Recombinant Bovine Bcl10-interacting CARD protein

What is Bovine Bcl10-interacting CARD protein and how does it function?

Bovine Bcl10-interacting CARD protein (such as bovine BinCARD) is a CARD-containing protein that interacts with BCL10 through heterotypic CARD-CARD interactions. This protein contains a caspase recruitment domain (CARD) that mediates protein-protein interactions critical for immune signaling pathways . Functionally, Bcl10-interacting CARD proteins like BinCARD act as negative regulators of BCL10-mediated NF-κB activation by suppressing the phosphorylation of BCL10 .

While human BinCARD has been shown to be ubiquitously expressed across tissues, bovine variants likely follow similar expression patterns with potential tissue-specific differences . The protein serves as an important regulatory component within immune signaling networks where BCL10 acts as a central mediator connecting various CARMA/CARD scaffold proteins to downstream effectors in multiple immune pathways .

What are the structural characteristics of Bcl10-interacting CARD domains?

CARD domains, including those found in Bcl10-interacting proteins, typically consist of a six-helix bundle structure that facilitates specific protein-protein interactions through both electrostatic and hydrophobic contacts. In BinCARD specifically, the CARD domain contains highly conserved leucine residues at positions 17 and 65 that are critical for its functionality . Mutations at these conserved leucine residues (Leu17 or Leu65) abolish the inhibitory effects of BinCARD on BCL10-induced NF-κB activation and phosphorylation .

The structural arrangement of CARD domains in Bcl10-interacting proteins enables specific heterotypic CARD-CARD interactions with BCL10. These interactions involve complementary charged surfaces, where one protein presents a basic CARD surface that engages with the negatively charged surface of its binding partner . This electrostatic complementarity ensures specificity in the assembly of signaling complexes and is likely conserved across species variants including bovine forms.

How is the expression of bovine Bcl10-interacting CARD proteins regulated?

The expression regulation of bovine Bcl10-interacting CARD proteins remains an area requiring further research, but based on known mechanisms for related proteins, regulation likely occurs at multiple levels:

• Transcriptional regulation: Tissue-specific transcription factors likely control basal expression patterns across different bovine tissues.

• Post-transcriptional regulation: mRNA stability and alternative splicing may generate tissue-specific isoforms with altered functionality.

• Post-translational modifications: Like other CARD-containing proteins, bovine Bcl10-interacting CARD proteins are likely subject to phosphorylation, ubiquitination, and other modifications that influence protein stability, localization, and activity .

• Protein turnover regulation: Degradation pathways including both proteasomal and autophagy-dependent mechanisms may regulate protein levels in response to cellular stimuli, similar to the regulation observed for BCL10 .

In human studies, BinCARD has been found to be ubiquitously expressed , suggesting that bovine variants may also have widespread expression with potential cell-type specific regulation in response to immune stimulation.

What are the optimal expression systems and purification strategies for recombinant bovine Bcl10-interacting CARD protein?

Expression and purification of recombinant bovine Bcl10-interacting CARD protein presents several challenges due to the protein's tendency to form higher-order structures. Based on research with similar CARD-containing proteins, the following methodological approach is recommended:

Expression Systems:

• Bacterial expression: E. coli BL21(DE3) with pET-based vectors containing an N-terminal His-tag can be used for initial attempts, preferably with reduced induction temperatures (16-18°C) to minimize inclusion body formation.

• Eukaryotic expression: Insect cell systems (Sf9, High Five) using baculovirus vectors may provide better folding and post-translational modifications compared to bacterial systems.

• Mammalian expression: HEK293 or CHO cells can be used when authentic mammalian post-translational modifications are required, though with lower yield than bacterial or insect systems.

Purification Strategy:

• Initial capture using immobilized metal affinity chromatography (IMAC)

• Ion exchange chromatography to separate charged variants

• Size exclusion chromatography to isolate monomeric protein and remove aggregates

When purifying CARD-containing proteins, buffer optimization is critical - inclusion of reducing agents (1-5 mM DTT or TCEP) and appropriate salt concentration (typically 150-300 mM NaCl) helps maintain protein solubility and prevent non-specific aggregation of the CARD domain.

