Recombinant Dicentrarchus labrax F-box/LRR-repeat protein 15 (fbxl15)

What is the basic structure and function of FBXL15 in cellular processes?

FBXL15 (F-Box and Leucine-Rich Repeat Protein 15) functions as a substrate recognition component within SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes. Its structure typically includes an F-box domain that mediates interaction with other SCF components and leucine-rich repeat (LRR) domains responsible for substrate recognition. Research demonstrates that FBXL15 forms a functionally active SCF complex that targets Smurf1 (an E3 ubiquitin ligase) for ubiquitination and proteasomal degradation .

The LRR domains of FBXL15 specifically interact with the HECT domain of Smurf1, binding to the large subdomain within the N-lobe. Through this interaction, FBXL15 promotes the ubiquitination of Smurf1 on specific lysine residues (K355 and K357) within the WW-HECT linker region . This targeting mechanism has significant downstream effects, as Smurf1 negatively regulates BMP signaling. By promoting Smurf1 degradation, FBXL15 positively regulates BMP signaling pathways that are essential for embryonic development and bone formation.

Experimental evidence from zebrafish models indicates that knockdown of fbxl15 causes embryonic dorsalization phenotypes similar to those observed in BMP-deficient mutants, while studies in rat bone tissues show that reduction of FBXL15 leads to significant bone mass loss . These findings demonstrate that FBXL15 plays a conserved role in vertebrate development and tissue homeostasis through its regulation of the ubiquitin-proteasome system.

How should recombinant Dicentrarchus labrax FBXL15 protein be designed for optimal expression?

When designing recombinant Dicentrarchus labrax FBXL15 for experimental purposes, researchers should consider several factors based on successful approaches with mammalian FBXL15 proteins. The design should incorporate appropriate expression systems, purification tags, and sequence considerations.

For expression systems, both mammalian and bacterial platforms have been successfully used with FBXL15 proteins from other species. HEK-293 cells have been employed for mouse FBXL15 expression , while E. coli-based systems using PET28a vectors have been utilized for human FBXL15 . For Dicentrarchus labrax FBXL15, researchers should consider testing both systems to determine which provides optimal yield and proper protein folding.

Regarding purification tags, histidine tags have proven effective for FBXL15 purification across species. Both standard His-tags and 6×His configurations have been successfully implemented. The tag position should be carefully considered, with N-terminal tags potentially interfering less with the C-terminal substrate-binding regions of the protein.

Sequence optimization should account for the codon usage bias of the expression system while maintaining the functional domains of FBXL15. Based on mammalian FBXL15 sequences, researchers should ensure preservation of:

• The F-box domain for interaction with SCF complex components

• The leucine-rich repeat (LRR) domains for substrate recognition

• Any conserved post-translational modification sites

Additionally, researchers may consider incorporating a protease cleavage site between the tag and the protein sequence to allow removal of the tag if it interferes with functional studies. This design approach should yield a recombinant protein suitable for biochemical and functional characterization.

What expression patterns of FBXL15 would be expected in different tissues of Dicentrarchus labrax?

While specific expression data for FBXL15 in Dicentrarchus labrax tissues is not directly reported in the search results, researchers can develop hypotheses and methodological approaches based on the protein's known functions in other vertebrates. The role of FBXL15 in BMP signaling, particularly during development and in bone formation , suggests tissues where its expression might be enriched.

For a comprehensive tissue expression profile, researchers should employ quantitative PCR (qPCR) analysis of FBXL15 mRNA levels across diverse tissues including:

• Developing skeletal structures (particularly during larval stages)

• Brain and neural tissues

• Muscle

• Liver

• Gills

• Reproductive organs

• Swim bladder (given the challenges with swimbladder development in sea bass larvae reported in )

Western blot analysis using antibodies specific to Dicentrarchus labrax FBXL15 (or cross-reactive antibodies if species-specific ones are unavailable) would complement the mRNA analysis to account for post-transcriptional regulation. For cellular localization within tissues, immunohistochemistry or in situ hybridization would be valuable, particularly during key developmental stages.

