Recombinant Xenopus laevis F-box/LRR-repeat protein 15 (fbxl15)

What is FBXL15 and what are its primary molecular functions?

FBXL15, also known as F-box and leucine-rich repeat protein 15 or FBXO37, is a substrate-recognizing subunit of the SCF (Skp1-Cullin1-F-box protein-Roc1) ubiquitin ligase complex. This protein plays a crucial role in protein degradation pathways by recognizing specific target proteins for ubiquitination and subsequent proteasomal degradation .

FBXL15 specifically targets the HECT-type ubiquitin ligase Smurf1 for degradation, thereby regulating Bone Morphogenetic Protein (BMP) signaling pathways. This regulation mechanism involves FBXL15 recognizing the large subdomain within the N-lobe of the Smurf1 HECT domain and promoting the ubiquitination of Smurf1 on lysine residues K355 and K357 within the WW-HECT linker region . Through this mechanism, FBXL15 functions as a positive regulator of BMP signaling in developmental processes and adult bone formation.

How is Xenopus laevis FBXL15 typically purified and stored for research applications?

Recombinant Xenopus laevis FBXL15 is typically provided as a lyophilized powder and requires reconstitution before use . The recommended reconstitution procedure is as follows:

• Briefly centrifuge the vial containing lyophilized protein to bring the contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) to the reconstituted protein

• Aliquot the solution for long-term storage

For storage conditions, the purified protein should be stored at -20°C where it remains stable for approximately one year after shipment . For long-term storage, temperatures of -20°C to -80°C are recommended. Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided as it may compromise protein integrity .

The purity of recombinant FBXL15 preparations typically exceeds 85% as determined by SDS-PAGE analysis .

How does FBXL15 regulate BMP signaling, and what experimental approaches reveal this interaction?

FBXL15 positively regulates BMP signaling through a mechanism involving the targeted degradation of Smurf1, a negative regulator of the BMP pathway. Experimental approaches to investigate this interaction include:

• Co-immunoprecipitation assays: These have demonstrated that FBXL15 interacts with Smurf1 both in vitro and endogenously in cultured cells. Importantly, this interaction occurs regardless of Smurf1's ligase activity, as FBXL15 interacts similarly with both wild-type Smurf1 and the ligase-inactive C699A mutant .

• Subcellular localization studies: Fluorescence microscopy has revealed that FBXL15 and Smurf1 are colocalized in the cytoplasm, supporting their functional interaction in cellular contexts .

• Ubiquitination assays: These have shown that FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of Smurf1's HECT domain and promotes ubiquitination of specific lysine residues (K355 and K357) within the WW-HECT linker region .

• In vivo functional studies: Knockdown of fbxl15 expression in zebrafish embryos using specific antisense morpholinos causes embryonic dorsalization that phenocopies BMP-deficient mutants, providing strong evidence for FBXL15's role in BMP signaling during embryonic development .

• Bone tissue analysis: Injection of FBXL15 siRNAs into rat bone tissues leads to significant loss of bone mass and decreased bone mineral density, highlighting FBXL15's importance in adult bone formation and maintenance .

These experimental approaches collectively demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signaling during both embryonic development and adult bone formation .

What methodologies are optimal for investigating FBXL15 protein-protein interactions?

To effectively investigate FBXL15 protein-protein interactions, researchers should consider the following methodological approaches:

• Affinity purification coupled with mass spectrometry (AP-MS): This approach can identify novel interaction partners of FBXL15 within the SCF complex or among its substrate proteins. Deep proteomics techniques similar to those used for X. laevis egg analysis can be adapted, involving protein digestion with LysC and Trypsin, fractionation with medium pH reverse-phase columns, and analysis by LC-MS .

• Domain mapping experiments: Using truncated versions of FBXL15 and its interaction partners helps identify specific domains responsible for protein-protein interactions. For FBXL15, focus should be on the leucine-rich repeat (LRR) domain, which has been shown to recognize specific substrates like Smurf1 .

