Recombinant Mouse F-box/LRR-repeat protein 13 (Fbxl13)

What is the molecular structure and basic function of mouse Fbxl13?

Mouse F-box/LRR-repeat protein 13 (Fbxl13) belongs to the F-box protein family characterized by an approximately 40 amino acid F-box motif and leucine-rich repeat domains. The protein functions as part of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex, which targets specific proteins for ubiquitination and subsequent proteasomal degradation .

Key structural details:

• Gene ID: 102862766

• Protein length: 477 amino acids (based on ortholog data)

• Functional domains: F-box domain and leucine-rich repeats

• Cellular localization: Primarily centrosomal

For experimental work, recombinant mouse Fbxl13 can be produced in expression systems similar to other F-box proteins, with the protein-coding region of the Fbxl13 cDNA ORF encoded by the open reading frame sequence .

How does Fbxl13 function within the ubiquitin-proteasome system?

Fbxl13 operates within the ubiquitin-proteasome pathway through the following mechanism:

• As an F-box protein, Fbxl13 serves as the substrate recognition component of the SCF complex

• The F-box domain interacts with Skp1 to integrate into the SCF complex

• The leucine-rich repeat domains recognize and bind specific substrate proteins

• Once bound, the SCF complex facilitates the transfer of ubiquitin to the target substrate

• Polyubiquitinated proteins are subsequently recognized and degraded by the 26S proteasome

Research demonstrates that Fbxl13 can interact directly with centrosomal proteins including Centrin-2, Centrin-3, CEP152, and CEP192, with biochemical assays confirming these as bona fide substrates . In particular, FBXL13 directly binds to CEP192 through interaction with amino acids 1-630, which in turn recruits CEP152 via the centriole binding region located between amino acids 221 and 1,319 .

What are the optimal expression systems for producing recombinant mouse Fbxl13?

For recombinant mouse Fbxl13 production, several expression systems have been evaluated, with their advantages and limitations outlined below:

Methodology for optimal expression in Pichia pastoris (recommended for functional studies):

• Clone the Fbxl13 cDNA into an appropriate yeast expression vector (e.g., vectors containing AOX1 promoter)

• Transform into competent P. pastoris cells

• Select transformants on appropriate media

• Induce expression using methanol

• Harvest and purify using affinity chromatography

• Validate identity through mass spectrometry and activity through functional assays

For studies requiring complex formation with other SCF components, co-expression systems in mammalian cells may provide the most physiologically relevant preparation .

What genetic knockout strategies have been used to study Fbxl13 function in mice?

Several knockout approaches have been employed to study Fbxl13 function in vivo, with CRISPR/Cas9 proving most effective:

The established knockout protocol involves:

• Designing guide RNAs targeting exons 1 and 7 of Fbxl13

• Introducing these gRNAs with Cas9 into embryonic stem cells

• Screening for clones with large deletions

• Injecting successful clones into 8-cell embryos

• Breeding chimeric mice to obtain heterozygous and then homozygous mutants

• Confirming gene deletion through genomic PCR and RT-PCR

A successful Fbxl13 knockout strategy documented in the literature incorporated:

• gRNAs targeting exon 1 and exon 7 to avoid cleavage of nearby genes

• Using pX459 V2.0 plasmid (#62988, Addgene) for delivery

• Transfection into EGR-G01 embryonic stem cells

• Screening 48 ES clones to identify 9 with large deletions

• Injection into ICR embryos

• Mating chimeric mice with B6D2F1 females to establish the line

After knockout generation, RT-PCR with primers designed within exon 1 and exon 20 confirmed the absence of Fbxl13 mRNA in knockout mouse testis .

What phenotypes are observed in Fbxl13 knockout mice?

Comprehensive phenotypic analysis of Fbxl13 knockout mice has revealed several important findings:

• General development: No overt abnormalities were observed in -/- Fbxl13 mice

• Reproductive function:

  • Male fertility: Fbxl13-/- male mice sired a comparable number of pups to wild-type males

  • Spermatogenesis: Normal testis morphology by PAS staining of seminiferous tubules

  • Sperm morphology: Normal head and tail morphology in spermatozoa from cauda epididymis

  • Sperm motility: Computer-Assisted Sperm Analysis (CASA) showed no significant differences in motility rates, progressive motility rates, or velocity parameters (VAP, VSL, VCL)

• Ciliogenesis:

  • Tracheal cilia: Scanning electron microscopy showed normal ciliary morphology

  • Ultrastructure: Transmission electron microscopy revealed normal '9+2' axonemal structure with both inner and outer dynein arms in tracheal cilia

This phenotypic analysis suggests that despite the presumed role of Fbxl13 in protein degradation pathways, it may have redundant functions with other F-box proteins or plays a role in specialized contexts not apparent under standard laboratory conditions.

How does Fbxl13 regulate centrosome function and cell motility?

