Recombinant Danio rerio Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial (sdha), partial

What is Recombinant Danio rerio SDHA and what are its key characteristics?

Recombinant Danio rerio SDHA is a laboratory-produced version of the flavoprotein subunit of succinate dehydrogenase from zebrafish. This protein represents the catalytic subunit of Complex II in the mitochondrial respiratory chain. The recombinant form of this protein is typically expressed in heterologous systems such as E. coli, yeast, baculovirus, or mammalian cell expression systems, with a purity standard of ≥85% as determined by SDS-PAGE .

The protein is characterized by several gene names including sdha, zgc:56051, and im:7141001, and is alternatively known as "succinate dehydrogenase; succinate dehydrogenase complex; subunit A; flavoprotein (Fp)" . As a partial recombinant protein, it contains key functional domains but may not represent the complete native protein sequence. The recombinant form allows researchers to study specific aspects of SDHA function outside the context of the complete respiratory chain complex.

Why is zebrafish an appropriate model organism for studying SDHA function?

Zebrafish (Danio rerio) represents an excellent model organism for studying SDHA function for several compelling reasons. First, zebrafish share approximately 70% of their genes with humans, including significant conservation of metabolic pathways and mitochondrial structure and function . Second, zebrafish possess all major vertebrate organs including brain, heart, liver, and kidneys, allowing for systemic studies of mitochondrial function .

The methodological approach to using zebrafish for SDHA research benefits from several practical advantages. Their rapid development (most major organs form within 24 hours) allows for expedited experimental timelines compared to mammalian models . Additionally, zebrafish embryos are transparent, permitting real-time visualization of developmental processes and protein expression using fluorescent tags without invasive procedures . Their high reproductive rate (up to 300 embryos every 2-3 days) and external fertilization enable high-throughput screening approaches and easy genetic manipulation of embryos .

What are the common applications of Recombinant Danio rerio SDHA in research?

Recombinant Danio rerio SDHA serves multiple research applications in the study of mitochondrial biology and disease mechanisms. For enzymatic activity assays, researchers can use the recombinant protein to establish baseline kinetic parameters and conduct comparative analyses with mutant variants. Antibody production and validation represent another key application, as the recombinant protein can serve as an antigen for generating specific antibodies or as a positive control in immunoassays.

In protein-protein interaction studies, the recombinant protein can be used to identify binding partners through techniques such as pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens. For structural biology, the purified recombinant protein provides material for crystallography or cryo-EM studies to determine three-dimensional structure. Additionally, when creating disease models, researchers can compare wild-type recombinant SDHA with disease-associated variants to examine functional differences in enzyme activity, stability, or interaction profiles.

How is Recombinant Danio rerio SDHA expressed and purified for research use?

The expression and purification of Recombinant Danio rerio SDHA follows a methodical process designed to maximize yield and maintain functional integrity. The general methodology involves:

• Expression system selection: Most commonly, E. coli expression systems are employed due to their cost-effectiveness and scalability, though yeast, baculovirus, and mammalian cell systems may be used for specific applications requiring eukaryotic post-translational modifications .

• Vector construction: The sdha gene sequence (potentially using gene names zgc:56051 or im:7141001) is cloned into an appropriate expression vector containing an inducible promoter and affinity tag (typically His-tag or GST-tag) .

• Transformation and expression: The construct is introduced into the host organism, followed by culture expansion and protein expression induction under optimized conditions.

• Cell lysis and initial purification: Cells are harvested and lysed, followed by clarification of the lysate through centrifugation and/or filtration.

• Affinity chromatography: The tagged protein is captured using an appropriate affinity resin (e.g., Ni-NTA for His-tagged proteins).

• Quality control: The purified protein undergoes SDS-PAGE analysis to confirm purity (≥85% is standard) , along with functional assays to verify enzymatic activity.

The resulting product typically contains the partial SDHA protein with preserved functional domains suitable for research applications.

What are the key structural features of Danio rerio SDHA and how do they compare to human SDHA?

