Recombinant Coturnix coturnix japonica Protein FAM65B (FAM65B), partial

What is FAM65B and what are its primary cellular functions?

FAM65B (Family with sequence similarity 65, member B) is a protein that plays a crucial role in cell quiescence and proliferation control. Research has established that FAM65B is a major target of the transcription factor FOXO1, which imposes quiescence in several cell types, including T lymphocytes . In its functional capacity, FAM65B acts as a molecular switch controlling quiescence in normal cells and proliferation in transformed cell lines .

When forcibly expressed in proliferating cells, FAM65B blocks mitosis by inducing defects in the mitotic spindle, which leads to G2/M cell cycle arrest and subsequent apoptosis. Flow cytometry analysis revealed that cells expressing FAM65B demonstrate significant G2/M accumulation after 3 days of culture (47 ± 7% of FAM65B-positive cells versus 22 ± 1.4% of control cells in G2/M phase) . This growth-inhibitory effect places FAM65B as a potential target for controlling cellular proliferation in both normal and transformed cells.

How is FAM65B expression regulated during T cell activation?

FAM65B expression is tightly regulated during T cell activation processes. In quiescent, naive T lymphocytes, FAM65B is highly expressed, serving as a molecular marker of the quiescent state . Upon T cell receptor (TCR) engagement, FAM65B is rapidly down-regulated, with complete loss observed 24 hours after the onset of stimulation .

This down-regulation appears to be functionally significant, as maintaining FAM65B expression during T cell activation (through transfection with FAM65B-GFP) completely inhibits proliferation induced by TCR engagement. Importantly, early TCR signaling events like ERK phosphorylation and CD69 expression remain unaffected by forced FAM65B expression, indicating that FAM65B acts downstream of initial activation signals to regulate proliferation rather than interfering with receptor signaling .

What protein interactions mediate FAM65B's cell cycle regulatory functions?

FAM65B forms a functionally significant tripartite molecular complex with two other proteins known to be involved in cell proliferation: HDAC6 deacetylase and the 14.3.3 scaffolding protein . This complex formation appears to be essential for FAM65B's anti-proliferative activity.

Interestingly, while FAM65B can bind directly to and inhibit RhoA (a protein involved in cytokinesis), this interaction does not appear to be responsible for FAM65B's anti-proliferative effects. Researchers demonstrated this by creating a mutant version of FAM65B (RL151-152AA) unable to bind RhoA. This mutant retained the ability to inhibit proliferation of Jurkat T cells as efficiently as wild-type FAM65B, indicating that FAM65B's anti-proliferative activity operates through mechanisms independent of RhoA inhibition .

How do FAM65B structural features relate to its functional domains?

The structural characteristics of FAM65B have been a subject of scientific debate. Conflicting analyses exist regarding whether FAM65B belongs to the family of phox homology (PX) domain-containing proteins. Some researchers have proposed a structural model suggesting homology between FAM65B's N-terminal region and sorting nexin (SNX) family members possessing phox homology (PX) and bin/amphiphysin/rvs (BAR) domains .

Attempts to replicate homology modeling of FAM65B N-terminal residues 1–300 using SwissModel (http://swissmodel.expasy.org) and Phyre2 server (www.sbg.bio.ic.ac.uk/phyre2) were unsuccessful in identifying PX-BAR proteins as structural templates . This contradictory evidence suggests researchers working with partial recombinant FAM65B should approach structural predictions with caution and verify domain identification through multiple analytical methods.

What expression systems are optimal for producing recombinant FAM65B?

The choice of expression system for producing recombinant Coturnix coturnix japonica FAM65B depends on the experimental requirements and downstream applications. Based on established protocols for similar proteins, several expression systems merit consideration:

When expressing partial FAM65B, researchers should carefully consider which domains are included in the construct and ensure proper protein folding through appropriate validation techniques (circular dichroism, limited proteolysis, etc.).

What purification strategies maintain FAM65B stability and function?

Purification of recombinant partial FAM65B requires strategies that maintain protein stability and biological activity. Based on successful approaches with similar proteins, a multi-step purification protocol is recommended:

• Initial capture: Affinity chromatography using tags (His, GST, or FLAG) permits efficient initial purification. Tag selection should consider the impact on protein folding and function.

• Intermediate purification: Ion exchange chromatography can separate charged variants and remove contaminants with different charge characteristics.

• Polishing step: Size exclusion chromatography helps remove aggregates and ensures monodispersity of the final product.

Throughout purification, buffer composition is critical. FAM65B stability may benefit from:

• Physiological pH (7.2-7.4)

• Moderate salt concentration (150-300 mM NaCl)

• Addition of reducing agents (1-5 mM DTT or TCEP) to maintain cysteine residues

• Stabilizing agents like glycerol (5-10%)

• Protease inhibitors during initial purification steps

For partial recombinant FAM65B, researchers should experimentally determine optimal conditions through stability screens and activity assays relevant to known FAM65B functions, such as its interaction with binding partners HDAC6 and 14.3.3 .

How can researchers validate FAM65B functionality after recombinant production?

Validating the functionality of recombinant partial FAM65B requires multiple complementary approaches:

• Structural integrity assessment: Circular dichroism spectroscopy and thermal shift assays can confirm proper folding and stability.

• Protein-protein interaction studies: Pull-down assays or surface plasmon resonance to verify binding to known partners like HDAC6 and 14.3.3 scaffolding protein . Co-immunoprecipitation experiments using cell lysates can confirm interaction with endogenous partners.

