Recombinant Xenopus laevis FAD-dependent oxidoreductase domain-containing protein 1 (foxred1)

What is FOXRED1 and what is its primary function in cellular metabolism?

FOXRED1 (FAD-dependent Oxidoreductase Domain-containing protein 1) is a protein containing an FAD-dependent oxidoreductase domain that localizes to the mitochondria. It functions primarily as a chaperone protein required for the assembly and proper function of mitochondrial complex I (NADH:ubiquinone oxidoreductase), the largest complex of the mitochondrial oxidative phosphorylation (OXPHOS) system. The protein plays a crucial role in maintaining complex I activity, which is essential for electron transport chain function and cellular energy production. Mutations in FOXRED1 are associated with mitochondrial complex I deficiency, highlighting its importance in cellular metabolism .

What are the advantages of using Xenopus laevis as a model system for FOXRED1 studies?

Xenopus laevis offers several significant advantages as a model system for studying FOXRED1:

• Evolutionary proximity to higher vertebrates in terms of physiology, gene expression, and organ development, making findings potentially translatable to human biology .

• Year-round availability of embryos through hormone-induced spawning, overcoming seasonal breeding limitations of other amphibian models .

• Rapid embryonic development with easily observable developmental stages, allowing for time-efficient experimental designs .

• The ability to access increasing resources of transgenic lines from repositories such as the National Xenopus Resource (NXR) and the European Xenopus Resource Centre (EXRC) .

• Compatibility with powerful gene expression modification techniques, including antisense morpholino oligonucleotides and CRISPR/Cas9 genome editing, enabling specific targeting of FOXRED1 .

• Large oocyte and embryo size facilitating microinjection techniques and biochemical analyses that require substantial material .

What are the optimal methods for isolating and culturing Xenopus laevis oocytes for FOXRED1 expression studies?

For optimal isolation and culture of Xenopus laevis oocytes for FOXRED1 expression studies, researchers should follow these methodological steps:

• Ovary Dissection: Harvest ovaries from young adult female Xenopus laevis (3-5 years old) following appropriate euthanasia protocols (submersion in 15% benzocaine for 15 minutes is standard) .

• Tissue Digestion: Treat ovaries with 3 mg/ml collagenase IA in Marc's modified Ringer's (MMR) buffer with gentle rocking for 30-45 minutes until dissociated oocytes are visible .

• Filtration: Pass the resulting mixture through two sets of filter meshes to separate oocytes by size and remove debris .

• Granulosa Cell Treatment: For experiments requiring intact granulosa cells, transfer oocytes directly to oocyte culture medium (OCM). For experiments requiring pure oocytes, remove granulosa cells by treating with 10 mg/ml trypsin in PBS for 1 minute, followed by MMR washes .

• Culture Conditions: Maintain oocytes in OCM at appropriate temperature (typically room temperature for short-term experiments) with careful monitoring of media pH and oxygenation .

• Validation: Confirm successful isolation and viability using standard markers or dyes such as Hoechst staining to verify granulosa cell removal when needed .

This protocol ensures high-quality oocyte preparations suitable for subsequent FOXRED1 expression or functional studies.

What protein detection methods are most effective for analyzing recombinant Xenopus FOXRED1 expression?

The most effective protein detection methods for analyzing recombinant Xenopus FOXRED1 expression include:

• SDS-PAGE Immunoblot Analysis: For detecting FOXRED1 protein levels, prepare mitochondrial fractions from cell lines expressing the recombinant protein. Heat samples at 70°C in the presence of β-Mercaptoethanol and separate on a 10% sodium dodecyl sulfate-polyacrylamide gel. This approach allows for precise quantification of FOXRED1 protein levels and assessment of potential post-translational modifications .

• Blue Native PAGE (BN-PAGE): For analyzing FOXRED1 incorporation into native mitochondrial complex I, solubilize native mitochondrial complexes with 2% detergent and perform BN-PAGE. This technique is particularly valuable for assessing whether recombinant FOXRED1 properly integrates into complex I and affects its assembly .

• Immunofluorescence Microscopy: For subcellular localization studies, use specific anti-FOXRED1 antibodies combined with mitochondrial markers to confirm proper targeting of the recombinant protein to mitochondria in Xenopus cells or embryos .

• Mass Spectrometry: For detailed protein characterization and interaction studies, employ techniques such as liquid chromatography-mass spectrometry (LC-MS/MS) to identify FOXRED1 binding partners and post-translational modifications.

These complementary approaches provide comprehensive analysis of recombinant FOXRED1 expression, processing, and function in the Xenopus system.

