Recombinant Rat F-box only protein 11 (Fbxo11), partial

What is the structural characterization of FBXO11 protein?

FBXO11 belongs to the F-box protein family, characterized by an approximately 40 amino acid motif called the F-box. It falls specifically into the Fbxs class of F-box proteins, which contain either different protein-protein interaction modules or no recognizable motifs, distinguishing it from other classes like Fbws (containing WD-40 domains) and Fbls (containing leucine-rich repeats). The protein is available in recombinant form from various expression systems, with confirmed purity levels of >85% as determined by SDS-PAGE analysis .

What are the primary cellular functions of FBXO11?

FBXO11 functions as a critical substrate recognition component of the SKP1-cullin-F-box (SCF) E3 ubiquitin ligase complex. This complex plays a central role in phosphorylation-dependent ubiquitination, targeting specific proteins for proteasomal degradation. Through this mechanism, FBXO11 participates in regulating multiple cellular processes by controlling protein turnover. Notably, FBXO11 has been implicated in immune regulation through its effects on MHC class II expression and may function as an emerging tumor suppressor in certain contexts .

How does FBXO11 expression vary across tissues and experimental models?

While the search results don't provide comprehensive tissue distribution data, FBXO11 function has been studied in several experimental models. Knockout mouse models (known as "Jeff mouse") exhibit phenotypes including reduced weight, deafness, and otitis media, suggesting important roles in growth, auditory function, and immune regulation . In cellular models, FBXO11 has been studied in myeloid cells where its deletion affects MHC-II expression, particularly in acute myeloid leukemia (AML) cell lines .

What are the optimal storage and handling conditions for recombinant FBXO11?

The stability and shelf life of recombinant FBXO11 depend on several factors including storage state, buffer composition, temperature, and the inherent stability of the protein itself. For optimal results:

• Liquid form: Store at -20°C/-80°C with a typical shelf life of 6 months

• Lyophilized form: Store at -20°C/-80°C with a typical shelf life of 12 months

• Working aliquots: Store at 4°C for up to one week only

• Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

What reconstitution protocols yield optimal FBXO11 activity?

For reconstitution of lyophilized FBXO11 protein:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Prepare small working aliquots to minimize freeze-thaw cycles

• Store reconstituted protein at -20°C/-80°C for long-term use

What experimental approaches best elucidate FBXO11's substrate recognition mechanisms?

Based on recent research methodologies, several approaches have proven effective:

• CRISPR/Cas9-mediated gene disruption to assess phenotypic consequences of FBXO11 loss

• RNA-sequencing to identify transcriptomic changes following FBXO11 depletion

• Chromatin immunoprecipitation (ChIP) analysis to investigate potential epigenetic functions

• Protein-protein interaction studies to identify binding partners and potential substrates

• Combined knockout approaches (e.g., with RREB1) to assess potential functional redundancy or synergy

Recent studies have revealed that, unlike some F-box proteins where substrate phosphorylation creates recognition sites, phosphorylation can unexpectedly inhibit degradation of FBXO11 substrates, suggesting more complex regulatory mechanisms .

How does FBXO11 dysfunction contribute to neurodevelopmental disorders?

De novo variants in FBXO11 have been identified in 20 individuals with variable neurodevelopmental disorders. These variants include two large deletions, ten likely gene-disrupting variants, and eight missense variants distributed throughout the FBXO11 gene. Clinical manifestations include intellectual disability, autism spectrum disorder, cleft lip or palate or bifid uvula (in 3 of 20 patients), and minor skeletal anomalies. These findings suggest that disruption of FBXO11-mediated protein degradation pathways can significantly impact neurodevelopment .

What role does FBXO11 play in cancer biology, particularly in immune regulation?

FBXO11 has emerged as a potential tumor suppressor that regulates immune response mechanisms. In acute myeloid leukemia (AML) cells:

• FBXO11 knockout induces surface MHC-II expression, which can be further enhanced by IFN-γ stimulation

• Combined knockout of FBXO11 and RREB1 (Ras-responsive element binding protein 1) further augments MHC-II expression

• This regulation appears to be independent of the Polycomb Repressive Complex 2 (PRC2) mechanism

• FBXO11 depletion affects transcription of only a small subset of genes (30-45 genes), including multiple classical MHC-II genes

These findings suggest that FBXO11 may influence tumor immunosurveillance by regulating antigen presentation machinery.