• Cell-based functional assays: Transfection of recombinant FAM65B into proliferating cells should reproduce the G2/M cell cycle arrest phenotype observed with endogenous FAM65B. Flow cytometry analysis should reveal G2/M accumulation consistent with previous findings (approximately 47% of FAM65B-positive cells in G2/M phase compared to 22% of control cells) .

• Assessment of apoptosis induction: Annexin V labeling can confirm the apoptotic effects associated with FAM65B overexpression .

When working with partial FAM65B constructs, researchers should determine whether the fragment contains the domains necessary for specific functions being tested. Different functional assays may be appropriate depending on which portion of the protein is expressed.

How should researchers interpret FAM65B expression patterns in comparative studies?

When analyzing FAM65B expression across different tissues or experimental conditions, researchers should consider several analytical factors:

• Reference gene selection: When performing qPCR analysis of FAM65B expression, careful selection of reference genes is critical. FAM65B expression fluctuates substantially during cellular activation states, so validation of reference gene stability across experimental conditions is essential.

• Isoform-specific analysis: FAM65B exists in multiple isoforms, and expression patterns may differ between them. In T cells, both major FAM65B isoforms show simultaneous loss upon activation . Researchers should employ primers or antibodies that can distinguish between isoforms or clearly state which isoforms are being detected.

• Temporal resolution: FAM65B expression changes rapidly during cellular activation, with complete loss observed 24 hours after T cell stimulation . Studies should include multiple time points to capture dynamic expression changes.

• Statistical approaches: When analyzing expression data, appropriate statistical methods should account for the potentially non-linear relationship between FAM65B levels and cellular phenotypes. Correlation analyses between FAM65B expression and functional outcomes (e.g., proliferation rates) should employ regression models suitable for potentially threshold-dependent effects.

Note: This table represents approximate expression patterns based on published findings in human T cells . Researchers should experimentally verify these patterns in Coturnix coturnix japonica cells.

What approaches address contradictions in FAM65B structure-function relationships?

Researchers face contradictory findings regarding FAM65B's structure and domain organization, particularly regarding its relationship to the PX domain family . To address these contradictions:

• Employ multiple bioinformatic approaches: Rather than relying on a single algorithm, use multiple sequence analysis tools and structural prediction methods. Compare results from BLAST, HMM-based methods, and threading approaches.

• Conduct domain mapping experiments: Generate a series of truncation mutants to experimentally map functional domains of FAM65B. This approach can resolve contradictions between computational predictions and actual protein function.

• Use direct structural studies: Where feasible, employ techniques like X-ray crystallography, cryo-EM, or NMR spectroscopy on recombinant protein fragments to resolve structural ambiguities.

• Validate structure-function relationships experimentally: For example, the finding that FAM65B's anti-proliferative activity is independent of its RhoA-binding capacity was established through site-directed mutagenesis (RL151-152AA mutant) . Similar approaches should be applied to test other structure-function hypotheses.

When publishing findings on recombinant Coturnix coturnix japonica FAM65B, researchers should explicitly acknowledge existing contradictions in the literature and explain how their methodological approach addresses these inconsistencies.

How can partial recombinant FAM65B be utilized to study avian T cell biology?

Recombinant partial FAM65B from Japanese quail represents a valuable tool for comparative immunology research. Future investigations might explore:

• Evolutionary conservation of T cell quiescence mechanisms: By comparing the function of quail FAM65B with mammalian orthologs, researchers can determine whether the molecular mechanisms controlling T cell quiescence are conserved across vertebrate lineages.

• Species-specific interaction partners: Proteomics approaches using partial recombinant FAM65B as bait could identify novel binding partners specific to avian species, potentially revealing unique aspects of T cell regulation in birds.

• Targeted manipulation of immune responses: Given FAM65B's role in setting T cell activation thresholds , recombinant protein could potentially be used to modulate immune responses in experimental settings, either by inhibiting FAM65B function to enhance responses or supplementing it to suppress unwanted activation.

• Biomarker development: The strong correlation between FAM65B expression and cellular quiescence suggests potential application as a biomarker for lymphocyte activation states in avian models of infection or immune dysfunction.

Research in these directions should employ the partial recombinant protein as both an experimental tool and a subject of study, with careful attention to which domains are represented in the partial construct.

What therapeutic implications arise from FAM65B's role in proliferation control?

The identification of FAM65B as "a potential new target for controlling proliferation of both transformed and normal cells" opens several therapeutic research directions:

• Cancer research applications: Given FAM65B's ability to induce G2/M arrest and apoptosis when expressed in proliferating cells, recombinant FAM65B (or derivatives) could be explored as potential anti-proliferative agents in cancer research. Experiments could assess species cross-reactivity to determine whether quail FAM65B can affect mammalian cancer cells.

• Immune modulation strategies: FAM65B's role in setting T cell activation thresholds suggests potential applications in autoimmunity research. Enhancing FAM65B function could theoretically raise activation thresholds and dampen excessive immune responses.

• Comparative oncology insights: Studying FAM65B across species may reveal conserved and divergent mechanisms of proliferation control, potentially identifying novel regulatory pathways that could be therapeutically targeted.

When pursuing these directions, researchers should be mindful that partial recombinant proteins may not recapitulate all functions of the full-length protein. Careful domain mapping and functional validation are essential prerequisites for therapeutic applications.