Given the importance of FBXL15 in embryonic development demonstrated in zebrafish , temporal expression patterns throughout Dicentrarchus labrax development would be particularly informative. Researchers should investigate expression during early embryogenesis, larval development (particularly during the critical first 45 days when mortality is high ), and metamorphosis to juvenile stages.

The data obtained would not only establish baseline expression patterns but also identify tissues for further functional studies and potentially reveal fish-specific expression patterns not observed in mammalian systems.

How do I optimize purification protocols for recombinant Dicentrarchus labrax FBXL15?

Optimizing purification protocols for recombinant Dicentrarchus labrax FBXL15 requires careful consideration of expression conditions, buffer composition, and purification techniques. Based on successful purification of FBXL15 from other species , the following methodological approach is recommended:

Expression and Initial Processing:

• Express His-tagged FBXL15 in the optimized expression system (either HEK-293 cells or E. coli)

• Harvest cells and lyse in buffer containing protease inhibitors to prevent degradation

• Clear lysate by centrifugation (high-speed centrifugation for bacterial systems; lower speed for mammalian cells)

Affinity Purification:

• Apply cleared lysate to Ni-NTA resin pre-equilibrated with binding buffer

• Wash extensively with binding buffer containing low concentrations of imidazole (10-20 mM) to reduce non-specific binding

• Elute with a gradient or step-wise increase of imidazole (up to 300 mM as used for human FBXL15 )

Further Purification (if needed):

• Apply affinity-purified protein to size exclusion chromatography to remove aggregates and improve homogeneity

• Consider ion exchange chromatography if contaminants remain

Quality Control:

• Assess purity by SDS-PAGE with Coomassie brilliant blue staining (target >90% purity )

• Confirm identity by Western blot using anti-His and/or anti-FBXL15 antibodies

• Verify proper folding using circular dichroism or functional binding assays

Storage Considerations:

• For lyophilization, use PBS with 5% trehalose and 5% mannitol as protectants

• For liquid storage, add glycerol (1:1) to prevent freeze-thaw damage

• Store at -20°C to -80°C for long-term stability

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

Optimization will likely require testing different buffer conditions, particularly pH ranges (typically 7.0-8.0) and salt concentrations (typically 150-300 mM NaCl) to identify conditions that maximize stability while maintaining functional activity. If the protein shows limited solubility, additives such as low concentrations of non-ionic detergents or stabilizing agents might improve results.

How does the SCF complex assembly with FBXL15 differ between Dicentrarchus labrax and mammalian systems?

The assembly of the SCF complex with FBXL15 represents a fundamental cellular process that may exhibit both conservation and species-specific adaptations between Dicentrarchus labrax and mammalian systems. While direct comparative data is not provided in the search results, researchers can investigate this question through several methodological approaches.

In mammalian systems, FBXL15 forms a complex with Cullin1, Skp1, and Roc1 to create a functional E3 ubiquitin ligase . The F-box domain of FBXL15 mediates interaction with Skp1, while Cullin1 serves as a scaffold linking Skp1/F-box proteins to the Roc1 RING finger protein that recruits E2 ubiquitin-conjugating enzymes. This architecture is likely conserved in Dicentrarchus labrax due to the fundamental nature of the ubiquitin-proteasome system across eukaryotes.

To investigate potential differences, researchers should:

• Perform sequence analysis of Dicentrarchus labrax SCF components compared to mammalian counterparts, focusing on interaction interfaces

• Conduct co-immunoprecipitation experiments with tagged Dicentrarchus labrax FBXL15 to identify associated proteins and compare to mammalian complexes

• Use yeast two-hybrid or in vitro binding assays to measure interaction affinities between FBXL15 and other SCF components across species

• Develop reconstitution assays with purified components to compare complex assembly kinetics and stability

Potential differences might include:

• Altered binding affinities that could affect complex dynamics and turnover

• Temperature-dependent assembly characteristics reflecting the poikilothermic nature of fish versus homeothermic mammals

• Species-specific regulatory mechanisms controlling complex formation

• Different subcellular localization patterns that might influence substrate accessibility

These experiments would provide insights into evolutionary conservation of E3 ligase complex assembly and potentially reveal adaptations specific to aquatic vertebrates that might be related to their unique physiology or developmental programs.