What experimental approaches can be used to investigate the interaction between bovine Bcl10 and Bcl10-interacting CARD proteins?

Several complementary approaches can be employed to study the CARD-CARD interactions between bovine BCL10 and its interacting partners:

In vitro interaction studies:

• Pull-down assays: Using recombinant tagged proteins to assess direct binding between purified components .

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics and affinity constants.

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of the interaction.

Cellular interaction studies:

• Co-immunoprecipitation: To detect protein-protein interactions in bovine cell lysates under native conditions .

• Proximity Ligation Assay (PLA): For visualizing interactions in situ within intact cells.

• Mammalian two-hybrid assay: To verify interactions in a cellular context .

Structural studies:

• X-ray crystallography: To determine atomic resolution structures of the CARD-CARD complex.

• Cryo-electron microscopy: Particularly useful for examining larger assemblies and filaments formed by these proteins .

• NMR spectroscopy: For studying dynamic aspects of the interaction.

Functional validation:

• Mutagenesis of key residues: Targeted mutations at conserved residues (e.g., Leu17, Leu65) to validate the functional importance of specific amino acids .

• NF-κB reporter assays: To assess the functional consequence of the interaction on downstream signaling .

How do post-translational modifications regulate the function of bovine Bcl10-interacting CARD proteins?

Post-translational modifications (PTMs) likely play crucial roles in regulating bovine Bcl10-interacting CARD protein function, similar to the extensive regulation documented for BCL10 itself:

Phosphorylation:
Phosphorylation events may regulate:

• Binding affinity to BCL10

• Subcellular localization

• Protein stability and turnover

• Inhibitory capacity on NF-κB signaling

Based on BCL10 regulation patterns, kinases such as IKKβ, CaMKII, and GSK3β may phosphorylate bovine Bcl10-interacting CARD proteins at serine/threonine residues . Different phosphorylation sites likely have distinct functional outcomes - some enhancing while others diminishing protein activity.

Ubiquitination:
Ubiquitination may regulate:

• Protein stability and degradation

• Signaling complex formation

• Subcellular trafficking

Both K48-linked ubiquitination (leading to proteasomal degradation) and K63-linked ubiquitination (typically involved in signaling complex assembly) may occur on bovine Bcl10-interacting CARD proteins .

Methodological approaches to study PTMs:

• Mass spectrometry-based proteomics to identify modification sites

• Phospho-specific antibodies for detecting specific phosphorylation events

• Site-directed mutagenesis of potential modification sites to assess functional relevance

• In vitro kinase/ubiquitination assays to identify enzymes responsible for specific modifications

What methods can be used to analyze the inhibitory effect of bovine Bcl10-interacting CARD protein on NF-κB signaling?

To analyze the inhibitory effects of bovine Bcl10-interacting CARD protein on NF-κB signaling, researchers can employ the following methodological approaches:

Cell-based reporter assays:

• Dual-luciferase NF-κB reporter assay: Transfect bovine cells with NF-κB responsive luciferase reporter along with recombinant bovine Bcl10-interacting CARD protein. Measure luciferase activity following stimulation with NF-κB activators .

• Dose-response studies: Express increasing amounts of bovine Bcl10-interacting CARD protein to determine the concentration-dependent inhibitory effect.

Biochemical analyses:

• Western blotting for phosphorylated BCL10: Detect changes in BCL10 phosphorylation states in the presence of recombinant bovine Bcl10-interacting CARD protein .

• IKK activity assays: Measure the kinase activity of the IKK complex in cell lysates with and without bovine Bcl10-interacting CARD protein expression.

• Immunoprecipitation of CBM complex components: Assess changes in the assembly of the CARMA/CARD-BCL10-MALT1 complex in the presence of bovine Bcl10-interacting CARD protein.

Functional readouts of NF-κB activity:

• EMSA (electrophoretic mobility shift assay): To directly measure NF-κB DNA binding activity.