• Yeast two-hybrid screening: This can be employed to screen for additional interaction partners of FBXL15, though results should be validated with orthogonal methods due to potential false positives.

• Co-immunoprecipitation with endogenous proteins: Using antibodies specific to FBXL15, such as the polyclonal antibody available commercially (e.g., 20895-1-AP), can help validate physiologically relevant interactions .

• Proximity labeling techniques: Methods such as BioID or APEX can identify proteins in close proximity to FBXL15 in living cells, potentially revealing transient or weak interactions that might be missed by traditional co-immunoprecipitation.

• Fluorescence resonance energy transfer (FRET): This technique can be used to study FBXL15 interactions in living cells, providing spatial and temporal information about protein-protein interactions.

When designing these experiments, consideration should be given to the expression system used for recombinant FBXL15 production, as different systems (E. coli, yeast, baculovirus, or mammalian cells) may affect protein folding and post-translational modifications that influence interaction capabilities .

How can researchers assess the functional differences between duplicated FBXL15 genes in Xenopus laevis?

Xenopus laevis, as an allotetraploid species, contains duplicate copies of many genes including potentially FBXL15, similar to the documented duplication of flt3 and flt3lg genes . To assess functional differences between duplicated FBXL15 genes, researchers can employ these methodological approaches:

These approaches collectively can determine whether duplicated FBXL15 genes in X. laevis have been retained through sub-functionalization, neo-functionalization, or functional redundancy, similar to the patterns observed with duplicated flt3 and flt3lg genes .

What are the optimal conditions for expressing recombinant Xenopus laevis FBXL15 in different expression systems?

The expression of recombinant Xenopus laevis FBXL15 can be achieved in multiple expression systems, each with specific considerations:

E. coli Expression System:

• Strain recommendations: BL21(DE3) or Rosetta for codon optimization

• Induction conditions: 0.5-1 mM IPTG at mid-log phase (OD600 0.6-0.8)

• Growth temperature: 16-18°C post-induction for 16-20 hours to enhance proper folding

• Solubility enhancement: Consider fusion tags such as SUMO, MBP, or GST to improve solubility

• Purification approach: Immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography

Yeast Expression System:

• Strain recommendations: Pichia pastoris or Saccharomyces cerevisiae

• Promoter selection: Strong inducible promoters like AOX1 for P. pastoris or GAL1 for S. cerevisiae

• Growth conditions: Carbon source-dependent induction (methanol for P. pastoris or galactose for S. cerevisiae)

• Post-translational modifications: Better glycosylation pattern than E. coli, though different from native Xenopus patterns

Baculovirus Expression System:

• Cell line recommendations: Sf9 or High Five insect cells

• Infection conditions: MOI of 2-5 for optimal protein production

• Harvest timing: 48-72 hours post-infection

• Advantages: Better post-translational modifications and protein folding than prokaryotic systems

• Considerations: More time-consuming and expensive than E. coli expression

Mammalian Cell Expression System:

• Cell line recommendations: HEK293 or CHO cells

• Transfection method: Lipid-based transfection or viral transduction

• Expression timing: Transient expression (3-5 days) or stable cell line development

• Advantages: Most authentic post-translational modifications and protein folding

• Considerations: Highest cost and longest timeline of all expression systems

For specialized applications requiring biotinylation, the E. coli biotin ligase (BirA) system can be employed to specifically attach biotin to an AviTag peptide integrated into the recombinant FBXL15 construct .

What are common challenges in studying FBXL15 interactions with the SCF complex, and how can they be addressed?

Studying FBXL15 interactions with the SCF complex presents several challenges that researchers should be prepared to address:

Challenge 1: Verifying Complete SCF Complex Formation

• Solution: Implement sequential co-immunoprecipitation assays targeting different components of the SCF complex (Skp1, Cullin1, FBXL15, Roc1) to confirm the presence of all components.

• Alternative approach: Use size exclusion chromatography to isolate and characterize the intact SCF(FBXL15) complex.