What is the role of FBXL15 in BMP signaling during Dicentrarchus labrax embryonic development?

Investigating the role of FBXL15 in BMP signaling during Dicentrarchus labrax embryonic development represents an important research direction given the crucial function of BMP pathways in vertebrate development. Based on evidence from zebrafish showing that fbxl15 knockdown causes embryonic dorsalization , a similar role might be expected in sea bass, though with species-specific features.

A comprehensive experimental approach would include:

• Expression analysis during development:

  • Temporal profiling of FBXL15 expression throughout embryogenesis using qPCR and in situ hybridization

  • Correlation with expression patterns of BMP pathway components (BMP ligands, receptors, Smads)

  • Spatial mapping in relation to developmental events where dorsoventral patterning occurs

• Functional manipulation:

  • Morpholino-mediated knockdown of FBXL15 during early development

  • CRISPR-Cas9 genome editing to generate FBXL15 mutants if technically feasible

  • Rescue experiments using mammalian FBXL15 to test functional conservation

• Pathway analysis:

  • Assessment of BMP signaling activity using phospho-Smad1/5/8 immunostaining

  • Analysis of BMP target gene expression in control versus FBXL15-depleted embryos

  • Investigation of Smurf1 protein levels and stability in relation to FBXL15 expression

• Phenotypic characterization:

  • Detailed morphological analysis of embryos with altered FBXL15 expression

  • Special attention to dorsoventral patterning, neural development, and early skeletal formation

  • Potential effects on swimbladder development, which is problematic in sea bass larvae

The experimental design should account for species-specific developmental timing and morphological features of Dicentrarchus labrax embryos. Additionally, researchers should consider the culture conditions for sea bass embryos, potentially using the closed recirculation systems with artificial seawater described in reference , which achieved 22% survival at day 45 of development.

This research would not only illuminate the evolutionary conservation of FBXL15 function in BMP signaling but might also provide insights relevant to aquaculture applications, particularly regarding early development challenges in sea bass.

How does substrate specificity of FBXL15 compare between fish and mammalian species?

Understanding the substrate specificity of FBXL15 across vertebrate lineages provides insights into both conserved cellular mechanisms and potential evolutionary adaptations. In mammalian systems, FBXL15 has been shown to specifically target Smurf1 for ubiquitination, recognizing the large subdomain within the N-lobe of the Smurf1 HECT domain via its leucine-rich repeat (LRR) domains . Whether this specificity is conserved in Dicentrarchus labrax and other fish species represents an important evolutionary question.

To compare substrate specificity between fish and mammalian FBXL15, researchers should implement a multi-faceted approach:

• Comparative biochemical analysis:

  • Express and purify recombinant Dicentrarchus labrax FBXL15 and potential substrates

  • Perform in vitro ubiquitination assays with reconstituted SCF^FBXL15 complexes from both fish and mammalian sources

  • Compare ubiquitination kinetics and patterns using purified components from different species

• Structural biology approaches:

  • Generate structural models of fish FBXL15 LRR domains based on mammalian templates

  • Identify potential differences in substrate-binding interfaces

  • Use mutagenesis to test the functional significance of divergent residues

• Proteomic identification of substrates:

  • Perform immunoprecipitation of FBXL15 from Dicentrarchus labrax cells followed by mass spectrometry

  • Compare ubiquitinome changes upon FBXL15 manipulation in fish versus mammalian cells

  • Validate candidate substrates through biochemical and functional assays

• Evolutionary analysis:

