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

Introduction to Recombinant Mouse Fbxo11

Fbxo11 belongs to the F-box protein family, which is characterized by an approximately 40 amino acid motif called the F-box. F-box proteins constitute one of the four subunits of the SCF ubiquitin protein ligase complex, which functions in phosphorylation-dependent ubiquitination . The F-box proteins are divided into three classes: Fbws containing WD-40 domains, Fbls containing leucine-rich repeats, and Fbxs containing either different protein-protein interaction modules or no recognizable motifs . Fbxo11 specifically belongs to the Fbxs class.

Recombinant partial mouse Fbxo11 protein refers to a laboratory-produced fragment of the full-length protein that contains specific domains or regions of interest. By focusing on particular segments of the protein, researchers can investigate specific aspects of Fbxo11 function without the complexities associated with the full-length protein. This approach has facilitated numerous research applications, including antibody production, protein interaction studies, and functional analyses that have significantly advanced our understanding of this important regulatory protein.

Gene and Protein Information

The mouse Fbxo11 gene is located on chromosomal region 17qE4, containing 23 exons and spanning approximately 75-kb of genomic DNA . The full-length Fbxo11 protein contains several functional domains, including the eponymous F-Box domain, which is important for interaction with S-phase kinase-associated protein 1 (SKP1) . This interaction facilitates the incorporation of Fbxo11 into the SCF complex, enabling its function in substrate recognition and ubiquitination.

Multiple isoforms of Fbxo11 have been identified, with the two primary variants being a larger 103 kDa isoform and a smaller 95 kDa isoform . Interestingly, co-immunoprecipitation studies have shown that only the larger isoform interacts with p53, suggesting functional specialization among the different isoforms . The protein can function as an arginine methyltransferase that symmetrically dimethylates arginine residues and acts as an adaptor protein to mediate the neddylation of p53 .

Biological Functions of Fbxo11

To understand the significance of recombinant partial Fbxo11 as a research tool, it is essential to comprehend the biological functions of the full-length protein.

Role in Ubiquitination and Protein Regulation

Fbxo11 serves as the substrate recognition component of the SCF ubiquitin ligase complex (SCF^FBXO11), identifying specific target proteins for ubiquitination . This post-translational modification typically signals for protein degradation via the 26S proteasome, though it can also regulate protein localization, activity, or interactions.

The SCF complex containing Fbxo11 consists of:

• SKP1 (S-phase kinase-associated protein 1)

• CUL1 (Cullin-1)

• RBX1 (RING-box protein 1)

• FBXO11 (F-box only protein 11)

Through co-immunoprecipitation studies in embryonic mouse lungs, researchers have confirmed that Fbxo11 interacts with both CUL1 and CUL4, indicating its incorporation into multiple cullin-based E3 ligase complexes . This suggests that Fbxo11 may have broader regulatory functions than initially appreciated.

Known Substrates and Interaction Partners

Several important substrates of Fbxo11 have been identified, highlighting its role in diverse cellular processes:

Snail Family Transcription Factors

Fbxo11 recognizes and promotes the ubiquitin-mediated degradation of multiple Snail family members, including Scratch . These transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT), a process crucial for embryonic development, wound healing, and cancer progression. The association between Fbxo11 and Snai1 appears to be independent of Snai1 phosphorylation in vitro, distinguishing this interaction from other F-box protein-substrate relationships that typically require substrate phosphorylation .

p53 Tumor Suppressor

Fbxo11 has been identified as an E3 ligase for p53, promoting its neddylation (conjugation of the ubiquitin-like protein NEDD8) . This modification suppresses p53's transcriptional activity. Co-immunoprecipitation studies in E15.5 wild-type lungs demonstrated that p53 co-immunoprecipitates with Fbxo11, and the size of the p53 band (71 kDa) suggested that the protein is modified, likely with NEDD8 (9 kDa) . Importantly, the Jeff mutation in Fbxo11 prevents this association with p53, potentially explaining some of the developmental phenotypes observed in Jeff mutant mice .

BCL6 Oncogene

Fbxo11 targets the oncoprotein BCL6 (B cell lymphoma 6) for degradation . BCL6 is a transcriptional repressor that plays critical roles in germinal center formation and antibody diversification. FBXO11 inactivation leads to abnormal germinal-center formation and increased BCL6 protein levels in germinal center B cells . This regulatory mechanism has significant implications for lymphoma development, as FBXO11 mutations are frequent in Burkitt lymphoma and contribute to increased BCL6 stabilization and lymphoma progression .

Table 2: Major Substrates of Fbxo11 and Their Functions

Signaling Pathway Regulation

Fbxo11 influences several key signaling pathways through its substrate targeting functions:

TGF-β/SMAD Pathway

Fbxo11 plays a crucial role in regulating transforming growth factor beta (TGF-β) signaling by controlling the levels of phosphorylated SMAD2 in epithelial cells . The mechanism appears to involve a complex interplay between Fbxo11, CDT2, and SET8. Fbxo11 regulates the TGF-β pathway in the embryonic lung via cross-talk with p53 . Both p53 homozygous mutants and double heterozygous mutants (Jf/+ p53/+) show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes .

EMT Regulation

By promoting the degradation of Snail family transcription factors, Fbxo11 serves as a critical inhibitor of epithelial-to-mesenchymal transition (EMT) . Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness, while depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion . This function positions Fbxo11 as a guardian of the epithelial state, with important implications for both development and disease progression.

Fbxo11 in Development and Disease Models

Animal models and cellular systems have provided valuable insights into the roles of Fbxo11 in development and disease.

Role in Lung Development

Studies of Fbxo11-deficient mouse models have revealed essential functions in lung development:

Fbxo11-/- homozygous mutant mice are born at the expected Mendelian ratios from heterozygous parents but die shortly after birth . Histological analysis of Fbxo11-/- lungs at E18.5 (saccular stage) shows an excessive number of subepithelial fibroblast-like cells surrounding the bronchi, resembling peri-bronchial fibrosis . Compared to wild-type control littermates, lungs from Fbxo11-/- homozygous mutant embryos show much thicker interstitial mesenchyme and less developed saccular structures .

Expression of the mesenchymal marker N-cadherin is detected in cells beneath epithelium in the control lung and is strongly enhanced in the lung of Fbxo11-/- homozygotes . These findings suggest that Fbxo11 plays a crucial role in regulating the balance between epithelial and mesenchymal development in the lung, potentially through its influence on EMT and TGF-β signaling.

The Jeff Mouse Model and Otitis Media

The Jeff mouse carries a mutation in the Fbxo11 gene and serves as an important model for studying otitis media (middle ear inflammation):

The Jeff mouse mutant heterozygous animals display conductive deafness due to the development of otitis media (OM) . The Fbxo11 locus is also associated with chronic otitis media with effusion (COME) and recurrent OM in humans . The Jeff mutation affects the ability of FBXO11 to stabilize p53, leading to perturbation in the TGF-beta/Smad2 signaling pathway important in immunity and inflammation .

Table 3: Comparison of Phenotypes in Different Fbxo11 Mutant Mouse Models

Immune System Effects

The Jeff mutation significantly affects immune cell content, as revealed by multicolor flow cytometry studies:

In blood of Jeff heterozygotes, researchers observed a significant increase in the number of NK, dendritic (CD11b+), neutrophils, and natural killer T (NKT) cells and a significant decrease in effector T-helper and B-lymphocytes compared to wild-type controls . The percentage of NK cells significantly decreased in the lungs of Jeff heterozygotes, with a concomitant reduction in B-lymphocytes and T-cytotoxic cells . In the spleen, Jeff heterozygotes displayed a significant decrease in mature B-lymphocytes, effector T-helper, and naïve T-cytotoxic cells .

Neutrophils, dendritic, and NKT cells dominated bulla fluid in Jeff heterozygote mice . Interestingly, similar analysis carried out on Fbxo11 tm2b/+ heterozygotes, which carry a null allele, showed no difference when compared to wild-type . This suggests that the Jeff mutation may have specific effects on immune function that differ from simple haploinsufficiency.

Role in Cancer and Lymphoma

Accumulating evidence suggests that Fbxo11 functions as a tumor suppressor in various types of cancer:

Epithelial Cancers

By promoting the degradation of Snail family proteins and thereby inhibiting EMT, Fbxo11 helps maintain the epithelial state and prevents cancer progression . Depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells . In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis .

Lymphomas and Leukemias

FBXO11 plays a critical role in preventing lymphomagenesis through its regulation of BCL6:

FBXO11 loss-of-function mutations are frequent in Burkitt lymphoma (BL), contributing to increased BCL6 stabilization and lymphoma progression . Deletion of one or both FBXO11 alleles strongly accelerated the development of lymphoma in Eμ-Myc mice . In human BL lines, FBXO11 deletion almost completely stabilized BCL6 expression, and targeting BCL6 in BL lines using BCL6 inhibitors or BCL6 degraders impaired BL growth in vitro and in vivo .

FBXO11 inactivation leads to abnormal germinal-center formation and increased BCL6 expression . In germinal center-specific FBXO11 knockout mice, FBXO11 reduction or loss led to an increased number of germinal center B cells, altered ratio of dark zone to light zone cells, and higher levels of BCL6 protein in germinal center B cells . B-cell receptor–mediated degradation of BCL6 was reduced in the absence of FBXO11, suggesting that FBXO11 contributes to the physiologic downregulation of BCL6 at the end of the germinal center reaction .

In myeloid malignancies, FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome (MDS) . Loss of the F-Box protein FBXO11 confers cytokine independent growth to MDS-L cells . FBXO11 expression is reduced in patients with secondary AML .