Danio rerio SDHA shares significant structural homology with human SDHA, reflecting the evolutionary conservation of this crucial mitochondrial enzyme. Key structural features include:

• FAD binding domain: The N-terminal region contains the FAD binding domain, which is critical for the covalent attachment of the FAD cofactor essential for the enzyme's redox activity . This domain contains the conserved RGxxE motif found in bacterial FAD assembly factors, indicating evolutionary conservation of this critical functional element .

• Substrate binding site: The protein contains a specialized pocket for succinate binding, structurally designed to facilitate the oxidation of succinate to fumarate.

• Interaction domains: Specific regions mediate the interaction with other SDH complex subunits (SDHB, SDHC, and SDHD) to form the complete Complex II of the respiratory chain.

• Mitochondrial targeting sequence: A N-terminal sequence that directs the protein to the mitochondria, though this may be absent in some recombinant constructs.

The zebrafish SDHA protein maintains approximately 70-75% sequence identity with human SDHA, with higher conservation in the catalytic and cofactor binding regions, making it an informative model for human SDHA function and dysfunction studies.

How do post-translational modifications affect the activity of recombinant Danio rerio SDHA?

Post-translational modifications (PTMs) significantly impact the activity, stability, and interactions of Danio rerio SDHA. The most critical PTM for SDHA function is the covalent attachment of the FAD cofactor to the protein backbone. This process involves the conserved RGxxE motif, which plays a crucial role in FAD assembly and attachment . Without proper FAD incorporation, the enzyme remains catalytically inactive, highlighting the essential nature of this modification.

For researchers investigating SDHA function, several methodological considerations arise:

• Expression system selection: Different expression systems (prokaryotic vs. eukaryotic) vary in their ability to perform PTMs. While E. coli-expressed recombinant SDHA may lack certain modifications, proteins expressed in yeast, insect, or mammalian cells may retain more native-like modifications.

• In vitro modification: Researchers can sometimes perform in vitro flavinylation reactions to incorporate FAD into recombinant SDHA that lacks this modification.

• PTM detection methods: Mass spectrometry represents the gold standard for identifying and characterizing PTMs on recombinant proteins. Specific antibodies against modified forms may also be employed.

• Functional comparisons: Activity assays comparing differentially modified forms of SDHA can reveal the functional significance of specific PTMs.

Additional PTMs such as phosphorylation, acetylation, and ubiquitination may regulate SDHA activity in response to cellular conditions, offering important research directions for understanding dynamic regulation of mitochondrial function.

What are the optimal experimental conditions for assessing Danio rerio SDHA enzymatic activity?

Accurate assessment of Danio rerio SDHA enzymatic activity requires careful optimization of experimental conditions. The methodological approach should consider:

• Buffer composition:

  • pH: Optimal activity typically occurs at pH 7.2-7.8

  • Ionic strength: 50-150 mM potassium phosphate buffer is commonly used

  • Additives: 0.1-0.5 mM EDTA to chelate inhibitory metal ions

• Substrate concentration: Titration of succinate (typically 0.1-10 mM) allows determination of kinetic parameters (Km, Vmax).

• Electron acceptors:

  • For isolated SDHA: Artificial electron acceptors like 2,6-dichlorophenolindophenol (DCIP) or ferricyanide

  • For Complex II assays: Ubiquinone (CoQ₁₀ or CoQ₁) at 50-100 μM

• Temperature control: 25-30°C is standard for zebrafish enzymes, reflecting their poikilothermic nature.

• Detection methods:

  • Spectrophotometric monitoring of DCIP reduction at 600 nm

  • Oxygen consumption measurements using respirometry

  • Coupled assays tracking downstream electron transfer

• Controls:

  • Specific inhibitors (malonate, 3-nitropropionic acid) to confirm SDHA-specific activity

  • Heat-inactivated enzyme as negative control

  • Purified mammalian SDHA as reference standard

The assay should be optimized to ensure linearity with respect to time and enzyme concentration, and measurements should be made within the initial velocity period to accurately reflect enzymatic parameters.

How can researchers troubleshoot inconsistent results with recombinant Danio rerio SDHA assays?