  • Analyze selection pressures on FBXL15 substrate-binding regions across vertebrate lineages

  • Identify lineage-specific adaptations that might reflect novel substrate interactions

  • Correlate with the evolution of key developmental and physiological processes

The results might reveal:

• Core conserved substrates (likely including Smurf1) that reflect fundamental aspects of cellular regulation

• Fish-specific substrates that could be related to adaptation to aquatic environments

• Differences in recognition mechanisms or ubiquitination patterns that might influence substrate fate

This comparative approach would contribute to our understanding of how E3 ubiquitin ligase networks have evolved across vertebrates while maintaining essential regulatory functions in development and physiology.

What methods are most appropriate for studying FBXL15 knockout effects in Dicentrarchus labrax?

Studying FBXL15 knockout effects in Dicentrarchus labrax presents distinct challenges compared to model organisms, necessitating creative methodological approaches. The longer generation time, limited genomic resources, and potential embryonic lethality of complete FBXL15 loss (suggested by zebrafish studies ) require researchers to consider alternative strategies to conventional knockout methods.

Recommended methodological approaches:

• Transient knockdown in embryos:

  • Morpholino antisense oligonucleotides targeting FBXL15 mRNA can be microinjected into fertilized eggs

  • CRISPR-Cas9 ribonucleoproteins can be injected for mosaic gene disruption

  • Phenotypic analysis should focus on early developmental processes, particularly dorsoventral patterning and BMP-dependent processes

• Cell culture models:

  • Establish primary cell cultures from Dicentrarchus labrax tissues

  • Use siRNA or shRNA approaches for transient FBXL15 knockdown

  • Analyze effects on BMP signaling, Smurf1 stability, and cellular ubiquitination patterns

• Conditional approaches:

  • Develop heat-shock or chemically inducible FBXL15 knockdown systems

  • Engineer dominant-negative FBXL15 constructs (e.g., F-box deletion mutants) for inducible expression

  • Target specific domains of FBXL15 to disrupt particular functions while preserving others

• Partial genomic modification:

  • Rather than complete gene deletion, target specific functional domains or regulatory elements

  • Generate hypomorphic alleles that reduce but don't eliminate FBXL15 function

  • Create specific mutations that affect particular aspects of FBXL15 function, such as substrate binding

• Pharmacological approaches:

  • Use small molecule inhibitors of SCF complex function as chemical surrogates for genetic manipulation

  • Develop targeted protein degradation approaches (PROTACs) specific to FBXL15

  • Combine with partial genetic approaches for enhanced specificity

For any approach, researchers should:

• Include appropriate controls to rule out off-target effects

• Validate knockdown/knockout efficiency at both mRNA and protein levels

• Perform rescue experiments to confirm phenotype specificity

• Consider compensatory mechanisms that might mask acute phenotypes

These methods can be implemented in the context of the closed recirculation systems with artificial seawater described for sea bass larval culture , which provide controlled conditions for developmental studies. The choice of approach should be guided by the specific research question, available resources, and ethical considerations regarding animal experimentation.

How can I design experiments to study the interaction between FBXL15 and the ubiquitin-proteasome system in Dicentrarchus labrax?

Designing experiments to study the interaction between FBXL15 and the ubiquitin-proteasome system (UPS) in Dicentrarchus labrax requires a systematic approach that addresses both biochemical mechanisms and physiological outcomes. Based on knowledge of mammalian FBXL15 function within SCF complexes , researchers can develop targeted experiments that reveal conserved and species-specific aspects of these interactions.