Challenge 2: Distinguishing Direct vs. Indirect Interactions

• Solution: Employ in vitro binding assays with purified recombinant proteins to establish direct interactions.

• Methodological consideration: Include appropriate controls using FBXL15 mutants lacking key functional domains to validate specificity.

Challenge 3: Capturing Transient or Dynamic Interactions

• Solution: Use chemical crosslinking coupled with mass spectrometry (XL-MS) to stabilize and identify transient interactions.

• Alternative approach: Employ proximity labeling methods (BioID, APEX) to identify proteins in close proximity to FBXL15 in living cells.

Challenge 4: Substrate Specificity Determination

• Solution: Develop global protein stability (GPS) profiling approaches to identify potential FBXL15 substrates on a proteome-wide scale.

• Validation method: Confirm identified targets through targeted degradation assays in cells with FBXL15 overexpression or knockdown.

Challenge 5: Functional Redundancy with Other F-box Proteins

• Solution: Implement comparative substrate binding assays with closely related F-box proteins.

• Complementary approach: Use combinatorial knockdown or knockout strategies to address potential compensatory mechanisms.

Challenge 6: Tissue-Specific Functions

• Solution: Employ tissue-specific expression systems or conditional knockouts in model organisms.

• Analytical approach: Compare FBXL15 function across different cell types and developmental stages, particularly in contexts where BMP signaling is known to be important .

What quality control measures should be implemented for recombinant Xenopus laevis FBXL15 before experimental use?

Before using recombinant Xenopus laevis FBXL15 in experimental applications, researchers should implement a comprehensive quality control workflow:

• Purity Assessment:

  • SDS-PAGE analysis with Coomassie staining (target: >85% purity)

  • Western blot verification using anti-FBXL15 antibody or tag-specific antibody

  • Mass spectrometry-based identification to confirm protein identity

• Structural Integrity Evaluation:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Size exclusion chromatography to confirm monodispersity and absence of aggregation

  • Dynamic light scattering (DLS) to verify size distribution and homogeneity

• Functional Verification:

  • In vitro SCF complex formation assay using purified components (Skp1, Cullin1, Roc1)

  • Substrate binding assay with known targets (e.g., Smurf1)

  • Ubiquitination activity assay to confirm catalytic functionality of the assembled SCF(FBXL15) complex

• Reproducibility Checks:

  • Batch-to-batch comparison of key parameters (purity, activity, etc.)

  • Stability assessment under various storage conditions (-80°C, -20°C, 4°C)

  • Freeze-thaw stability testing to establish optimal handling procedures

• Application-Specific Controls:

  • For protein-protein interaction studies: Include binding-deficient mutants as negative controls

  • For functional assays: Compare with commercially available FBXL15 preparations

  • For cell-based experiments: Validate cellular uptake and localization using fluorescently labeled protein

A standardized quality control report should document these parameters for each batch of recombinant FBXL15, ensuring experimental reliability and reproducibility.

How does FBXL15 function differ between developmental stages and adult tissues in Xenopus laevis?

The function of FBXL15 varies across developmental stages and adult tissues in Xenopus laevis, reflecting its diverse roles in embryonic development and tissue homeostasis:

Embryonic Development:
During embryonic development, FBXL15 plays a crucial role in BMP signaling regulation, which is essential for proper dorsal-ventral axis formation. This has been demonstrated in zebrafish, where knockdown of fbxl15 expression using specific antisense morpholinos causes embryonic dorsalization that phenocopies BMP-deficient mutants . Similar mechanisms likely operate in Xenopus laevis embryos, where proper BMP signaling gradients are essential for normal development.

