Recombinant Mouse Bcl10-interacting CARD protein

What is the basic structure of mouse Bcl10 protein?

Mouse Bcl10 is a 233 amino acid protein that contains an N-terminal CARD domain (approximately residues 13-103), which is critical for its signaling function. The protein forms filaments through CARD-CARD interactions that are essential for downstream signaling. The full-length sequence includes both the CARD domain and C-terminal regions that mediate interactions with additional proteins in the signaling cascade . The CARD domain adopts a characteristic six-helix bundle structure, similar to other CARD-containing proteins, with helices 1-6 arranged in a specific orientation that facilitates protein-protein interactions .

What are the primary functions of Bcl10 in immune signaling?

Bcl10 functions as a key adaptor protein that bridges CARD domain-containing proteins to immune activation pathways. It channels both adaptive and innate immune signaling downstream of CARD domain-containing proteins including CARD9, CARD11, and CARD14 to activate NF-kappa-B and MAP kinase p38 (MAPK11, MAPK12, MAPK13, and/or MAPK14) pathways . These pathways ultimately stimulate the expression of genes encoding pro-inflammatory cytokines and chemokines. In adaptive immunity, Bcl10 is activated by CARD11 downstream of T-cell receptor (TCR) and B-cell receptor (BCR) signaling, while in innate immunity, it is activated by CARD9 downstream of C-type lectin receptors, which are essential for antifungal immunity .

How does Bcl10 interact with CARD domain-containing proteins?

Bcl10 interacts with CARD domain-containing proteins through heterotypic CARD-CARD interactions. The process involves homooligomerized CARD domain-containing proteins forming a nucleating helical template that recruits Bcl10 via CARD-CARD interaction . This promotes polymerization of Bcl10, subsequent recruitment of MALT1, and formation of a CBM (CARD-BCL10-MALT1) complex . Structural studies combined with molecular modeling have demonstrated that the CARD of proteins like CARD11 can function as a seed to nucleate the assembly of Bcl10 CARD filaments. While in vitro Bcl10 filaments can form in the absence of CARD11, CARD11 decreases the lag period of Bcl10 polymerization and thus appears to function as an initiator of the process .

What domains mediate the interaction between Bcl10 and MALT1?

The association between Bcl10 and MALT1 involves complex interactions between multiple protein domains. Deletional analysis has shown that Bcl10 amino acids 1-114 are sufficient to mediate NF-κB activation and to associate with MALT1 . Specifically, the Bcl10 CARD (approximately residues 13-103) comprises the majority of this fragment and contributes significantly to binding with MALT1 . Additional studies have revealed that the region between amino acids 107-119 is also important, with specific conserved residues in this region affecting MALT1 binding efficiency . Molecular modeling and mutagenesis studies have identified charged or polar residues (including Asp 80, Glu 84, Lys 90, and Asp 101) on the helix 5-6 face of the Bcl10 CARD that may be important for the interaction with MALT1 .

What are the most effective methods for expressing and purifying recombinant mouse Bcl10 protein?

Recombinant Bcl10 protein can be effectively expressed in Escherichia coli expression systems, similar to the human Bcl10 protein described in the search results . A typical approach includes:

• Cloning the full-length mouse Bcl10 cDNA (amino acids 1-233) into a suitable expression vector with a histidine tag

• Transforming the construct into an E. coli strain optimized for protein expression

• Inducing protein expression with IPTG

• Lysing cells and purifying the protein using affinity chromatography with nickel columns

• Further purifying by size-exclusion chromatography to ensure >85% purity

The purified protein should be suitable for downstream applications such as SDS-PAGE and mass spectrometry analysis . Researchers should be aware that Bcl10 has a tendency to form filaments, which can affect purification strategies and subsequent experimental applications.

How can I design optimal mutagenesis studies to investigate Bcl10-CARD interactions?

Based on structural and functional studies, an effective mutagenesis approach should target:

How can CRISPR/Cas9 technology be utilized to study Bcl10-CARD protein interactions in mouse models?

CRISPR/Cas9 technology provides powerful approaches for studying Bcl10-CARD protein interactions in mouse models:

• Generation of knockout cell lines: Create Bcl10 or CARD protein (e.g., CARD11) knockout cell lines using sgRNAs targeting specific exons. For Bcl10, an effective strategy involves targeting exon1-intron1, using two sgRNAs to create a deletion .

• Verification of knockout efficiency: Confirm the loss of protein expression through Western blot analysis using appropriate antibodies .

• Functional reconstitution studies: Perform lentiviral transduction with wild-type or mutant Bcl10 or CARD proteins to assess functional complementation in knockout cells .

• Chimeric protein approach: Design fusion proteins (e.g., Bcl10-CARD11) to bypass inducible recruitment and investigate specific aspects of signaling. Include appropriate mutations (e.g., MALT1 cleavage-resistant mutations like Bcl10 R228A) to prevent indirect effects .