Experimental Design Framework:

• Biochemical Characterization:

  • Reconstitute the SCF^FBXL15 complex using purified recombinant components from Dicentrarchus labrax

  • Test ubiquitination activity with different E2 enzymes (UbcH5c and UbcH7 have been shown to work with mammalian FBXL15 )

  • Identify ubiquitination sites on candidate substrates (particularly Smurf1) using mass spectrometry

  • Compare ubiquitin chain topologies (K48 vs. K63 linkages) to determine degradation vs. signaling outcomes

• Cellular Analysis:

  • Develop sea bass cell culture systems amenable to transfection

  • Generate fluorescent reporters for monitoring proteasome activity

  • Track substrate protein turnover using cycloheximide chase experiments

  • Measure FBXL15-dependent changes in global ubiquitination patterns

• Physiological Studies:

  • Manipulate FBXL15 expression during specific developmental windows

  • Monitor effects on BMP-dependent processes (particularly those relevant to sea bass development )

  • Investigate tissue-specific consequences of altered UPS activity

  • Examine environmental influences (temperature, salinity) on FBXL15-UPS interactions

Technical Considerations:

Controls and Validation:

• Use proteasome inhibitors (MG132, bortezomib) to confirm involvement of the proteasome

• Include ligase-inactive FBXL15 mutants as negative controls

• Perform parallel experiments in mammalian systems for comparison

• Validate key findings using multiple complementary approaches

This experimental framework would provide insights into both the mechanistic details of FBXL15 function within the UPS and the physiological consequences of this regulation in Dicentrarchus labrax, potentially revealing adaptations specific to fish biology.

What challenges should be anticipated when investigating post-translational modifications of FBXL15 in Dicentrarchus labrax?

Investigating post-translational modifications (PTMs) of FBXL15 in Dicentrarchus labrax presents several technical and conceptual challenges that researchers should anticipate and address in their experimental design. While the search results don't provide specific information about FBXL15 PTMs, this represents an important research frontier for understanding regulatory mechanisms.

Key Challenges and Mitigation Strategies:

• Limited species-specific reagents:

  • Challenge: Lack of Dicentrarchus labrax-specific antibodies for detecting modified FBXL15.

  • Strategy: Develop custom antibodies against predicted PTM sites or use tag-based approaches (e.g., epitope-tagged FBXL15 expressed in sea bass cells).

• PTM site identification:

  • Challenge: Insufficient protein sequence database coverage for Dicentrarchus labrax.

  • Strategy: Use de novo sequencing approaches in mass spectrometry and cross-reference with better-annotated fish genomes.

• Environmental influences on PTMs:

  • Challenge: Fish PTM patterns may vary with environmental conditions (temperature, salinity, oxygen levels).

  • Strategy: Design experiments that systematically control and vary environmental parameters to assess their impact on FBXL15 modifications.

• Low abundance of modified forms:

  • Challenge: PTM-bearing proteins often represent a small fraction of the total protein pool.

  • Strategy: Implement enrichment techniques such as phosphopeptide enrichment (TiO₂, IMAC) or ubiquitin remnant antibodies before analysis.

• PTM crosstalk and dynamics:

  • Challenge: Multiple PTMs may interact functionally and show complex temporal dynamics.

  • Strategy: Use time-course experiments and multiple PTM detection methods in parallel.

Methodological Approach:

• Prediction and conservation analysis:

  • Identify potential PTM sites in Dicentrarchus labrax FBXL15 based on sequence conservation with mammalian counterparts

  • Focus on key regulatory regions such as the F-box domain and substrate-binding regions

• Mass spectrometry-based detection:

  • Express and purify FBXL15 from Dicentrarchus labrax cells under various conditions

  • Perform comprehensive PTM mapping using high-resolution mass spectrometry

  • Use different protease digestions to improve sequence coverage

• Functional validation:

  • Generate site-specific mutants of predicted PTM sites (e.g., Ser/Thr to Ala for phosphorylation sites)

  • Test effects on FBXL15 stability, localization, and substrate recognition

  • Identify enzymes responsible for PTM addition and removal

• Comparative analysis:

  • Compare PTM patterns between Dicentrarchus labrax FBXL15 and mammalian counterparts

  • Correlate differences with functional or environmental adaptations

This research would provide insights into how PTMs regulate FBXL15 function in fish species and potentially reveal evolutionary adaptations in ubiquitin ligase regulation across vertebrate lineages. Understanding these modifications could also help explain tissue-specific or developmental-stage-specific functions of FBXL15 in sea bass.