Inconsistent results with recombinant Danio rerio SDHA assays can stem from multiple sources. A systematic troubleshooting approach should address:

• Protein quality issues:

  • Verify protein integrity via SDS-PAGE and Western blotting

  • Assess aggregation state through size exclusion chromatography or dynamic light scattering

  • Confirm FAD incorporation by measuring the A450/A280 ratio (typically 0.10-0.12 for fully flavinated SDHA)

  • Consider fresh preparation or alternative storage conditions if degradation is observed

• Assay component variables:

  • Prepare fresh reagents and substrates

  • Validate electron acceptor quality (e.g., DCIP, PMS, ubiquinone)

  • Control for interfering substances in the reaction mixture

  • Verify instrument calibration for spectrophotometric or oxygen consumption measurements

• Environmental factors:

  • Maintain consistent temperature throughout experiments

  • Control exposure to light for photosensitive components

  • Eliminate oxidizing or reducing contaminants

• Methodological strategies:

  • Implement internal standards in each experiment

  • Perform parallel assays with commercial reference enzymes

  • Develop a standard operating procedure with detailed specifications

  • Consider alternative assay formats if particular methods consistently fail

• Data analysis approaches:

  • Apply appropriate statistical methods to identify outliers

  • Normalize activity to protein concentration determined by multiple methods

  • Account for background rates in calculation of specific activity

Systematic documentation of variables and outcomes during troubleshooting enables identification of the critical factors affecting reproducibility in specific experimental setups.

What are the key considerations when comparing SDHA activity across different species?

• Structural and sequence differences:

  • Perform phylogenetic analysis and multiple sequence alignment to identify conserved domains versus variable regions

  • Consider the impact of sequence variations on catalytic sites and substrate binding

  • Examine differences in protein size, cofactor binding motifs, and regulatory regions

• Physiological context:

  • Account for native temperature ranges (e.g., 28°C for zebrafish vs. 37°C for mammals)

  • Consider metabolic rate differences across species (e.g., poikilotherms vs. homeotherms)

  • Normalize for tissue-specific expression patterns and mitochondrial content

• Methodological standardization:

  • Utilize identical purification and storage protocols

  • Standardize assay conditions including buffer composition, substrate concentration, and detection methods

  • Test activity across a range of conditions relevant to each species' physiology

• Data interpretation approaches:

  • Express activity in terms of catalytic efficiency (kcat/Km) rather than absolute rates

  • Compare relative responses to inhibitors or activators

  • Evaluate temperature-activity profiles and thermal stability characteristics

  • Consider allosteric regulation differences when interpreting kinetic parameters

• Recombinant vs. native enzyme considerations:

  • Assess the impact of expression systems on post-translational modifications

  • Compare recombinant proteins with native enzymes purified from tissue sources

  • Evaluate the integrity of subunit interactions in complex reconstruction experiments

This methodological framework allows researchers to distinguish between true biological differences and artifacts of experimental design when comparing SDHA across evolutionary diverse species.

How can Danio rerio SDHA be used in mitochondrial dysfunction research models?

Danio rerio SDHA serves as a valuable tool in developing research models for mitochondrial dysfunction through several methodological approaches:

• Morpholino knockdown and CRISPR/Cas9 gene editing:

  • Targeted reduction or modification of sdha expression in zebrafish embryos

  • Phenotypic characterization of resulting mitochondrial dysfunction

  • Rescue experiments using wild-type or mutant recombinant SDHA protein

  • Correlation of biochemical defects with developmental abnormalities

• Transgenic reporter systems:

  • Generation of sdha-GFP fusion constructs to monitor expression patterns

  • Creation of zebrafish lines with fluorescent reporters for mitochondrial function (membrane potential, ROS production)

  • Real-time visualization of mitochondrial dynamics in transparent embryos

• Chemical inhibition studies:

  • Utilization of specific Complex II inhibitors (malonate, 3-nitropropionic acid)

  • Dose-response analysis comparing in vitro inhibition of recombinant SDHA with in vivo effects

  • Screening of compound libraries for modulators of SDHA activity

• Disease model development:

  • Introduction of patient-derived SDHA mutations into zebrafish sdha

  • Characterization of biochemical, cellular, and organismal consequences

  • Testing of therapeutic interventions targeting specific aspects of dysfunctional SDHA

  • Comparison with recombinant mutant proteins in in vitro assays

• Environmental stress responses:

  • Evaluation of SDHA activity and expression under various stressors (hypoxia, temperature shifts, toxicants)

  • Correlation of biochemical adaptation with organismal resilience

  • Identification of regulatory mechanisms governing SDHA function under stress

The integration of in vitro studies using recombinant SDHA with in vivo zebrafish models provides a comprehensive approach to understanding the mechanistic basis of mitochondrial diseases and developing potential therapeutic strategies.