Developmental stage-specific analysis would likely reveal:

• Early cleavage stages: Maternal FBXL15 protein presence in the egg, as suggested by deep proteomics studies of the Xenopus laevis egg that have identified thousands of proteins

• Gastrulation: Critical role in dorsal-ventral patterning through regulation of BMP signaling

• Neurulation: Potential involvement in neural development and neural crest formation

• Organogenesis: Tissue-specific functions, particularly in developing skeletal elements

Adult Tissues:
In adult tissues, FBXL15 functions appear to be tissue-specific, with particularly important roles in:

• Bone and Cartilage: Based on studies in mammalian systems, FBXL15 significantly influences bone homeostasis through positive regulation of BMP signaling. Injection of FBXL15 siRNAs into rat bone tissues leads to significant loss of bone mass and decreased bone mineral density . In Xenopus, this role would be particularly relevant in the adult skeleton.

• Reproductive Tissues: The presence of FBXL15 in Xenopus laevis eggs suggests potential roles in gametogenesis or early embryonic development .

• Immune System: Potential involvement in immune regulation, as F-box proteins often regulate signaling pathways involved in immune responses.

To thoroughly investigate these stage- and tissue-specific functions, researchers should employ:

• Temporal expression profiling across developmental stages

• Spatial expression analysis using in situ hybridization

• Tissue-specific conditional knockdown or knockout approaches

• Comparative functional assays in different tissue contexts

What methodologies can be used to investigate post-translational modifications of FBXL15 and their impact on function?

Post-translational modifications (PTMs) of FBXL15 likely play important roles in regulating its function, stability, and interactions. Here are methodological approaches to investigate these modifications:

1. Identification of PTMs:

2. Functional Analysis of PTMs:

3. Structural Impact Analysis:

To understand how PTMs affect FBXL15 structure and function:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to assess conformational changes

• X-ray crystallography or cryo-EM of modified vs. unmodified FBXL15

• In silico molecular dynamics simulations to predict structural impacts of specific PTMs

4. Temporal Dynamics Assessment:

To investigate when and where PTMs occur:

• Synchronize cells and analyze PTM patterns through cell cycle phases

• Assess PTM changes following specific stimuli (e.g., BMP pathway activation)

• Use FRET-based biosensors to monitor PTM dynamics in real-time in living cells

5. Enzymatic Regulation Identification:

To identify enzymes that regulate FBXL15 PTMs:

• Kinase/phosphatase substrate screens

• E3 ligase/deubiquitinase interaction proteomics

• CRISPR screens to identify enzymes affecting FBXL15 stability or function

How can CRISPR-Cas9 gene editing be optimized for studying FBXL15 function in Xenopus laevis?

CRISPR-Cas9 gene editing in Xenopus laevis presents unique considerations due to the species' allotetraploid genome, containing both L (long) and S (short) chromosomes. Here's a comprehensive approach for optimizing CRISPR-Cas9 studies of FBXL15:

1. Guide RNA Design and Validation:

2. Delivery Methods Optimization:

3. Phenotypic Analysis Strategies:

Given FBXL15's role in BMP signaling and development, employ these assessment methods:

• Morphological analysis focusing on dorsal-ventral patterning defects

• In situ hybridization for BMP pathway components and targets

• Immunostaining for phosphorylated Smad1/5/8 to assess BMP pathway activity

• Skeletal staining protocols to assess potential bone and cartilage phenotypes

• RNA-seq analysis to identify global transcriptional changes

4. Addressing Xenopus-Specific Challenges:

5. Functional Validation Approaches:

• Rescue experiments using mRNA injection coding for FBXL15 (resistant to sgRNA targeting)

• Domain-specific mutant rescue to map functional regions

• Smurf1 overexpression to test pathway specificity of phenotypes

• Small molecule manipulation of BMP pathway components to confirm mechanism

By optimizing these parameters, researchers can effectively use CRISPR-Cas9 gene editing to study FBXL15 function in Xenopus laevis, revealing its roles in developmental processes, BMP signaling regulation, and potential sub-functionalization of duplicated genes in this allotetraploid species .

How does Xenopus laevis FBXL15 compare structurally and functionally to its orthologs in other vertebrate species?

