Recombinant Xenopus laevis Adrenodoxin-like protein, mitochondrial (fdx1l), partial

What is Xenopus laevis fdx1l and what is its function in mitochondria?

Xenopus laevis fdx1l (ferredoxin 1-like) is a mitochondrial iron-sulfur protein that likely serves similar functions to mammalian ferredoxins. Based on homology with human ferredoxins (FDX1 and FDX2), fdx1l likely functions as an electron donor in various mitochondrial processes.

Current research on human ferredoxins indicates two distinct but related proteins: FDX1 primarily functions in steroid hormone biosynthesis by transferring electrons to cytochrome P450 enzymes, while FDX2 is essential for iron-sulfur cluster assembly and heme A biosynthesis . The specific functions of X. laevis fdx1l may align with either of these roles, though comprehensive characterization studies comparing its activity to mammalian counterparts are still emerging.

Why use Xenopus laevis as a model system for studying fdx1l?

Xenopus laevis provides several distinct advantages as a model system for studying mitochondrial proteins like fdx1l:

• Developmental accessibility: X. laevis embryos develop externally with large, easily manipulated eggs (>1mm diameter) and well-characterized developmental stages .

• Experimental tractability: The system permits various genetic manipulation techniques including microinjection of mRNA, morpholino oligonucleotides, or CRISPR-Cas9 components .

• Evolutionary conservation: As a vertebrate model, X. laevis shares significant conservation of mitochondrial pathways with mammals, making findings potentially translatable to human biology .

• Cost-effectiveness: Maintaining X. laevis colonies is relatively inexpensive compared to mammalian models, allowing for larger-scale experimental approaches .

• Cell culture advantages: X. laevis cells are easily obtained, maintained without special conditions, and are suitable for long periods of live imaging .

How does fdx1l differ from human FDX1 and FDX2?

While comprehensive comparative studies of X. laevis fdx1l against human ferredoxins are still developing, we can infer several key differences based on current knowledge:

Human FDX1 and FDX2 share only 33% protein sequence identity despite structural similarities . X. laevis fdx1l may represent an evolutionary intermediate or possess unique characteristics adapted to amphibian physiology.

What are the most effective methods for expressing recombinant Xenopus laevis fdx1l?

For successful expression of recombinant X. laevis fdx1l, researchers should consider these methodological approaches:

Bacterial Expression Systems:

• Use E. coli BL21(DE3) or Rosetta strains for high-yield expression

• Consider adding a solubility tag (MBP, SUMO, or GST) to enhance protein solubility

• Express at lower temperatures (16-18°C) to improve proper folding

• Include iron and sulfur sources in the medium for proper Fe-S cluster formation

• Purify under anaerobic or low-oxygen conditions to maintain Fe-S cluster integrity

Xenopus Expression Systems:
For in vivo expression within X. laevis:

• Microinjection of mRNA into embryos remains the gold standard for expression in developmental studies

• For later stage expression, targeted electroporation has shown success in X. laevis embryos

• Viral vectors, particularly vesicular stomatitis virus (VSV), have proven effective for gene delivery in adult X. laevis neurons, whereas adeno-associated virus (AAV) and lentivirus have shown limited efficiency

When designing constructs, inclusion of appropriate mitochondrial targeting sequences is crucial for proper localization.

How can I determine if recombinant X. laevis fdx1l contains the proper Fe-S cluster?

Verification of proper Fe-S cluster incorporation is critical for functional studies of fdx1l. Recommended analytical approaches include:

• UV-visible spectroscopy: Characteristic absorption peaks at approximately 415, 455, and 480 nm indicate properly formed [2Fe-2S] clusters .

• Electron paramagnetic resonance (EPR): Fe-S clusters show distinctive EPR signals in their reduced state, confirming proper electronic structure.

• Circular dichroism (CD) spectroscopy: Provides information about secondary structure and proper protein folding.

• NMR spectroscopy: Can reveal hyperfine 1H NMR signals indicating electron delocalization patterns in the Fe-S cluster, as demonstrated with human ferredoxins .

• Activity assays: Functional Fe-S clusters can be assessed through electron transfer capacity using cytochrome c reduction assays, similar to those described for human Adx .

• Iron quantification: Colorimetric assays or ICP-MS can determine iron:protein ratios, with a 2:1 ratio expected for [2Fe-2S] clusters.

The protein should appear reddish-brown when properly folded with intact Fe-S clusters.

What are the optimal conditions for biophysical characterization of X. laevis fdx1l interactions?