How does FBXL15 structure and function compare across vertebrate species?

Comparative analysis of FBXL15 across vertebrate species provides valuable insights into both conserved functional mechanisms and lineage-specific adaptations. Based on available data, we can construct a comparative framework for FBXL15 research across species.

The primary structure of FBXL15 shows significant conservation in key functional domains across vertebrates. Mammalian FBXL15 proteins are approximately 300 amino acids in length , with highly conserved F-box and leucine-rich repeat (LRR) domains. While specific Dicentrarchus labrax FBXL15 sequence data is not provided in the search results, we can analyze the available mammalian sequences as reference points.

Table 1: Comparative Analysis of FBXL15 Proteins Across Species

At the functional level, FBXL15 demonstrates a conserved role in BMP signaling regulation across vertebrates. In zebrafish, knockdown of fbxl15 causes embryonic dorsalization phenotypes similar to BMP-deficient mutants , suggesting that the regulatory relationship between FBXL15 and BMP signaling emerged early in vertebrate evolution and has been maintained.

The molecular mechanism of FBXL15 action involves several conserved features:

• Formation of SCF E3 ubiquitin ligase complexes with Cullin1, Skp1, and Roc1

• Recognition of Smurf1 via the LRR domains, specifically binding to the large subdomain of the HECT N-lobe

• Promotion of Smurf1 ubiquitination on specific lysine residues (K355 and K357 in mammalian Smurf1)

• Enhancement of BMP signaling through Smurf1 degradation

This molecular mechanism likely represents a core conserved function of FBXL15 across vertebrates, though species-specific variations in substrate recognition, regulation, or additional functions may exist, particularly in lineages that diverged early such as fish.

Further comparative research could reveal important evolutionary insights into the ubiquitin-proteasome system and its role in developmental signaling pathways across vertebrate species.

What methodological approaches are optimal for studying FBXL15 in sea bass larval development?

Given the challenges in studying Dicentrarchus labrax larval development and the specific research interest in FBXL15, researchers need carefully optimized methodological approaches. The high mortality rate during larval stages and developmental challenges such as swimbladder abnormalities make this a technically demanding research area.

Optimal Culture Conditions:

Based on successful sea bass larval culture systems described in the search results, researchers should implement:

• Completely closed recirculation systems with artificial seawater

• Controlled water quality parameters (see Table 2)

• Carefully managed bacterial populations to reduce pathogenic load

• Precise staging of developmental processes

Table 2: Optimal Water Quality Parameters for Sea Bass Larval Culture

FBXL15 Analysis During Development:

To study FBXL15 specifically during sea bass development, researchers should employ:

• Expression analysis:

  • Quantitative PCR at defined developmental stages

  • Whole-mount in situ hybridization to visualize spatial expression patterns

  • Protein detection using custom antibodies or epitope-tagged constructs

• Functional perturbation:

  • Microinjection of antisense morpholinos into fertilized eggs

  • CRISPR-Cas9 delivery for targeted gene editing

  • mRNA injection for rescue or overexpression studies

• Phenotypic assessment:

  • Focus on processes known to involve BMP signaling

  • Special attention to dorsoventral patterning

  • Detailed analysis of swimbladder development, given its problematic nature in sea bass larvae

  • Assessment of skeletal development based on FBXL15's role in bone formation

• Molecular pathway analysis:

  • Monitor Smurf1 protein levels and stability

  • Assess BMP signaling activity through phospho-Smad detection

  • Analyze expression of BMP target genes

These approaches should be adapted to the specific challenges of working with sea bass larvae, including their relatively large size compared to model fish species, potentially different developmental timing, and species-specific technical requirements for manipulation and culture.

By combining optimal larval culture conditions with molecular and genetic approaches tailored to Dicentrarchus labrax, researchers can effectively investigate FBXL15 function during sea bass development.