How can I optimize recombinant expression of functional Xenopus FOXRED1 protein?

To optimize recombinant expression of functional Xenopus FOXRED1 protein, consider implementing these methodological approaches:

• Expression System Selection:

  • For in vitro studies: Use E. coli systems with specialized vectors containing mitochondrial targeting sequence-deleted constructs

  • For cellular studies: Consider baculovirus-insect cell systems which better handle post-translational modifications

  • For in vivo studies: Employ Xenopus embryo microinjection of mRNA encoding FOXRED1

• Construct Design:

  • Include appropriate purification tags (His, GST) positioned to avoid interference with the FAD-binding domain

  • Optimize codon usage for the expression system

  • Consider including the mitochondrial targeting sequence for cellular studies or removing it for bacterial expression

• Expression Conditions:

  • For bacterial systems: Lower temperature (16-18°C) expression often improves folding of complex proteins

  • Include FAD cofactor in media or lysis buffer to improve stability

  • Optimize induction parameters (inducer concentration, OD at induction, duration)

• Purification Strategy:

  • Employ gentle lysis methods to maintain protein integrity

  • Include protease inhibitors and reducing agents throughout purification

  • Consider detergent solubilization methods similar to those used for human FOXRED1

  • Implement multiple purification steps (affinity, ion exchange, size exclusion) for highest purity

• Functional Validation:

  • Assess FAD binding through spectroscopic methods

  • Verify proper folding through circular dichroism

  • Test enzymatic activity using oxidoreductase activity assays

  • Confirm ability to rescue complex I assembly in FOXRED1-deficient systems

These optimizations should result in higher yields of properly folded, functionally active recombinant Xenopus FOXRED1 protein suitable for downstream applications.

How does FOXRED1 contribute to mitochondrial complex I assembly and function in Xenopus?

FOXRED1 plays a critical role in mitochondrial complex I assembly and function in Xenopus through several mechanisms:

• Assembly Factor Activity: As in other vertebrates, Xenopus FOXRED1 functions as a dedicated assembly factor for complex I, facilitating the proper integration of subunits during the complex's biogenesis. This chaperone-like function ensures correct protein folding and subunit interactions during the multi-step assembly process of this large 44-subunit complex .

• FAD Cofactor Utilization: The FAD-dependent oxidoreductase domain in FOXRED1 likely participates in redox reactions necessary for proper complex I maturation, potentially modifying specific subunits or cofactors to enable correct assembly .

• Quality Control: FOXRED1 may participate in quality control mechanisms that prevent the incorporation of damaged or improperly folded subunits into the complex, thus maintaining functional integrity of complex I.

• Tissue-Specific Regulation: During Xenopus development, FOXRED1 activity may be differentially regulated across tissues, contributing to the established pattern of variable complex I activity observed in different Xenopus tissues and developmental stages .

• ROS Management: Given the relationship between complex I function and reactive oxygen species (ROS) production, FOXRED1's role in ensuring proper complex I assembly indirectly contributes to cellular ROS homeostasis, which is particularly important in Xenopus oocytes that maintain ROS-free mitochondrial metabolism .

Defects in FOXRED1 function result in impaired complex I assembly and activity, leading to mitochondrial dysfunction similar to that observed in human mitochondrial disorders .

What is the relationship between FOXRED1 and reactive oxygen species (ROS) management in Xenopus oocytes?

The relationship between FOXRED1 and reactive oxygen species (ROS) management in Xenopus oocytes represents a fascinating area of mitochondrial biology:

• ROS-Free Environment: Xenopus oocytes maintain a remarkably ROS-free mitochondrial environment, particularly in early developmental stages. This protection is critical for preserving mtDNA integrity for future generations .

• FOXRED1 and Complex I Regulation: FOXRED1's role in complex I assembly and function directly impacts ROS production, as complex I is a major source of mitochondrial ROS. In early Xenopus oocytes (stage I), complex I activity is very low, which correlates with negligible levels of ROS as measured by peroxiredoxin 3 (Prdx3) dimerization .

• Developmental Regulation: As oocytes mature from early (stage I) to late stages (stage VI), complex I activity increases, accompanied by increased Prdx3 dimerization, indicating rising ROS levels. This suggests FOXRED1 activity may be developmentally regulated to control complex I assembly and subsequent ROS production .

• Redox Indicators: The reduced cellular redox state in early oocytes, as evidenced by a 20-fold higher ratio of reduced glutathione to oxidized glutathione compared to late-stage oocytes, further supports this relationship .