• ChIP (chromatin immunoprecipitation): To assess NF-κB subunit recruitment to target gene promoters.

• qRT-PCR and ELISA: To measure expression of NF-κB target genes and secreted cytokines.

What are the experimental challenges in studying filament formation by Bcl10 and its regulation by Bcl10-interacting CARD proteins?

Studying BCL10 filament formation and its regulation by Bcl10-interacting CARD proteins presents several experimental challenges:

Technical challenges in structural biology:

• Sample preparation for cryo-EM: Obtaining homogeneous preparations of BCL10 filaments with consistent structural properties is challenging due to their dynamic nature .

• Reconstitution of physiological filaments in vitro: Creating experimental conditions that recapitulate the cellular environment where these filaments naturally form.

• Resolving heterogeneity: BCL10 filaments may exist in multiple conformational states, complicating structural analysis .

Functional analysis challenges:

• Real-time monitoring: Developing systems to observe filament dynamics in real-time within living cells.

• Quantification: Accurately measuring filament formation, elongation rates, and disassembly.

• Separating effects: Distinguishing between inhibition of filament nucleation versus elongation or stability when studying regulatory factors.

Recommended methodological approaches:

• Fluorescence microscopy: Using tagged proteins to visualize filament formation in cells .

• Light scattering techniques: To monitor polymerization kinetics in vitro.

• Mutagenesis: Strategic mutations at the CARD-CARD interfaces to understand filament assembly mechanisms .

• In vitro reconstitution: Combining purified components to rebuild minimal systems for filament formation.

• Super-resolution microscopy: To examine detailed structures below the diffraction limit.

How can recombinant bovine Bcl10-interacting CARD protein be used in comparative immunology studies?

Recombinant bovine Bcl10-interacting CARD protein offers valuable opportunities for comparative immunology research:

Cross-species comparison of immune signaling mechanisms:

• Comparing the structural and functional conservation of CARD-mediated signaling between bovine, human, and other mammalian systems.

• Identifying species-specific adaptations in immune signaling cascades that may reflect different pathogen pressures.

• Evaluating differences in regulatory mechanisms controlling NF-κB activation across species.

Methodological approaches:

• In vitro signaling reconstitution: Using purified components from different species to compare biochemical properties and interaction specificities.

• Cross-complementation experiments: Testing whether bovine Bcl10-interacting CARD protein can functionally replace its human ortholog in cellular assays.

• Structural biology: Comparing the three-dimensional structures of CARD domains across species to identify conserved and divergent features.

• Evolutionary analysis: Conducting phylogenetic studies to trace the evolutionary history of these signaling components and identify signatures of positive selection.

Potential research applications table:

What are the promising future research directions for bovine Bcl10-interacting CARD proteins?

Several promising research directions could advance our understanding of bovine Bcl10-interacting CARD proteins:

Structural characterization:

• High-resolution structures of bovine Bcl10-interacting CARD proteins alone and in complex with BCL10.

• Comparative structural analysis with human orthologs to identify species-specific features.

• Cryo-EM studies of higher-order assemblies formed by these proteins.

Functional characterization in bovine-specific immune contexts:

• Role in bovine-specific immune responses to relevant pathogens.

• Tissue-specific expression patterns and functions in specialized bovine immune compartments.

• Species-specific signaling pathways and binding partners.

Therapeutic potential:

• Exploitation of the inhibitory effect on NF-κB signaling for therapeutic applications in bovine inflammatory diseases.

• Development of peptide inhibitors based on CARD-CARD interaction interfaces.

• Investigation of bovine Bcl10-interacting CARD proteins as potential targets for veterinary medicine.

Systems biology approaches:

• Integration of bovine Bcl10-interacting CARD proteins into comprehensive models of immune signaling networks.

• Comparative systems analysis of CBM complex regulation across species.

• Computational prediction of species-specific regulatory mechanisms.

Research challenges to address:

• Limited availability of bovine-specific research tools and reagents.

• Complexity of studying protein-protein interactions in physiologically relevant contexts.

• Translating in vitro findings to in vivo significance in bovine immunity.