Based on studies with human ferredoxins, the following conditions are recommended for characterizing X. laevis fdx1l interactions:

NMR Spectroscopy:

• Prepare uniformly 15N-labeled fdx1l at 0.2-0.5 mM in 20 mM sodium phosphate buffer (pH 7.4) with 50 mM NaCl

• Maintain sample temperature at 25°C

• Consider both oxidized and reduced states of fdx1l, as they will show significant spectral differences

• Use 1H-15N TROSY-HSQC experiments for optimal resolution

Interaction Studies:

• For protein-protein interactions, use substoichiometric quantities of interaction partners to prevent severe line broadening in NMR experiments

• Consider microscale thermophoresis (MST) for determining binding constants between fdx1l and potential interaction partners

• Study both oxidized and reduced states, as redox state significantly affects interaction profiles

Buffer Conditions:

• 20-100 mM potassium phosphate buffer (pH 7.4)

• 50-150 mM NaCl

• For in vitro activity assays, include 1.1 mM NADPH as the electron source

How can I investigate the role of X. laevis fdx1l in Fe-S cluster biogenesis?

To investigate fdx1l's role in Fe-S cluster biogenesis, consider these advanced experimental approaches:

In vitro Fe-S Cluster Assembly:

• Reconstitute a minimal Fe-S cluster assembly system using purified components:

  • Recombinant X. laevis fdx1l (oxidized and reduced forms)

  • Cysteine desulfurase complex ([Acp]2:[ISD11]2:[NFS1]2)

  • Iron-sulfur cluster scaffold protein (ISCU)

  • Frataxin (FXN)

  • An electron source (NADPH + adrenodoxin reductase)

• Monitor Fe-S cluster formation on ISCU spectroscopically, tracking characteristic absorption changes at 456 nm .

• Compare assembly rates with fdx1l versus human FDX1 and FDX2 under identical conditions to assess functional equivalence.

In vivo Approaches:

What experimental design would best elucidate potential roles of fdx1l in Xenopus embryonic development?

To investigate fdx1l's developmental roles, a comprehensive experimental design should include:

Spatiotemporal Expression Analysis:

• Perform quantitative RT-PCR across developmental stages from oocyte to tadpole.

• Use whole-mount in situ hybridization to determine tissue-specific expression patterns.

• Generate transgenic reporter lines with fdx1l promoter driving fluorescent protein expression.

Loss-of-Function Studies:

• Design targeted CRISPR-Cas9 knockout or morpholino-based knockdown strategies.

• Deliver reagents via microinjection at the 1-2 cell stage for ubiquitous effects.

• Use targeted injections into specific blastomeres for tissue-restricted analysis.

• Analyze phenotypes across key developmental stages, documenting mortality, morphological abnormalities, and behavioral defects.

Gain-of-Function and Rescue Studies:

• Overexpress wild-type or mutant fdx1l via mRNA microinjection.

• Perform domain-swap experiments with human FDX1/FDX2 to identify critical functional regions.

• Combine knockdown with rescue constructs to validate specificity.

Mechanistic Analysis:

• Assess mitochondrial function in developing embryos using ratiometric imaging of mitochondrial membrane potential.

• Measure activities of Fe-S-dependent enzymes throughout development.

• Analyze energetic status using ATP/ADP ratios and metabolomic profiling.

• Investigate potential roles in steroid hormone biosynthesis during critical developmental windows.

How does the function of fdx1l in Xenopus laevis compare to its homologs in other model organisms?

A comprehensive comparative analysis would involve:

For thorough comparative analysis:

• Perform sequence and structural alignments of fdx1l with homologs across species.

• Use complementation assays in yeast Gal-YAH1 cells (where YAH1 expression can be regulated) to test functional conservation, as previously done with human FDX1 and FDX2 .

• Generate expression constructs of X. laevis fdx1l with mutations corresponding to known disease-causing mutations in human FDX2 to assess conservation of functional residues.

• Compare biochemical properties (redox potential, interaction partners) across species to identify conserved and divergent features.

Why might recombinant X. laevis fdx1l be expressed in an inactive form?

Several factors can lead to inactive recombinant fdx1l:

Fe-S Cluster Formation Issues:

• Insufficient iron or sulfur sources: Include ferrous ammonium sulfate and L-cysteine in the expression medium.

• Oxidative damage: Express and purify under low-oxygen conditions or add reducing agents like DTT or β-mercaptoethanol.

• Improper scaffold proteins: Co-express with iron-sulfur cluster assembly proteins (ISC machinery) for improved cluster insertion.