• Metabolic Adaptation: FOXRED1 likely participates in the metabolic adaptation observed throughout oogenesis, where early oocytes must maintain low ROS levels while still supporting essential mitochondrial functions.

This relationship highlights FOXRED1's importance in the delicate balance between mitochondrial energy production and ROS management during Xenopus oocyte development.

How do mutations in FOXRED1 affect mitochondrial function in model systems?

Mutations in FOXRED1 have profound effects on mitochondrial function in model systems, revealing the protein's essential role in cellular energy metabolism:

Mechanistically, FOXRED1 mutations lead to:

• Disrupted Protein Structure: Mutations can alter the conformation of FOXRED1, as demonstrated by in silico structural analysis using tools like SWISS-MODEL . For example, the missense mutation p.Gly307Glu changes a non-polar glycine to an acidic negatively charged glutamic acid, likely causing local conformational changes that impact function .

• Assembly Defects: Both human patient studies and model systems show that FOXRED1 mutations result in impaired complex I assembly, visualized through techniques like blue native PAGE immunoblot analysis .

• Respiratory Chain Dysfunction: Functional studies reveal decreased complex I-dependent respiration and altered oxygen consumption rates in cells with FOXRED1 mutations .

• Compensatory Responses: Systems with FOXRED1 mutations often show metabolic reprogramming, with increased reliance on alternative energy-generating pathways.

These findings highlight FOXRED1's essential role in mitochondrial function across species and suggest that the Xenopus system could provide valuable insights into the pathophysiology of human FOXRED1-related disorders.

How can CRISPR/Cas9 genome editing be optimized for studying FOXRED1 function in Xenopus laevis?

Optimizing CRISPR/Cas9 genome editing for studying FOXRED1 function in Xenopus laevis requires careful consideration of several technical aspects:

• Guide RNA (gRNA) Design:

  • Target conserved exonic regions of FOXRED1, particularly within the FAD-binding domain

  • Account for Xenopus laevis' allotetraploid genome by designing gRNAs that target both homeologs (L and S chromosomes)

  • Use Xenopus-specific gRNA design tools that consider the species' codon usage and genomic features

  • Validate gRNA specificity using Xenopus genome databases to minimize off-target effects

• Delivery Method Optimization:

  • For early developmental studies: microinject Cas9 protein (or mRNA) together with gRNA into fertilized eggs at the one-cell stage

  • For tissue-specific studies: consider electroporation of Cas9-gRNA ribonucleoprotein complexes into target tissues

  • Optimize injection volumes (typically 2-5 nl) and concentrations (250-500 ng/μl for Cas9 mRNA; 200-400 ng/μl for gRNAs)

• Efficacy Validation:

  • Employ T7 endonuclease I assay or high-resolution melt analysis to assess editing efficiency

  • Use targeted deep sequencing to characterize the spectrum and frequency of induced mutations

  • Verify protein reduction through western blotting with anti-FOXRED1 antibodies

• Phenotypic Analysis:

  • Assess complex I activity using biochemical assays in isolated mitochondria

  • Measure oxygen consumption rates in FOXRED1-edited tissues

  • Evaluate mitochondrial morphology using electron microscopy or live-cell imaging

  • Analyze ROS levels using probes such as MitoSOX Red or MitoTracker Red CM-H2Xros

• Control Strategies:

  • Include rescue experiments with wild-type FOXRED1 mRNA to confirm specificity of observed phenotypes

  • Generate homozygous mutant lines through F1 crossing of founder animals

  • Create tissue-specific conditional knockouts using inducible Cas9 systems

These optimized approaches leverage Xenopus laevis' strengths as a model organism while addressing the specific challenges of FOXRED1 functional studies .

What are the key experimental considerations when comparing FOXRED1 function across developmental stages in Xenopus?

When comparing FOXRED1 function across developmental stages in Xenopus, researchers should address several key experimental considerations:

• Stage-Specific Sampling:

  • Precisely stage embryos according to established Xenopus developmental tables

  • For oocyte studies, carefully classify according to established stages (I-VI)

  • Maintain consistent sampling methods across developmental timepoints

  • Consider both chronological and developmental timing in experimental design

• Normalization Strategies:

  • Account for dramatic changes in cell number, tissue composition, and metabolic activity across development

  • Normalize FOXRED1 expression to appropriate reference genes that remain stable across the developmental stages being studied

  • For protein studies, use multiple loading controls and consider whole-protein normalization methods

• Technical Adaptations:

  • Adjust protein extraction protocols to accommodate the varying yolk content across developmental stages

