Appropriate homogenization: Use gentle methods that preserve enzyme activity

• Subcellular fractionation: Isolate mitochondria for enriched LIAS activity assessment

• Careful normalization: Account for differences in mitochondrial content between samples

For zebrafish embryo experiments, standardized protocols such as those used in toxicity testing provide frameworks for consistent sample collection and processing . Consider using protocols that maintain sample viability at 26.0 ± 1.0°C under controlled light/dark cycles .

How can zebrafish LIAS research contribute to understanding human metabolic disorders?

Zebrafish LIAS studies can provide insights into human disorders through:

• Modeling disease mutations: The R249H mutation in human LIAS causes severe disease ; equivalent mutations can be introduced in zebrafish

• High-throughput screening: Zebrafish embryos enable screening of compounds that might rescue LIAS deficiency

• Mechanistic studies: Transparent zebrafish allow visualization of metabolic consequences of LIAS dysfunction

• Developmental impact: Assessing how LIAS deficiency affects early development may provide insights into congenital disorders

The translational value of zebrafish is enhanced by similarities in lipid metabolism and oxidative stress responses between zebrafish and humans . Zebrafish respond to many pathological factors with robust oxidative stress, making them excellent models for studying how LIAS dysfunction affects cellular metabolism .

What advanced analytical techniques are being developed for studying iron-sulfur cluster dynamics in enzymes like LIAS?

Cutting-edge techniques for studying iron-sulfur clusters include:

• Time-resolved spectroscopy: Capturing cluster assembly and degradation kinetics

• Cryo-electron microscopy: Visualizing cluster environments at near-atomic resolution

• Advanced mass spectrometry: Identifying post-translational modifications and protein interactions

• Hydrogen-deuterium exchange mass spectrometry: Probing conformational changes during catalysis

While not specific to zebrafish LIAS, these techniques can be applied to understand fundamental aspects of LIAS function. Current LC-MS methods used for human LIAS activity assays and the comprehensive lipidomic analyses demonstrated in zebrafish brain studies provide frameworks for these advanced applications.

How might the mechanisms of iron-sulfur cluster transfer to LIAS differ between zebrafish and human systems?

This question explores potential species-specific differences in iron-sulfur cluster biogenesis:

• Donor protein preferences: Studies with human LIAS identified both ISCU and ISCA2 as effective cluster donors , but zebrafish may show different preferences

• Transfer efficiency: Kinetics of cluster transfer may vary between species due to structural differences

• Regulatory mechanisms: Control of iron-sulfur cluster biogenesis may have species-specific elements

• Subcellular localization: Differences in mitochondrial organization may affect cluster transfer

While direct comparisons between zebrafish and human LIAS cluster transfer mechanisms are not available in the search results, the established methodologies for human LIAS reconstitution provide a foundation for investigating these potential differences .