Recombinant Mouse F-box only protein 10 (Fbxo10), partial

What is FBXW10 and what is its role in cellular processes?

FBXW10 (F-Box and WD Repeat Domain Containing 10) functions as a probable substrate-recognition component of an SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex. This complex mediates the ubiquitination and subsequent proteasomal degradation of target proteins. Research indicates that overexpression of FBXW10 leads to the degradation of chromobox proteins CBX5 and CBX1, suggesting its involvement in chromatin regulation pathways . The protein contains both an F-box motif, which interacts with SKP1, and WD-repeat domains, which are involved in substrate recognition for ubiquitination .

How does FBXW10 differ from other F-box family proteins?

FBXW10 belongs to the FBXW subfamily of F-box proteins that contain WD repeat domains, distinguishing it from FBXO proteins (F-box only) and FBXL proteins (containing leucine-rich repeats). Like other F-box proteins, FBXW10 forms part of an SCF complex, but its specific WD-repeat structure determines its unique substrate selectivity. Unlike the well-characterized F-box protein Skp2, which targets cell cycle regulators and is itself regulated during the cell cycle, FBXW10 has been associated with spermatogenic failure and primary failure of tooth eruption . Additionally, FBXW10 is involved in Class I MHC mediated antigen processing and presentation pathways, suggesting roles in immune function that differentiate it from other F-box proteins .

What is the significance of using a "partial" recombinant protein for research?

The partial recombinant mouse FBXW10 protein (amino acids 701-1030) covers a specific functional region of the full protein while excluding others. This partial construct contains critical domains involved in protein-protein interactions necessary for substrate recognition. Working with partial proteins offers technical advantages including improved solubility, stability, and expression efficiency compared to full-length proteins which may be difficult to express in prokaryotic systems. Researchers should note that this partial construct (Expression Region: 701-1030aa) represents approximately 30% of the complete protein sequence and includes functional domains critical for studying substrate interactions .

What are the optimal storage and handling conditions for recombinant FBXW10?

Recombinant mouse FBXW10 protein stability is highly dependent on proper storage and handling. For optimal results, store the protein in its supplied Tris-based buffer with 50% glycerol at -20°C/-80°C, where liquid formulations maintain stability for approximately 6 months, while lyophilized forms remain stable for up to 12 months . Avoid repeated freeze-thaw cycles, as these can significantly reduce protein activity through structural degradation. For ongoing experiments, prepare working aliquots stored at 4°C that should be used within one week. When handling the protein, maintain cold chain conditions and minimize exposure to proteases by using protease inhibitors when appropriate during experimental procedures .

How can I verify the activity of recombinant FBXW10 in experimental setups?

Verification of FBXW10 activity requires assessment of its ability to form functional SCF complexes and mediate substrate ubiquitination. Design an in vitro ubiquitination assay containing E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, ubiquitin, ATP, and potential substrates such as CBX5 or CBX1 . Monitor substrate ubiquitination using western blotting with anti-ubiquitin antibodies. Additionally, perform co-immunoprecipitation experiments to confirm FBXW10's interaction with other SCF components (SKP1, CUL1, and ROC1/RBX1). For cellular systems, overexpress FBXW10 and measure the degradation rates of known substrates, comparing these with proteasome inhibitor-treated controls to confirm the specificity of the degradation pathway .

What experimental controls should be included when studying FBXW10-mediated ubiquitination?

When investigating FBXW10-mediated ubiquitination, implement a comprehensive set of controls to ensure reliable results. Include a reaction omitting ATP to confirm energy-dependent ubiquitination, reactions lacking E1, E2, or FBXW10 to verify the requirement of each component, and reactions with proteasome inhibitors (such as MG132 or LLnL) to demonstrate proteasome-dependent degradation of ubiquitinated substrates . For substrate specificity controls, use mutant versions of putative substrates with altered recognition sites. Additionally, incorporate F-box mutant versions of FBXW10 that cannot bind SKP1 and form functional SCF complexes as negative controls. For cellular experiments, use both wild-type and F-box mutant FBXW10 to distinguish between SCF-dependent and independent effects, similar to approaches used with other F-box proteins like Skp2 .

How does FBXW10 substrate specificity compare to other F-box proteins like Skp2?