  • Modify mitochondrial isolation procedures based on tissue composition changes

  • Adapt imaging parameters when studying different sized cells/tissues

• Contextual Analysis:

  • Correlate FOXRED1 function with developmental changes in mitochondrial activity, such as the progression from low complex I activity in early oocytes to higher activity in later stages

  • Consider the changing redox environment, as indicated by glutathione ratios and peroxiredoxin dimerization patterns across development

  • Evaluate FOXRED1 in the context of changing metabolic demands throughout development

• Experimental Controls:

  • Include stage-matched controls for all experiments

  • Consider parallel analysis of other complex I assembly factors to distinguish FOXRED1-specific effects

  • Validate findings using multiple techniques (e.g., protein levels, mRNA expression, functional assays)

By addressing these considerations, researchers can generate more reliable and interpretable data when studying the dynamic role of FOXRED1 throughout Xenopus development.

How can proteomic approaches be used to identify FOXRED1 interaction partners in Xenopus mitochondria?

Proteomic approaches offer powerful tools for identifying FOXRED1 interaction partners in Xenopus mitochondria, providing insights into its functional networks:

• Co-Immunoprecipitation (Co-IP) Coupled with Mass Spectrometry:

  • Generate Xenopus-specific anti-FOXRED1 antibodies or use epitope-tagged recombinant FOXRED1

  • Isolate highly purified mitochondria from Xenopus tissues or embryos

  • Solubilize mitochondrial membranes using mild detergents (digitonin or n-dodecyl β-D-maltoside)

  • Perform immunoprecipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Use label-free quantification or stable isotope labeling techniques for comparative analyses

• Proximity Labeling Approaches:

  • Express FOXRED1 fused to biotin ligases (BioID or TurboID) in Xenopus embryos

  • Allow in vivo biotinylation of proximal proteins

  • Isolate mitochondria and purify biotinylated proteins using streptavidin

  • Identify interaction candidates through mass spectrometry

  • Validate spatial proximity through complementary approaches

• Cross-Linking Mass Spectrometry (XL-MS):

  • Apply membrane-permeable crosslinkers to intact mitochondria

  • Isolate FOXRED1-containing complexes

  • Digest and analyze by specialized XL-MS workflows

  • Identify both stable and transient interaction partners

  • Map interaction interfaces at amino acid resolution

• Comparative Interaction Profiling:

  • Compare FOXRED1 interactomes across developmental stages

  • Analyze interaction changes in response to mitochondrial stress

  • Contrast wild-type versus mutant FOXRED1 interaction networks

  • Study interactome differences between oocytes with varying levels of ROS

• Validation and Functional Characterization:

  • Confirm key interactions through reciprocal Co-IP

  • Visualize co-localization using immunofluorescence microscopy

  • Assess functional relevance through knockdown/knockout of interaction partners

  • Reconstitute interactions using purified components

These proteomic strategies can reveal FOXRED1's role in complex I assembly and potential moonlighting functions in Xenopus mitochondria, providing comparative insights to mammalian systems.

How does Xenopus FOXRED1 research contribute to understanding human mitochondrial disorders?

Xenopus FOXRED1 research provides valuable insights into human mitochondrial disorders through several translational mechanisms:

• Evolutionary Conservation and Functional Parallels:

  • The high conservation of FOXRED1 between Xenopus and humans enables direct extrapolation of functional findings

  • Xenopus studies reveal fundamental mechanisms of complex I assembly that apply across vertebrates

  • Specific pathogenic variants identified in human patients can be modeled and studied in the Xenopus system

• Developmental Context:

  • Xenopus provides an excellent model for studying developmental aspects of FOXRED1 function

  • The progression of mitochondrial maturation during Xenopus development offers insights into tissue-specific manifestations of human FOXRED1-related disorders

  • Embryonic lethality of severe FOXRED1 mutations in mammals can be bypassed using the Xenopus system, allowing study of fundamental mechanisms

• Experimental Advantages:

  • The ability to generate large numbers of embryos facilitates high-throughput screening of potential therapeutic compounds

  • Xenopus oocytes' unique ROS-free environment provides a controlled system to study FOXRED1's role in ROS management, relevant to oxidative stress in human mitochondrial disorders

  • The external development and optical clarity of Xenopus embryos allow real-time visualization of mitochondrial dynamics in FOXRED1-deficient conditions

• Disease Modeling:

  • Human patient mutations (such as c.920G>A/p.Gly307Glu) can be introduced into Xenopus FOXRED1 to study mechanisms of pathogenicity

  • Biochemical defects in complex I assembly and function observed in human patients can be recapitulated and studied mechanistically in Xenopus

  • The effects of environmental factors on FOXRED1-related phenotypes can be readily assessed in the Xenopus system

These translational aspects make Xenopus FOXRED1 research a valuable complement to mammalian models and clinical studies of mitochondrial complex I deficiency.