Research Applications of Recombinant Partial Fbxo11

The recombinant partial mouse Fbxo11 protein serves as a valuable tool for various research applications.

Antibody Production and Immunological Techniques

One of the primary uses of recombinant partial Fbxo11 is as an antigen for antibody production. Mouse monoclonal antibodies have been raised against a partial recombinant FBXO11, such as clone 4C12 from Abnova, which was generated using FBXO11 (NP_079409, 744 a.a. ~ 843 a.a) partial recombinant protein with GST tag .

These antibodies have diverse applications:

• Western blotting for detecting Fbxo11 in protein lysates

• Immunohistochemistry on fixed tissues to localize Fbxo11 expression

• Immunofluorescence for subcellular localization studies

• ELISA for quantitative measurement of Fbxo11 levels

• Immunoprecipitation for studying protein-protein interactions

The availability of specific antibodies against Fbxo11 has facilitated numerous studies investigating its expression, localization, and function in various tissues and disease states.

Protein Interaction Studies

Recombinant partial Fbxo11 enables detailed investigation of protein-protein interactions:

• Pull-down assays can identify binding partners by incubating GST-tagged recombinant Fbxo11 with cell lysates

• Surface plasmon resonance (SPR) measures binding kinetics between Fbxo11 and potential substrates

• Co-immunoprecipitation studies can confirm interactions in cellular contexts

These approaches have been instrumental in elucidating Fbxo11's interactions with substrates like p53, Snail family proteins, and BCL6, as well as its incorporation into the SCF complex through interaction with SKP1.

Functional Assays

The recombinant protein facilitates various functional studies:

Ubiquitination and Neddylation Assays

In vitro ubiquitination assays using recombinant Fbxo11 help define its substrate specificity and the mechanisms of ubiquitin chain formation. Similarly, neddylation assays can evaluate Fbxo11's ability to promote NEDD8 conjugation to substrates like p53, as demonstrated in the finding that FBXO11 promotes the neddylation of p53 at Lys320 and Lys321 .

Therapeutic Development

Research using recombinant Fbxo11 contributes to therapeutic development in several ways:

• Screening for small molecules that modulate Fbxo11 activity

• Identification of downstream effectors as potential drug targets

• Development of targeted therapies for conditions associated with Fbxo11 dysfunction

For example, studies have shown that targeting BCL6 in Burkitt lymphoma cell lines using BCL6 inhibitors or degraders impairs growth in vitro and in vivo, identifying BCL6 as an actionable therapeutic target in BL . Additionally, the antitumoral effects of MYC inhibition were further enhanced by BCL6 degradation, suggesting that targeting of MYC and BCL6 could represent an efficacious therapeutic approach in BL .

Recent Advances and Future Directions

Research involving Fbxo11 continues to evolve, with several recent advances and promising future directions.

Generation of Advanced Mouse Models

The development of conditional knockout mouse models has enabled tissue-specific studies of Fbxo11 function:

C57BL/6 mouse embryonic stem cells heterozygous for a targeted allele of FBXO11 were obtained from the International Knockout Mouse Consortium (EUCOMM) . The resulting FBXO11 conditional allele has 2 loxP sites inserted into intronic regions upstream and downstream of exon 4 that allow disruption of the FBXO11 sequence after Cre recombination .

Similarly, researchers have generated mice in which a "STOP" cassette was inserted into the Fbxo11 genomic locus, resulting in a null allele . This cassette was flanked by FRT sites and could be removed by FLP recombinase-mediated recombination, creating a conditional "floxed" allele where exon 4 was flanked by LoxP sites .

These mouse models have been invaluable for studying the tissue-specific functions of Fbxo11, particularly in B cells and lung development.

CRISPR/Cas9 Applications

The advent of CRISPR/Cas9 gene editing has revolutionized Fbxo11 research:

Researchers have used CRISPR/Cas9 to generate Fbxo11-deficient cell lines, including Burkitt lymphoma lines. For example, RAJI and DAUDI cell lines were transduced with lentiviral particles carrying sgRNAs targeting FBXO11, followed by selection and confirmation of FBXO11 deficiency . These cellular models have facilitated functional studies of FBXO11's role in cancer progression and therapeutic response.

Emerging Roles in Disease

Recent research has identified new roles for Fbxo11 in various diseases:

• FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome

• Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism

• Spliceosome mutations commonly found in MDS/AML are rare in patients with low FBXO11 expression

These findings suggest that Fbxo11 may have broader regulatory functions than initially appreciated, particularly in the context of hematological malignancies.

Future Research Directions

Several promising areas for future research include:

1. Comprehensive characterization of the substrate landscape of Fbxo11 using proteomics approaches

2. Investigation of tissue-specific functions using conditional knockout models

3. Detailed structural analysis of Fbxo11-substrate interactions

4. Development of selective modulators of Fbxo11 activity

5. Exploration of Fbxo11's role in additional developmental processes and disease contexts

6. Elucidation of the specific roles of different Fbxo11 isoforms