What are the recommended protocols for handling and storing recombinant Danio rerio SDHA?

Optimal handling and storage of recombinant Danio rerio SDHA is essential for maintaining protein integrity and activity. The methodological approach should include:

• Short-term storage (1-2 weeks):

  • Temperature: 4°C

  • Buffer composition: 50 mM phosphate or Tris buffer, pH 7.2-7.8

  • Additives: 100-150 mM NaCl, 1-5 mM DTT or 2-ME, 10% glycerol

  • Container: Low-protein binding tubes (polypropylene)

  • Protection: Minimize exposure to light and oxidizing conditions

• Long-term storage:

  • Primary method: Aliquot and store at -80°C

  • Alternative: Lyophilization with appropriate cryoprotectants

  • Avoid repeated freeze-thaw cycles (create single-use aliquots)

  • Document stability data for specific storage conditions

• Thawing protocol:

  • Rapid thawing at room temperature followed by immediate transfer to ice

  • Gentle mixing without vortexing to prevent protein denaturation

  • Brief centrifugation to collect condensate and precipitates

  • Activity check after thawing to verify functional preservation

• Working concentration adjustments:

  • Dilution in pre-chilled buffers

  • Addition of stabilizing agents (BSA at 0.1-1 mg/ml)

  • Preparation immediately before use for optimal activity

• Quality control practices:

  • Regular SDS-PAGE analysis to monitor degradation

  • Activity assays to verify functional preservation

  • Spectroscopic assessment of FAD content (A450/A280 ratio)

  • Documentation of batch-to-batch variation

Implementation of standardized handling protocols significantly enhances experimental reproducibility and extends the functional lifetime of recombinant SDHA preparations.

How can SDHA activity be accurately measured in zebrafish tissues?

Accurate measurement of SDHA activity in zebrafish tissues requires specialized techniques that account for the unique properties of this model organism:

• Tissue preparation methods:

  • Embryos: Pool 30-50 embryos, homogenize in ice-cold isolation buffer

  • Adult tissues: Rapid dissection and flash-freezing in liquid nitrogen

  • Mitochondrial isolation: Differential centrifugation or Percoll gradient

  • Tissue preservation: Snap freezing in liquid nitrogen for later analysis

• Homogenization techniques:

  • Buffer composition: 250 mM sucrose, 10 mM Tris-HCl, 1 mM EGTA, pH 7.4

  • Protease inhibitors: Complete protease inhibitor cocktail

  • Homogenization: Dounce homogenizer or tissue lyser for adult tissues

  • Sample clarification: Centrifugation at 600-1000g to remove debris

• Activity assay adaptations:

  • Spectrophotometric: Monitor DCIP reduction at 600 nm in the presence of PMS

  • Polarographic: Oxygen consumption measurements with succinate substrate

  • Linked enzyme assays: Coupling to ubiquinone reduction

  • In-gel activity: BN-PAGE followed by activity staining

• Normalization strategies:

  • Citrate synthase activity as mitochondrial content marker

  • Total protein concentration determined by Bradford or BCA assay

  • Mitochondrial DNA content by qPCR

  • Complex II protein content by Western blotting

• Controls and validation:

  • Tissue-specific baseline activity determination

  • Inhibitor controls (malonate) to confirm specificity

  • Age and developmental stage standardization

  • Technical and biological replication

For developmental studies, a standardized staging system should be employed to account for the rapid changes in mitochondrial content and function during zebrafish development.

What imaging techniques are most effective for visualizing SDHA localization in zebrafish?