FBXW10 and Skp2 exhibit distinct substrate recognition mechanisms despite sharing the core F-box domain that mediates interaction with the SCF complex. While Skp2 predominantly targets cell cycle regulators like p27 through phosphorylation-dependent recognition, FBXW10 has been shown to target chromatin regulators including CBX5 and CBX1 . The substrate specificity of FBXW10 is determined by its WD-repeat domains, which form a β-propeller structure that creates a binding platform for specific substrate proteins. Unlike Skp2, which shows cell cycle-dependent expression and activity, current data doesn't indicate strong cell cycle regulation of FBXW10 . Researchers investigating FBXW10 substrate specificity should consider employing protein-protein interaction studies, mass spectrometry-based approaches, and cellular degradation assays to identify and validate novel FBXW10 substrates distinct from those targeted by other F-box proteins .

What structural elements of FBXW10 are critical for its function in the SCF complex?

Critical structural elements of FBXW10 include the F-box domain and WD-repeat regions, each serving distinct functions within the SCF complex architecture. The F-box domain (approximately 40 amino acids) mediates binding to SKP1, which acts as an adapter to connect FBXW10 to the CUL1 scaffold protein . The WD-repeat regions form a β-propeller structure that creates a substrate recognition interface essential for target protein binding. Analysis of other F-box proteins suggests that disruption of the F-box domain prevents incorporation into functional SCF complexes, while mutations in substrate recognition domains maintain SCF assembly but impair substrate binding . Researchers investigating FBXW10 structure-function relationships should consider generating specific mutants: F-box domain mutants to disrupt SKP1 binding, WD-repeat mutants to alter substrate specificity, and combinatorial mutants to understand interdomain dependencies .

How can mass spectrometry be optimized for identifying novel FBXW10 substrates?

Optimizing mass spectrometry for novel FBXW10 substrate identification requires a multi-faceted approach. Begin by expressing tagged FBXW10 in an appropriate cell system and treat cells with proteasome inhibitors (MG132 or LLnL) to stabilize ubiquitinated substrates . Perform immunoprecipitation of FBXW10 under conditions that preserve protein-protein interactions, followed by on-bead tryptic digestion or elution and subsequent digestion. For comparison, use both wild-type FBXW10 and a substrate-binding deficient mutant (with altered WD-repeats) to differentiate genuine substrates from background interactors. Employ quantitative approaches such as SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling to compare protein abundance between experimental conditions. During data analysis, focus on proteins enriched in wild-type samples and apply ubiquitination site mapping to confirm direct regulation. Validate candidates through follow-up biochemical assays including in vitro ubiquitination and cellular degradation experiments .

What strategies can overcome poor solubility of recombinant FBXW10 in experimental buffers?

Poor solubility of recombinant FBXW10 is a common technical challenge that can be addressed through multiple approaches. First, optimize buffer conditions by testing various pH ranges (6.5-8.5), salt concentrations (150-500 mM NaCl), and additions of solubility enhancers like 5-10% glycerol, 0.1% Triton X-100, or 1-5 mM DTT to prevent aggregation . Consider using the partial construct (amino acids 701-1030) with the N-terminal 6xHis-SUMO tag, which significantly improves solubility compared to full-length protein . For expression, lower induction temperatures (16-18°C) and reduced IPTG concentrations can promote proper folding. If purifying from inclusion bodies is necessary, use denaturing conditions followed by step-wise dialysis for refolding. Alternatively, consider switching to eukaryotic expression systems like insect or mammalian cells for complex proteins. During experimental procedures, maintain the protein in its optimized buffer and only dilute into experimental buffers immediately before use .

How can researchers address inconsistent results in FBXW10-mediated ubiquitination assays?

Inconsistent results in FBXW10-mediated ubiquitination assays can stem from multiple factors that require systematic troubleshooting. First, verify protein quality through SDS-PAGE and activity assays, as degraded or improperly folded FBXW10 will yield variable results . Ensure all components of the ubiquitination machinery (E1, E2, ubiquitin, ATP) maintain consistent activity between experiments by using fresh reagents and conducting parallel control reactions with well-characterized F-box proteins like Skp2 . Standardize experimental conditions including temperature, incubation times, and component concentrations, and consider time-course experiments to identify optimal reaction durations. For cellular assays, account for variations in transfection efficiency, protein expression levels, and cell cycle distribution, particularly since F-box protein activity can be cell cycle-dependent . Finally, validate results using multiple detection methods, such as western blotting with different antibodies and ubiquitin chain-specific antibodies to characterize ubiquitination patterns .