What phenotypes are observed in FBXO11 knockout models?

The "Jeff mouse" model with Fbxo11 mutation/knockout exhibits several distinctive phenotypes:

These phenotypes collectively suggest broad developmental and physiological roles for FBXO11 beyond its biochemical function in protein ubiquitination .

How do post-translational modifications affect FBXO11 substrate selectivity?

Unlike the typical model where substrate phosphorylation creates recognition sites for F-box proteins, phosphorylation has been shown to inhibit the degradation of FBXO11 substrates. This suggests alternative regulatory mechanisms that may include:

• Regulation of FBXO11 localization or accessibility to substrates

• Conformational changes affecting binding interfaces

• Competition with other binding partners

• Additional modifications such as lysine methylation, lysine acetylation, or tyrosine phosphorylation

Future research should investigate these mechanisms to fully understand the complexity of FBXO11-mediated protein regulation.

What is the mechanistic relationship between FBXO11 and MHC class II regulation?

The molecular pathway connecting FBXO11 to MHC-II expression remains incompletely understood. Current evidence suggests:

• FBXO11 regulation of MHC-II appears independent of H3K27me3 deposition, as ChIP-seq data reveals minimal H3K27me3 at MHC-II pathway genes

• This indicates that FBXO11 likely operates through PRC2-independent mechanisms

• FBXO11 may target transcriptional regulators of MHC-II genes for degradation

• The CtBP complex has been implicated in this pathway, with several components identified in targeted screens alongside FBXO11

Understanding these mechanisms could lead to therapeutic approaches that enhance tumor immunogenicity through modulation of FBXO11 activity.

How might emerging structural biology approaches advance our understanding of FBXO11?

Advanced structural characterization of FBXO11 could reveal:

• The precise binding interface between FBXO11 and its substrates

• Conformational changes associated with substrate recognition

• Structural basis for the unexpected inhibitory effect of phosphorylation on substrate degradation

• Potential allosteric sites for therapeutic targeting

Technologies such as cryo-electron microscopy, hydrogen-deuterium exchange mass spectrometry, and computational modeling could provide valuable insights into FBXO11's structural biology and mechanism of action.

What techniques can resolve contradictory findings about FBXO11's role in different cellular contexts?

To address potential contradictions in FBXO11 function across different cell types and conditions, researchers should consider:

• Cell type-specific conditional knockout models to assess context-dependent functions

• Temporal regulation systems (e.g., inducible CRISPR) to distinguish between acute and chronic effects

• Proteome-wide analyses to comprehensively identify cell-specific FBXO11 substrates

• Single-cell approaches to account for heterogeneity within populations

• Integration of multiple -omics data types to build comprehensive regulatory networks

These approaches could help resolve apparent contradictions by precisely defining FBXO11's role in specific cellular and physiological contexts.

What strategies can address poor activity of recombinant FBXO11 in functional assays?

When facing challenges with recombinant FBXO11 activity:

• Verify protein integrity by SDS-PAGE (expected purity >85%)

• Consider alternative expression systems (both yeast and E.coli sources are available)

• Optimize buffer conditions, particularly salt concentration and pH

• Ensure proper complex formation with other SCF components if studying ubiquitination activity

• Include protease inhibitors to prevent degradation during experimental procedures

How can researchers distinguish between direct and indirect effects of FBXO11 manipulation?

To differentiate direct FBXO11 effects from secondary consequences:

• Perform acute vs. chronic depletion experiments

• Use substrate-binding mutants that maintain structural integrity but lack specific interaction capabilities

• Conduct rescue experiments with wild-type vs. mutant FBXO11

• Employ direct biochemical assays to confirm physical interactions with putative substrates

• Utilize proximity labeling approaches to identify proteins in the immediate vicinity of FBXO11

These approaches help establish causality and differentiate primary effects from downstream consequences of FBXO11 manipulation.