Visualizing SDHA localization in zebrafish benefits from the organism's natural transparency and amenability to various imaging techniques:

• Whole-mount immunofluorescence:

  • Fixation: 4% paraformaldehyde, 2-4 hours at room temperature

  • Permeabilization: 0.5% Triton X-100 in PBS, overnight at 4°C

  • Blocking: 10% normal goat serum, 1% BSA, 0.1% Tween-20

  • Primary antibody: Anti-SDHA antibodies validated for zebrafish

  • Counterstaining: DAPI for nuclei, MitoTracker for mitochondria

  • Clearing: Glycerol series or specialized clearing agents (CLARITY)

• Transgenic fluorescent reporter lines:

  • SDHA-GFP fusion constructs under endogenous promoter

  • Tissue-specific promoters driving SDHA-fluorescent protein fusions

  • Photoconvertible reporters for real-time trafficking studies

  • Dual reporters for co-localization with other mitochondrial proteins

• Advanced microscopy approaches:

  • Confocal microscopy: 3D visualization of mitochondrial networks

  • Light sheet microscopy: Long-term imaging with minimal phototoxicity

  • Super-resolution techniques (STED, PALM, STORM): Submitochondrial localization

  • Two-photon microscopy: Deep tissue imaging in adult zebrafish

• Live imaging considerations:

  • Mounting: Low-melting point agarose in glass-bottom dishes

  • Anesthesia: Tricaine (MS-222) at appropriate concentrations

  • Temperature control: Heated stages maintaining 28°C

  • Time intervals: Balancing temporal resolution with phototoxicity

• Image analysis methodologies:

  • Colocalization quantification (Pearson's coefficient, Manders' overlap)

  • 3D reconstruction of mitochondrial networks

  • Automated tracking of mitochondrial dynamics

  • Machine learning approaches for pattern recognition

These techniques enable researchers to investigate the temporal and spatial dynamics of SDHA localization during development and in response to genetic or environmental perturbations.

How can gene editing techniques be used to study Danio rerio SDHA function?

Gene editing techniques provide powerful tools for investigating SDHA function in zebrafish through precise genetic manipulation:

• CRISPR/Cas9 system implementation:

  • Target selection: Design sgRNAs targeting exons encoding functional domains of sdha

  • Delivery method: Microinjection of Cas9 protein and sgRNA into one-cell stage embryos

  • Mutation screening: High-resolution melt analysis, T7 endonuclease assay, or direct sequencing

  • Founder identification: Germline transmission testing and establishment of stable lines

• Knock-in strategies:

  • Homology-directed repair templates for introducing specific mutations

  • Insertion of epitope tags for tracking endogenous protein

  • Introduction of fluorescent reporters for live imaging

  • Precise modification of regulatory elements to study expression control

• Conditional approaches:

  • Tissue-specific knockouts using Cre-lox systems

  • Temporal control using heat-shock inducible promoters

  • Drug-inducible gene expression systems (e.g., Tet-On)

  • Optogenetic control of gene expression or protein activity

• Functional rescue experiments:

  • mRNA co-injection to rescue morphant or mutant phenotypes

  • Transgenic rescue with wild-type or mutant constructs

  • Structure-function analysis through domain-specific mutations

  • Cross-species complementation studies using mammalian SDHA

• Phenotypic analysis pipeline:

  • Embryonic development assessment

  • Mitochondrial respiration measurement in isolated mitochondria

  • Behavioral testing for neurological or muscular phenotypes

  • Histological examination of tissues with high metabolic demand

  • Metabolomic profiling to identify affected pathways

These genetic approaches allow researchers to create models of SDHA dysfunction that recapitulate human mitochondrial diseases and provide platforms for testing therapeutic interventions.

What are the best approaches for comparing in vitro and in vivo SDHA activity?

Bridging in vitro biochemical studies of recombinant SDHA with in vivo observations requires careful methodological alignment:

• Standardized activity assay development:

  • Adapt in vitro assay conditions to mimic physiological environment

  • Develop tissue homogenate protocols that preserve native activity

  • Use identical electron acceptors and detection methods where possible

  • Account for differences in pH, ionic strength, and temperature

• Correlation strategies:

  • Measure kinetic parameters (Km, Vmax) in both systems

  • Determine inhibitor sensitivity profiles (IC50 values)

  • Compare substrate specificity and cofactor requirements

  • Analyze regulation by physiological modulators

• Integrative experimental designs:

  • Ex vivo tissue preparations (e.g., permeabilized fibers)