What methods can be used to compare FOXRED1 function between Xenopus and mammalian systems?

To effectively compare FOXRED1 function between Xenopus and mammalian systems, researchers should employ multiple complementary methodological approaches:

• Comparative Sequence and Structure Analysis:

  • Conduct phylogenetic analysis of FOXRED1 sequences across species

  • Use homology modeling to compare predicted protein structures

  • Identify conserved functional domains and species-specific variations

  • Map human disease mutations onto Xenopus FOXRED1 sequence

• Cross-Species Functional Complementation:

  • Express Xenopus FOXRED1 in FOXRED1-deficient mammalian cells to assess functional conservation

  • Perform reciprocal experiments with human FOXRED1 in Xenopus systems

  • Quantify rescue efficiency through complex I activity assays and oxygen consumption measurements

  • Create chimeric proteins to map species-specific functional domains

• Parallel Experimental Systems:

  • Conduct identical biochemical assays in both systems under standardized conditions

  • Use SDS-PAGE and BN-PAGE immunoblot analysis to compare complex I assembly patterns

  • Employ identical ROS detection methods (e.g., MitoTracker Red CM-H2Xros, MitoSOX Red)

  • Measure mitochondrial function parameters using consistent protocols

• Comparative -Omics Approaches:

  • Compare FOXRED1 interactomes between species using standardized proteomic workflows

  • Analyze transcriptional responses to FOXRED1 deficiency across species

  • Conduct metabolomic profiling to identify conserved and divergent metabolic consequences

  • Use systems biology approaches to map species-specific network differences

• Evolutionary Medicine Perspective:

  • Analyze the impact of equivalent mutations across species

  • Compare tissue-specific expression patterns and developmental regulation

  • Evaluate differences in compensatory mechanisms between species

  • Assess response to potential therapeutic interventions across systems

These approaches enable rigorous cross-species comparison while accounting for inherent biological differences between Xenopus and mammalian systems, providing insights into both conserved and species-specific aspects of FOXRED1 function.

What emerging technologies might advance our understanding of FOXRED1 in mitochondrial biology?

Several emerging technologies hold significant promise for advancing our understanding of FOXRED1 in mitochondrial biology:

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM, SIM) to visualize FOXRED1 localization within mitochondrial subcompartments at nanoscale resolution

  • Live-cell imaging with fluorescent protein-tagged FOXRED1 to track its dynamics during complex I assembly

  • Correlative light and electron microscopy (CLEM) to connect FOXRED1 function with ultrastructural changes

  • Expansion microscopy to physically enlarge specimens for improved visualization of mitochondrial protein complexes

• Next-Generation Biochemical Approaches:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic conformational changes in FOXRED1

  • Cryo-electron microscopy to determine high-resolution structures of FOXRED1 alone and in complex with interaction partners

  • Native mass spectrometry to analyze intact FOXRED1-containing complexes

  • Microfluidic approaches for single-mitochondrion analysis of FOXRED1 function

• Advanced Genetic Engineering:

  • Base editing and prime editing technologies for precise FOXRED1 modification without double-strand breaks

  • Inducible degradation systems (e.g., AID, dTAG) for temporal control of FOXRED1 levels

  • Mitochondrially targeted CRISPR systems for organelle-specific genome editing

  • Synthetic biology approaches to create minimal FOXRED1 variants with engineered functions

• Real-Time Metabolic Sensing:

  • Genetically encoded biosensors for mitochondrial NAD+/NADH ratios to correlate with FOXRED1 activity

  • Fluorescent ROS sensors with improved sensitivity for detecting subtle changes in oxidative stress

  • FRET-based proximity sensors to monitor FOXRED1 interactions in real-time

  • NMR-based metabolic flux analysis to quantify FOXRED1's impact on mitochondrial metabolism

• Integrative Multi-Omics:

  • Single-cell multi-omics to reveal cell-to-cell variability in FOXRED1 function

  • Spatial transcriptomics and proteomics to map FOXRED1 activity across tissues

  • Machine learning approaches to integrate diverse datasets and predict FOXRED1 functions

  • Comparative mitochondrial proteomics across evolutionary distant species to identify core FOXRED1 functions

These emerging technologies, particularly when applied in the advantageous Xenopus model system , promise to reveal new insights into FOXRED1's role in mitochondrial biology and complex I assembly.