Protein Folding Problems:

• Expression temperature: Lower to 16-18°C to allow proper folding.

• Solubility tags: Use fusion partners like MBP or SUMO to enhance solubility.

• Chaperone co-expression: Co-express with chaperones like GroEL/ES to facilitate folding.

Truncation or Mutation Issues:

• Codon optimization: Ensure codon usage is optimized for the expression system.

• Verify sequence integrity: Confirm absence of mutations or premature stop codons.

• Check for proteolysis: Add protease inhibitors during purification.

Functional Assay Considerations:

• Redox state: Ensure protein is in the correct redox state for activity assays.

• Interaction partners: Some activities may require partner proteins like adrenodoxin reductase.

• Buffer conditions: Test various pH and salt conditions for optimal activity.

Studies with human ferredoxins indicate that removing the Fe-S cluster results in partially unfolded proteins with reduced spectral dispersion in NMR experiments , highlighting the importance of proper cluster incorporation.

How can contradicting results regarding fdx1l function in different experimental systems be reconciled?

Contradicting results about fdx1l function may stem from several factors:

System-Specific Differences:

• Expression contexts: Recombinant protein studies versus in vivo knockdown may yield different results due to compensatory mechanisms in living systems.

• Species differences: Xenopus fdx1l may have evolved species-specific functions distinct from mammalian ferredoxins.

• Developmental stage: Function may vary throughout development or across tissues.

Methodological Approaches:

• Use multiple experimental systems: Combine in vitro biochemistry, cell culture, and in vivo studies.

• Perform dose-response studies: Contradictions may arise from different expression levels or activity thresholds.

• Assess temporal dynamics: Some functions may only be apparent under specific temporal conditions.

Analytical Framework:

• Dual functionality model: Consider that fdx1l may serve both FDX1 and FDX2-like functions, with context-dependent dominance of either role.

• Redundancy and compensation: Investigate potential redundant systems that may mask phenotypes in certain contexts.

• Interaction network mapping: Identify all interaction partners in each experimental system to explain context-specific behaviors.

For example, human FDX1 and FDX2 have both been reported to function in iron-sulfur cluster assembly in vitro, though with different efficiencies , yet in vivo studies suggest more specialized roles . Such discrepancies can be reconciled through careful consideration of experimental context and comprehensive validation across multiple systems.

What are the most common pitfalls in analyzing fdx1l knockout phenotypes in Xenopus laevis?

Researchers analyzing fdx1l knockout phenotypes should be aware of these common pitfalls:

Technical Challenges:

• Incomplete knockdown: Morpholinos may not achieve complete protein elimination; verify knockdown efficiency by Western blot.

• Off-target effects: Both morpholinos and CRISPR-Cas9 can produce unintended effects; use appropriate controls and rescue experiments.

• Mosaicism in F0 CRISPR animals: First-generation knockouts may be mosaic, confounding phenotypic analysis; consider analyzing F1 generation or use quantitative assessment of editing efficiency.

Biological Complexities:

• Pseudotetraploidy of X. laevis: This species underwent genome duplication, potentially resulting in gene redundancy and compensation .

• Maternal contribution: Eggs contain maternal mRNAs and proteins that may mask early developmental phenotypes; consider generating maternal-zygotic knockouts.

• Developmental stage specificity: Phenotypes may only manifest at specific developmental stages; conduct thorough temporal analysis.

Interpretative Challenges:

• Secondary vs. primary effects: Mitochondrial dysfunction can cause pleiotropic effects; distinguish direct from indirect consequences.

• Metabolic adaptation: Cells may adapt metabolically to chronic fdx1l deficiency, masking acute phenotypes.

• Context dependency: Phenotypes may vary with environmental conditions (temperature, stress, etc.).

To address these challenges, employ robust experimental designs including:

• Multiple knockout/knockdown approaches

• Comprehensive rescue experiments with wild-type and mutant constructs

• Tissue-specific manipulation where possible

• Detailed temporal analysis across developmental stages

• Biochemical validation of expected molecular consequences

What is the potential role of X. laevis fdx1l in copper metabolism and elesclomol response?

Recent research on human FDX1 has revealed unexpected roles in copper metabolism that may be conserved in X. laevis fdx1l:

Copper Release Mechanism:
Evidence suggests that human FDX1 is essential for releasing copper from the copper-transporting drug elesclomol (ES) within mitochondria . In this role, FDX1 appears to catalyze the reduction of Cu(II) to Cu(I) in the ES-Cu complex, facilitating copper dissociation. This function may be conserved in X. laevis fdx1l.