Fbxl13 plays a critical role in centrosome regulation through its interaction with key centrosomal proteins:

• Substrate targeting: FBXL13 specifically targets CEP192 isoform 3 for degradation, while not affecting Centrin-2 and Centrin-3 despite binding to them

• Ubiquitination mechanism:

  • The SCF^FBXL13 complex polyubiquitylates CEP192 isoform 3

  • This requires an intact F-box domain for recruitment of the ubiquitination machinery

  • Pull-down experiments demonstrated that FBXL13 downregulates centrosomal CEP192 and γ-tubulin

• Functional impact on cell motility:

  • FBXL13 knockdown significantly reduces the migration rate of U2OS cells in scratch assays

  • This phenotype can be rescued by expressing siRNA-resistant wild-type FBXL13

  • Mutant forms lacking the F-box domain fail to rescue the migration defect

  • Slight overexpression of FBXL13 increases migration compared to control cells

The regulation of CEP192 levels by FBXL13 appears to be a critical mechanism for controlling centrosome function and, consequently, cell motility. This suggests potential applications in studying cell migration in developmental and disease contexts.

How is Fbxl13 implicated in circadian rhythm regulation and what experimental approaches can address this?

Evidence suggests Fbxl13 may function in circadian rhythm regulation, similar to its family member Fbxl3:

• Genetic association:

  • A genome-wide association study (GWAS) of 89,283 individuals identified a genetic variant near FBXL13 (rs3972456, P=6.0×10^-9) associated with self-reported morningness

  • This intronic variant of FAM185A is 16 kb away from FBXL13 and is located in a DNase I hypersensitive site for 8 cell types

  • The variant is known to alter three regulatory motifs

• Mechanistic hypothesis:

  • FBXL13 may have a circadian role similar to FBXL3, which mediates the degradation of CRY1 and CRY2 proteins

  • Mutant FBXL3 mice show an extended circadian period, suggesting F-box proteins can significantly impact circadian rhythms

Experimental approaches to investigate Fbxl13's role in circadian regulation:

• Molecular rhythm analysis:

  • Real-time monitoring of PER2::LUC oscillations in tissues from Fbxl13 knockout mice

  • Determination of free-running period in Fbxl13^-/- mice using wheel-running activity

• Biochemical interactions:

  • Co-immunoprecipitation to detect interactions between Fbxl13 and circadian clock proteins

  • In vitro ubiquitination assays to assess if Fbxl13 can target clock proteins for degradation

• Tissue-specific knockout studies:

  • Conditional deletion of Fbxl13 in the suprachiasmatic nucleus (SCN) to assess central clock function

  • Analysis of peripheral clock function in tissue-specific knockouts

What is the relationship between Fbxl13 and neuropsychiatric disorders?

Emerging evidence links Fbxl13 to neuropsychiatric conditions through multiple mechanisms:

• Genetic associations:

  • FBXL13 has been highlighted as a core gene involved in bipolar disorder

  • It is associated with Class 1 MHC-mediated antigen processing and presentation, suggesting a potential immune component to its role in psychiatric disorders

• Potential molecular mechanisms:

  • Ubiquitin-proteasome pathway dysregulation is implicated in several neuropsychiatric disorders

  • As a component of this pathway, Fbxl13 may contribute to protein homeostasis in neurons

  • Potential connection to circadian rhythm disruptions, which are common in bipolar disorder and depression

• Experimental approaches to investigate this relationship:

  • Analysis of Fbxl13 expression in post-mortem brain tissue from patients with psychiatric disorders

  • Behavioral phenotyping of Fbxl13 knockout mice for relevant endophenotypes (anxiety, depression-like behavior, altered circadian patterns)

  • Investigation of Fbxl13 substrates in neural tissues to identify pathways affected by its dysfunction

• Methodological considerations:

  • Cell-type specific analysis is crucial as ubiquitin ligase function may vary between neurons, glia, and other cell types

  • Integration of genetics, proteomics, and behavioral data is necessary for a comprehensive understanding

  • Pharmacological modulation of Fbxl13 activity could provide insights into therapeutic potential

How can researchers overcome challenges in maintaining Fbxl13 stability and activity in experimental systems?

Recombinant Fbxl13 presents several stability challenges that researchers should address:

• Storage conditions optimization:

  • Store lyophilized protein at -80°C for long-term storage

  • After reconstitution, add carrier protein (0.1% BSA minimum) to prevent adsorption

  • Aliquot to minimize freeze-thaw cycles

  • For reconstituted protein, store at -20°C for up to 3 months or at 4°C for up to 2 weeks

• Maintaining SCF complex integrity:

  • Consider co-expressing Skp1, Cul1, and Rbx1 with Fbxl13 for functional studies

  • For in vitro ubiquitination assays, isolate the entire SCF complex rather than Fbxl13 alone

  • Validate complex formation via size-exclusion chromatography or native PAGE

• Activity verification methods:

  • Develop a CEP192 ubiquitination assay as a positive control for Fbxl13 activity

  • Monitor substrate levels (e.g., CEP192) via western blotting as a functional readout

  • Consider fluorescence-based ubiquitination assays for quantitative assessment

Common problems and solutions:

What are the critical controls and validation steps for Fbxl13 functional studies?