What experimental considerations are important when comparing mouse FBXW10 function to human FBXW10?

When comparing mouse and human FBXW10 functions, researchers must account for several key considerations to ensure valid cross-species comparisons. Although both proteins share conserved F-box and WD-repeat domains, sequence variations may affect substrate specificity, binding affinity, and regulation. Conduct detailed sequence alignment and structural analysis to identify conserved and divergent regions that might influence function . For experimental design, express both species' proteins under identical conditions and compare their biochemical properties including stability, complex formation with SCF components, and substrate binding profiles. When examining substrate targeting, test both proteins against potential substrates from each species to identify any species-specific interactions. In cellular systems, ensure appropriate cellular contexts by using matched cell lines from each species or by complementing knockout systems. Finally, consider potential differences in post-translational modifications, expression patterns, and regulation networks between species that might affect experimental outcomes .

How might FBXW10 contribute to disease mechanisms based on its known functions?

Based on current knowledge, FBXW10's role in the ubiquitin-proteasome pathway suggests several potential disease mechanisms worthy of investigation. The association of FBXW10 with Spermatogenic Failure 64 indicates its importance in reproductive biology, potentially through the regulation of proteins essential for spermatogenesis . Its involvement in primary failure of tooth eruption suggests developmental roles that might extend to other tissues and organs . FBXW10's ability to target chromatin regulators CBX5 and CBX1 for degradation implies possible roles in epigenetic dysregulation, which could contribute to developmental disorders or cancer progression through altered gene expression patterns . Additionally, FBXW10's connection to Class I MHC antigen processing and presentation pathways suggests potential immunological functions that, when dysregulated, might contribute to autoimmune conditions or immune evasion in cancer . Future research should explore these connections through animal models, patient-derived samples, and comprehensive substrate identification studies to develop potential therapeutic approaches targeting FBXW10-dependent pathways.

What genomic and proteomic approaches would best identify the complete set of FBXW10 substrates?

Identifying the complete set of FBXW10 substrates requires an integrated genomic and proteomic approach. Begin with proteomic strategies including proximity-dependent biotin identification (BioID) or APEX2 labeling coupled with FBXW10 to identify proteins in close proximity to FBXW10 in living cells. Follow with global protein stability profiling using methods like GPS (Global Protein Stability) or MUGC (Multiplexed Global Ubiquitination Correlation) to identify proteins whose stability is affected by FBXW10 expression or depletion . On the genomic side, perform RNA-seq and ChIP-seq following FBXW10 manipulation to identify transcriptional changes and altered chromatin states, particularly focusing on regions where CBX5 and CBX1 (known FBXW10 substrates) normally bind . Complement these approaches with computational prediction methods that identify potential degron sequences recognized by FBXW10's WD-repeat domains. Finally, integrate these multi-omic datasets using systems biology approaches to build comprehensive models of FBXW10-regulated networks, prioritizing candidates for validation through biochemical and cellular assays .

How might engineering FBXW10 variants advance targeted protein degradation technologies?

Engineering FBXW10 variants holds promising potential for advancing targeted protein degradation technologies, particularly in therapeutic applications. By modifying the substrate recognition domains (WD-repeats) while maintaining the F-box domain, researchers could create chimeric FBXW10 proteins that recruit specific disease-relevant proteins to the SCF complex for ubiquitination and degradation . This approach could be further enhanced by combining FBXW10 engineering with PROTAC (Proteolysis Targeting Chimera) technology, where the engineered FBXW10 substrate-binding domain is fused to ligands for target proteins, creating bifunctional molecules that bring specific targets to the degradation machinery . Additionally, developing conditionally active FBXW10 variants through incorporation of drug-responsive domains or light-sensitive switches would enable temporal control of protein degradation in research and therapeutic settings. The partial FBXW10 construct (aa 701-1030) provides an excellent starting point for such engineering efforts, as it contains the critical domains while offering improved expression and stability characteristics compared to the full-length protein .