Experimental Approaches to Investigate This Function:

• In vitro copper release assays: Using purified recombinant fdx1l with ES-Cu complexes and the colorimetric Cu-specific chelator bathocuproine disulfonate (BCS) .

• CRISPR knockout studies: Generating fdx1l-deficient X. laevis embryos or cell lines and testing their response to elesclomol and copper deficiency.

• Mitochondrial respiration analysis: Assessing whether fdx1l is required for ES-mediated rescue of mitochondrial respiration in copper-depleted X. laevis cells.

• Structural studies: Investigating potential copper-binding sites in fdx1l structure using site-directed mutagenesis.

Physiological Relevance:
This function may connect fdx1l to copper-dependent enzymes like cytochrome c oxidase (COX), suggesting fdx1l could play a broader role in mitochondrial metalloproteins beyond iron-sulfur clusters. The requirement for FDX1 in elesclomol-mediated copper delivery appears to be specific to mitochondria, as non-mitochondrial copper enzymes can receive copper from elesclomol through FDX1-independent mechanisms .

How might X. laevis fdx1l be involved in stress responses and mitophagy regulation?

Recent studies suggest human FDX1 may play unexpected roles in cellular stress responses that could be conserved in X. laevis fdx1l:

FDX1 and Mitophagy:
Downregulation of human FDX1 has been shown to activate mitophagy , suggesting a potential role in mitochondrial quality control. This connection could represent an important adaptive response linking iron-sulfur cluster biogenesis to mitochondrial turnover.

Potential Mechanisms:

• ROS-mediated signaling: FDX1 downregulation may increase reactive oxygen species (ROS), triggering mitophagy through established ROS-sensitive pathways.

• PI3K/AKT pathway activation: Human FDX1 downregulation activates the PI3K/AKT signaling pathway , which has known roles in autophagy regulation.

• Fe-S protein dysfunction: Loss of Fe-S cluster biogenesis may lead to accumulation of damaged Fe-S proteins, triggering selective mitophagy.

Experimental Approaches:

• Live imaging of mitophagy: Use mRFP-GFP-LC3 reporters in X. laevis cells or embryos with manipulated fdx1l levels.

• Electron microscopy: Analyze mitochondrial morphology and autophagosome formation in fdx1l-deficient samples.

• Stress resistance assays: Test whether fdx1l manipulation affects resistance to various stressors.

• Signaling pathway analysis: Investigate activation of stress-responsive pathways like PINK1/Parkin, AMPK, and PI3K/AKT.

Developmental Context:
These stress response functions may be particularly relevant during developmental transitions or environmental challenges in X. laevis, linking mitochondrial function to broader cellular adaptations.

What is the potential of X. laevis fdx1l as a target for translational research in disease models?

X. laevis fdx1l research could have significant translational implications based on emerging roles of human ferredoxins in disease:

Cancer Biology Applications:
Human FDX1 has been implicated in cancer biology through several mechanisms:

• Metabolic reprogramming: FDX1 downregulation contributes to metabolic shifts in cancer cells .

• Resistance to proteotoxic stress: FDX1 is a targetable vulnerability in cancers resistant to proteotoxic stress .

• Immune response modulation: FDX1 expression correlates with immune checkpoint gene expression and may influence immunotherapy effectiveness in melanoma .

Copper Deficiency Disorders:
The role of FDX1 in copper metabolism suggests potential applications in:

• Therapeutic copper delivery: Understanding how fdx1l facilitates copper release could inform better treatments for copper deficiency disorders.

• Biomarker development: FDX1 expression levels might predict responsiveness to copper-based therapies.

Mitochondrial Disease Models:
X. laevis embryos with manipulated fdx1l could serve as models for:

• Iron-sulfur cluster assembly disorders: Including multiple mitochondrial dysfunction syndromes.

• Friedreich's ataxia: Where frataxin deficiency disrupts iron-sulfur cluster biogenesis.

Experimental Approaches:

• High-throughput drug screening: Using X. laevis embryos or cells with fdx1l mutations to identify therapeutic compounds.

• Disease-mimicking mutations: Introducing mutations corresponding to human disease-causing variants in FDX1/FDX2.

• Xenopus tumor models: Developing X. laevis cancer models to study fdx1l's role in tumor growth and response to therapy.

• Patient-derived xenografts: Testing personalized treatment approaches in X. laevis models based on patient-specific FDX1/FDX2 alterations.