Comparative analysis of FBXL15 across vertebrate species reveals important insights into its evolutionary conservation and functional specialization:

Structural Comparison:

Functional Conservation:

FBXL15's role in BMP signaling regulation appears to be evolutionarily conserved across vertebrates:

• Zebrafish: Knockdown of fbxl15 in zebrafish embryos causes dorsalization that phenocopies BMP-deficient mutants, indicating a conserved role in dorsal-ventral patterning through BMP pathway regulation .

• Mammals: In rat models, FBXL15 knockdown leads to significant bone mass loss and decreased bone mineral density, consistent with its role in BMP signaling which is crucial for bone formation and homeostasis .

• Xenopus laevis: While specific functional studies in X. laevis are less documented, the high sequence conservation suggests similar roles in developmental processes and BMP signaling.

Evolutionary Insights:

• Gene Duplication: In X. laevis, the allotetraploid genome may contain duplicated FBXL15 genes (similar to the documented duplication of flt3 and flt3lg genes ), potentially leading to sub-functionalization.

• Sequence Divergence: Comparative sequence analysis could reveal regions under different selective pressures, indicating functionally important domains.

• Expression Pattern Conservation: Similar developmental expression patterns across species would further support functional conservation.

To further investigate evolutionary aspects of FBXL15, researchers should:

• Perform comprehensive phylogenetic analysis of FBXL15 across vertebrate lineages

• Compare substrate specificity (particularly toward Smurf1) across species

• Assess cross-species rescue experiments to test functional equivalence

• Investigate potential differences in post-translational regulation between species

These comparative approaches can reveal how FBXL15 function has been conserved or diversified throughout vertebrate evolution, providing insights into both fundamental mechanisms and species-specific adaptations in BMP signaling regulation .

What insights can be gained from studying the evolution and potential sub-functionalization of duplicated FBXL15 genes in Xenopus laevis?

Studying the evolution and potential sub-functionalization of duplicated FBXL15 genes in Xenopus laevis can provide valuable insights into genome evolution, gene regulation, and developmental biology:

1. Mechanisms of Gene Duplication Retention:

The allotetraploid nature of Xenopus laevis has resulted in the presence of two subgenomes (L and S chromosomes) with duplicated genes. Similar to documented cases of duplicated genes like flt3 and flt3lg , duplicated FBXL15 genes may have been retained through various mechanisms:

• Sub-functionalization: Division of ancestral functions between duplicates

• Neo-functionalization: Acquisition of novel functions by one copy

• Dosage selection: Retention to maintain proper protein expression levels

• Complementary degenerative mutations: Mutations in regulatory regions leading to differential expression patterns

2. Expression Pattern Divergence:

Analysis of differential expression patterns between FBXL15 duplicates might reveal:

3. Functional Divergence Analysis:

Comparing the functional properties of duplicated FBXL15 proteins could reveal:

• Differences in substrate recognition specificity (particularly toward Smurf1)

• Alterations in SCF complex formation efficiency

• Differences in protein stability or post-translational regulation

• Varying abilities to regulate BMP signaling

4. Evolutionary Rate Comparison:

5. Comparative Genomic Context:

Examining the genomic context of duplicated FBXL15 genes might reveal:

• Differential loss of neighboring genes

• Variations in regulatory elements

• Chromosomal environment influences on expression

• Conservation of synteny with other duplicated genes

6. Implications for Understanding Xenopus Biology:

This research holds significance for:

• Understanding the evolution of immune system components and developmental processes

• Elucidating mechanisms of sub-functionalization after whole-genome duplication

• Providing insights into the unique biology of polyploid organisms

• Generating models for studying the consequences of gene duplication in vertebrate evolution

By employing approaches similar to those used in studying duplicated flt3 and flt3lg genes , researchers can gain comprehensive insights into how genome duplication has shaped the evolution of FBXL15 genes and their functions in Xenopus laevis.

What are promising new methodologies that could advance our understanding of FBXL15 function in development and disease?