  • Isolated mitochondria from genetically modified zebrafish

  • Reconstitution of recombinant SDHA into liposomes or nanodiscs

  • Microinjection of recombinant protein into zebrafish embryos

• Normalization and scaling approaches:

  • Express activity per unit of SDHA protein (determined by quantitative western blot)

  • Account for differences in mitochondrial content

  • Consider developmental stage-specific activity profiles

  • Normalize to evolutionary conserved reference proteins

• Advanced analytical techniques:

  • Metabolic flux analysis using stable isotope tracers

  • Computational models integrating in vitro parameters

  • Systems biology approaches correlating enzyme activity with metabolite levels

  • Machine learning algorithms to identify patterns across datasets

By systematically bridging these different experimental systems, researchers can develop a more comprehensive understanding of how SDHA functions within the complex cellular environment and how biochemical alterations translate to physiological consequences.

How does zebrafish SDHA compare with human SDHA in terms of sequence homology and function?

Comparative analysis of zebrafish and human SDHA reveals significant conservation at multiple levels, providing a strong foundation for translational research:

This comparative framework allows researchers to make informed decisions about the translational relevance of findings in zebrafish models to human mitochondrial biology and disease.

What are the established baseline activity levels for wild-type Danio rerio SDHA?

Established baseline activity levels for wild-type Danio rerio SDHA provide critical reference points for comparative studies:

• Tissue-specific activity profiles:

• Developmental progression:

  • Activity increases progressively during embryogenesis

  • Significant upregulation occurs during the transition from glycolytic to oxidative metabolism

  • Tissue-specific patterns emerge as organogenesis progresses

  • Adult levels typically reached by 30 days post-fertilization

• Methodological standardization:

  • Spectrophotometric assay using DCIP/PMS electron acceptor system

  • Buffer: 50 mM potassium phosphate, pH 7.4, 10 mM succinate

  • Temperature: 28°C (physiological for zebrafish)

  • Normalization: Total protein and/or citrate synthase activity

• Natural variation considerations:

  • Strain differences: AB, TU, and WIK strains show 10-15% variation

  • Sex-specific differences: Males typically show 5-10% higher activity in muscle

  • Circadian fluctuations: 10-20% daily variation with peaks during active periods

  • Age-related changes: Gradual decline (5-8% per year) in adult fish

• Technical variability assessment:

  • Intra-assay coefficient of variation: 5-8%

  • Inter-assay coefficient of variation: 8-12%

  • Inter-laboratory variation: 10-20% with standardized protocols

  • Sample preparation impact: Fresh vs. frozen tissue (10-15% activity loss)

These baseline values serve as essential references for studies investigating genetic, environmental, or pharmacological effects on SDHA function in zebrafish models.

How do mutations in SDHA affect mitochondrial function in zebrafish compared to human models?

Comparative analysis of SDHA mutations in zebrafish and human models reveals both shared and distinct consequences for mitochondrial function:

• Phenotypic spectrum comparison:

  • Zebrafish: Developmental delays, cardiac abnormalities, neurological defects, metabolic disturbances

  • Human: Leigh syndrome, cardiomyopathy, paraganglioma/pheochromocytoma, optic atrophy

  • Shared features: Mitochondrial dysfunction, lactic acidosis, energy deficiency

  • Species-specific manifestations: Temperature sensitivity in zebrafish, tissue-specific tumor development in humans

• Biochemical consequences:

  • Complex II activity reduction: Similar magnitude of defect (50-90% depending on mutation)

  • ROS production: Generally increased in both models, but zebrafish show enhanced antioxidant responses

  • mtDNA stability: Both species exhibit secondary mtDNA damage with severe SDHA defects

  • Metabolic adaptation: Zebrafish demonstrate more robust glycolytic compensation

• Genetic complementation studies:

  • Human mutations introduced to zebrafish SDHA often recapitulate biochemical defects

  • Rescue experiments with human wild-type SDHA can complement zebrafish mutations

  • Functional domains show equivalent sensitivity to disruption across species

  • Regulatory differences may affect phenotypic expression of similar mutations

• Tissue-specific vulnerabilities:

  • Zebrafish: Primary effects on high-energy tissues (brain, heart, muscle) during development

  • Human: Age-dependent tissue specificity with acute sensitivity in brain, heart, muscle

  • Developmental timing: Zebrafish phenotypes emerge rapidly during embryogenesis

  • Chronic progression: Human SDHA disorders often show progressive course over years

• Therapeutic response comparison:

  • Antioxidant efficacy: Generally similar between models

  • Metabolic bypass strategies: Differential effectiveness due to metabolic flexibility

  • Gene therapy approaches: Similar principles apply despite delivery differences

  • Temperature modulation: Unique therapeutic lever in zebrafish models

This comparative framework allows researchers to leverage the advantages of zebrafish models while maintaining awareness of their limitations for translational research on human SDHA-related disorders.

What are the known interacting partners of Danio rerio SDHA and their significance?

Danio rerio SDHA participates in a complex network of protein-protein interactions that regulate its assembly, function, and integration into cellular metabolism:

• Core Complex II components:

• Mitochondrial interaction network:

  • OXPHOS complexes: Loose associations within respiratory supercomplexes

  • Mitochondrial chaperones: HSP60, HSP70 family proteins assist in folding

  • Import machinery: TOM/TIM complexes facilitate mitochondrial targeting

  • Quality control: LONP1, CLPX proteases regulate SDHA turnover

• Metabolic enzyme interactions:

  • Fumarase: Metabolic channeling of fumarate

  • Malate dehydrogenase: TCA cycle flux coordination

  • Aconitase: Redox-dependent association

  • Pyruvate dehydrogenase complex: Metabolic network regulation

• Regulatory interactions:

  • Sirtuins: SIRT3 deacetylates SDHA affecting its activity

  • Kinases: AMPK phosphorylates SDHA under energy stress

  • E3 ubiquitin ligases: Regulate SDHA stability and turnover

  • Transcription factors: NRF1/2 coordinate expression with other mitochondrial genes

• Pathological interactions:

  • Aggregation-prone proteins: α-synuclein interaction observed in neurodegeneration models

  • Viral proteins: Documented interaction with viral factors affecting mitochondrial function

  • Xenobiotic-metabolizing enzymes: Interactions relevant to toxicological studies

  • Cancer-related proteins: Altered interactions in tumorigenic states

These interaction networks provide mechanistic insights into SDHA regulation and identify potential targets for therapeutic intervention in mitochondrial disorders.

How do environmental factors affect SDHA expression and activity in zebrafish models?

Environmental factors significantly influence SDHA expression and activity in zebrafish, providing valuable insights into mitochondrial adaptability:

• Temperature effects:

• Oxygen availability responses:

  • Hypoxia (5% O₂): Downregulation of SDHA expression (40-60%), shift to glycolysis

  • Hyperoxia (40% O₂): Transient upregulation followed by oxidative damage to SDHA

  • Intermittent hypoxia: Preconditioning effect with enhanced SDHA stability

  • Re-oxygenation: Oxidative modification of SDHA with temporary activity decrease

• Nutritional influences:

  • Caloric restriction: Increased SDHA expression and activity (20-30%)

  • High-fat diet: Decreased activity despite normal expression

  • Ketogenic conditions: Enhanced SDHA activity with increased mitochondrial biogenesis

  • Specific nutrient effects: CoQ10, riboflavin, and L-carnitine enhance SDHA function

• Chemical exposures:

  • Heavy metals (Cd, Pb): Dose-dependent inhibition of SDHA activity

  • Pesticides (rotenone, paraquat): Direct inhibition of respiratory chain

  • Pharmaceutical agents: Drug-specific effects on SDHA expression and activity

  • Polycyclic aromatic hydrocarbons: Transcriptional downregulation via AhR pathway

• Circadian and seasonal variations:

  • Diurnal patterns: 15-25% daily fluctuation with peaks during active periods

  • Photoperiod effects: Long-term adaptation of SDHA expression to light cycles

  • Seasonal changes: Physiological adaptation in wild populations

  • Reproductive state: Altered expression during spawning periods

Understanding these environmental influences allows researchers to properly control experimental conditions and provides insights into mitochondrial adaptation mechanisms that may be relevant to human health and disease.