For rigorous Fbxl13 functional studies, implement these critical controls and validation steps:

• Protein quality controls:

  • SDS-PAGE analysis for purity and expected molecular weight

  • Mass spectrometry validation of protein identity

  • Circular dichroism to confirm proper folding

  • Size-exclusion chromatography to assess aggregation state

• Functional validation:

  • In vitro ubiquitination assay using known substrates (e.g., CEP192)

  • Co-immunoprecipitation with Skp1 and Cul1 to confirm complex formation

  • F-box domain mutant as a negative control for SCF formation

  • Leucine-rich repeat domain mutants to evaluate substrate specificity

• Cell-based assay controls:

  • siRNA-resistant wild-type FBXL13 for rescue experiments

  • F-box deletion mutant as a dominant-negative control

  • Empty vector controls for overexpression studies

  • Proper mRNA and protein level verification after knockdown/overexpression

• In vivo experiment controls:

  • Littermate controls for knockout studies

  • Tissue-specific expression verification

  • Functional redundancy assessment with other F-box proteins

  • Physiological substrate level monitoring

Proper experimental design should incorporate substrate specificity validation, as Fbxl13 shows selective targeting of CEP192 isoform 3 while binding but not degrading Centrin-2 and Centrin-3 .

How might proteomics approaches be used to identify novel substrates and functions of Fbxl13?

Advanced proteomics strategies offer powerful approaches to uncovering the complete substrate repertoire of Fbxl13:

• Proximity-dependent biotin identification (BioID):

  • Fusion of Fbxl13 with a promiscuous biotin ligase (BirA*)

  • Expression in relevant cell types to biotinylate proximal proteins

  • Streptavidin pull-down and mass spectrometry identification

  • Comparison between wild-type and F-box mutant to distinguish substrates from interactors

• Quantitative diGly proteomics:

  • Treatment of cells with proteasome inhibitors

  • Comparison between Fbxl13 knockout and wild-type conditions

  • Enrichment of ubiquitinated peptides using anti-K-ε-GG antibodies

  • Mass spectrometry to identify differentially ubiquitinated proteins

• Protein stability profiling:

  • Global protein turnover analysis using pulse-chase SILAC or TMT labeling

  • Comparison between Fbxl13-deficient and wild-type cells

  • Identification of proteins with altered half-lives

  • Validation of direct targeting using in vitro ubiquitination assays

• Tissue-specific substrate identification:

  • Analysis of ubiquitinome changes in relevant tissues from Fbxl13 knockout mice

  • Particular focus on centrosome-associated proteins and potential circadian regulators

  • Integration with phosphoproteomics to identify regulation by post-translational modifications

These approaches would extend beyond the currently known CEP192 substrate and potentially reveal tissue-specific functions of Fbxl13 that might explain the limited phenotypes observed in knockout mice.

What therapeutic potential exists in targeting or modulating Fbxl13 function?

The potential therapeutic applications of Fbxl13 modulation span several disease contexts:

• Cancer and cell migration:

  • Given Fbxl13's role in regulating cell motility through CEP192 degradation , inhibition might reduce cancer cell migration and metastasis

  • Methodological approach: Small molecule screening for compounds that disrupt Fbxl13-substrate interactions while preserving SCF complex integrity

• Neuropsychiatric disorders:

  • The association of FBXL13 with bipolar disorder suggests potential as a therapeutic target

  • Modulation might normalize protein homeostasis in affected neural circuits

  • Research approach: Conditional Fbxl13 modulation in specific brain regions to assess behavioral effects

• Circadian rhythm disorders:

  • If Fbxl13 functions analogously to Fbxl3 in circadian regulation , it could be targeted to adjust circadian period

  • This has implications for sleep disorders, jet lag, and shift work adaptation

  • Experimental strategy: Small molecule screens for compounds that modify Fbxl13 activity with circadian readouts

• Challenges and considerations:

  • Substrate specificity: Ensuring selective modulation of Fbxl13 without affecting other F-box proteins

  • Tissue specificity: Developing approaches to target Fbxl13 in relevant tissues while minimizing off-target effects

  • Timing of intervention: Determining optimal temporal windows for Fbxl13 modulation in circadian contexts

• Validation approaches:

  • CRISPR activation/inhibition systems for temporal control of Fbxl13 expression

  • Structure-based drug design targeting specific protein-protein interactions

  • In vivo models of relevant diseases to evaluate efficacy and safety profiles