Several emerging methodologies hold significant promise for advancing our understanding of FBXL15 function in development and disease:

1. Advanced Genome Editing and Functional Genomics:

2. Advanced Protein Analysis Technologies:

3. Systems Biology Approaches:

• Multi-omics integration combining proteomics, transcriptomics, and metabolomics data to place FBXL15 in broader cellular networks

• Mathematical modeling of BMP signaling incorporating FBXL15-mediated regulation

• Computational prediction of FBXL15 substrates based on structural and sequence features

• Network analysis to identify central nodes in FBXL15-regulated pathways

4. Advanced Developmental Biology Tools:

5. Therapeutic Applications:

• Development of small molecules targeting the FBXL15-Smurf1 interaction for potential bone disease therapies

• Gene therapy approaches to modulate FBXL15 levels in disorders with dysregulated BMP signaling

• Biomarker development based on FBXL15 pathway activity in developmental disorders

• Xenopus FBXL15 as a model for human disease-associated variants

6. Translational Research Opportunities:

Expanding our understanding of FBXL15 could have implications for:

• Skeletal disorders, given its role in bone homeostasis

• Developmental abnormalities involving dorsal-ventral patterning

• Potential roles in cancer through regulation of cell cycle and protein metabolism

• Regenerative medicine applications through modulation of BMP signaling pathways

These methodologies represent the cutting edge of molecular and developmental biology research and hold significant promise for advancing our understanding of FBXL15's complex roles in various biological processes.

What are the most important unanswered questions regarding FBXL15 function and regulation in Xenopus laevis?

Despite growing knowledge about FBXL15, several critical questions remain unanswered, particularly in the context of Xenopus laevis biology:

1. Developmental Function and Regulation:

• How are FBXL15 expression and activity regulated throughout Xenopus development?

• Does FBXL15 have maternal effects in early Xenopus embryos, given its presence in the egg proteome ?

• How does FBXL15 function interact with other key developmental signaling pathways beyond BMP?

• What is the specific spatial-temporal expression pattern of FBXL15 during Xenopus development?

2. Evolutionary and Genome Duplication Questions:

• Have the duplicated FBXL15 genes in the allotetraploid X. laevis genome undergone sub-functionalization or neo-functionalization?

• How do the expression patterns and functions of FBXL15.L and FBXL15.S compare?

• What selective pressures have maintained both copies in the genome?

• How does FBXL15 function in X. laevis compare to its diploid relative X. tropicalis?

3. Molecular Mechanism Questions:

• What is the full spectrum of FBXL15 substrates in Xenopus beyond Smurf1?

• How is FBXL15 itself regulated post-translationally in Xenopus cells?

• What are the structural determinants of substrate specificity in Xenopus FBXL15?

• How does FBXL15 function change in different cellular compartments or tissues?

4. Technical and Methodological Questions:

• What are the optimal methods for studying FBXL15 in the context of a tetraploid genome?

• How can CRISPR-Cas9 approaches be optimized for efficient targeting of both FBXL15 homoeologs?

• What reporter systems could best reveal FBXL15 activity dynamics in vivo?

• How can the effects of FBXL15 manipulation be separated from general disruptions to the SCF complex?

5. Comparative Biology Questions:

• How conserved is the FBXL15-Smurf1-BMP signaling axis between amphibians and mammals?

• Are there amphibian-specific functions of FBXL15 related to unique aspects of amphibian development?

• Does FBXL15 play roles in amphibian-specific processes like metamorphosis?

• How do FBXL15 functions compare between aquatic and terrestrial life stages of Xenopus?

6. Translational Research Questions:

• Could insights from Xenopus FBXL15 function inform therapeutic approaches for human developmental or skeletal disorders?

• What insights about human FBXL15 function can be uniquely obtained from the Xenopus model system?

• How do environmental factors affect FBXL15 function in Xenopus, and might this inform environmental health research?

Addressing these questions requires integrative approaches combining genome editing, proteomics, developmental biology techniques, and comparative analyses. The answers would significantly advance our understanding of both fundamental mechanisms of cell signaling regulation and the unique aspects of Xenopus development and physiology.