• Readouts for functional analysis: Monitor downstream signaling events, such as NF-κB activation or cleavage of MALT1 substrates (e.g., CYLD, A20) .

What are the critical technical considerations when designing fusion proteins to study Bcl10-CARD interactions?

When designing fusion proteins to study Bcl10-CARD interactions, consider the following technical aspects:

• Orientation of fusion: The orientation can significantly affect protein function. For example, fusing Bcl10 through its C-terminus to the N-terminus of CARD11 can create a functional chimera that bypasses inducible CARD11-Bcl10 association .

• Incorporation of mutations: Include specific mutations to prevent unwanted effects, such as the MALT1 cleavage-resistant Bcl10 R228A mutation to avoid indirect reconstitution of CARD11 from cleaved fusion proteins .

• Expression level control: Design constructs that allow for expression at near-endogenous levels, as overexpression can lead to constitutive activation of downstream pathways .

• Inclusion of functional mutations: Incorporate mutations that disrupt specific interactions (e.g., BCL10 R42E to prevent BCL10 oligomerization, CARD11 R35A to disrupt CARD function) to study their individual contributions to signaling .

• Detection tags: Include appropriate epitope tags that do not interfere with protein function but allow for detection and purification of the fusion proteins .

What are common pitfalls in Bcl10-CARD protein interaction studies and how can they be overcome?

Common challenges and solutions in Bcl10-CARD protein interaction studies include:

• Protein instability: Bcl10 fusion proteins with a functional CARD domain are often expressed at lower levels compared to mutants that prevent Bcl10 oligomerization . Solution: Optimize expression conditions, include proteasome inhibitors during extraction, and consider using cleavage-resistant mutations.

• Non-specific aggregation: The tendency of Bcl10 to form filaments can lead to non-specific aggregation in vitro. Solution: Include appropriate controls to distinguish between functional oligomerization and non-specific aggregation, such as mutations that specifically disrupt known interaction interfaces.

• Interference from endogenous proteins: Endogenous Bcl10 or CARD proteins can complicate the interpretation of results. Solution: Use knockout cell lines generated by CRISPR/Cas9 technology as clean genetic backgrounds for reconstitution studies .

• Variability in activation readouts: NF-κB activation assays can show variability. Solution: Use multiple readouts, including direct assessment of protein interactions by co-immunoprecipitation and monitoring of downstream substrate cleavage events.

How can I optimize co-immunoprecipitation assays for studying Bcl10-CARD protein interactions?

To optimize co-immunoprecipitation (co-IP) assays for Bcl10-CARD protein interactions:

• Lysis conditions: Use mild detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions while effectively lysing cells.

• Salt concentration: Optimize salt concentration in buffers (typically 150-300 mM NaCl) to reduce non-specific binding while maintaining specific interactions.

• Controls for specificity: Include appropriate negative controls, such as:

  • Mutants known to disrupt specific interactions (e.g., Bcl10 G78R for CARMA1 binding)

  • Deletion mutants lacking interaction domains (e.g., Bcl10 Δ107-119 for MALT1 binding)

• Expression level variation: Account for differences in expression levels by performing titration experiments to ensure that co-IP efficiency is not simply due to higher expression levels .

• Antibody selection: Choose antibodies with high specificity and affinity for the target proteins, and consider using epitope tags (FLAG, HA, etc.) when specific antibodies are not available.

How should I interpret discrepancies between in vitro and cellular studies of Bcl10-CARD interactions?

When faced with discrepancies between in vitro and cellular studies:

• Consider context-dependent regulation: Bcl10-CARD interactions may be regulated differently in cellular environments due to post-translational modifications, localization, or additional binding partners not present in simplified in vitro systems.

• Evaluate protein concentration effects: In vitro studies often use protein concentrations that differ significantly from physiological levels. Bcl10 filaments can form spontaneously in vitro at high concentrations but may require nucleation by CARD proteins like CARD11 at physiological concentrations .

• Assess the impact of experimental tags and fusion proteins: Tags used for purification or detection can affect protein folding, interactions, or functional activity. Compare results obtained with differently tagged constructs.

• Analyze the role of post-translational modifications: Phosphorylation and ubiquitination of Bcl10 occur in cells and regulate its function, but these modifications may be absent in recombinant proteins used for in vitro studies.

• Combine multiple approaches: Use complementary techniques (structural studies, biochemical assays, cellular assays, and in vivo models) to build a more comprehensive understanding of the interactions.

What statistical approaches are most appropriate for analyzing Bcl10-CARD protein interaction data?

When analyzing Bcl10-CARD protein interaction data:

• Quantification of co-IP efficiency: Normalize co-immunoprecipitated proteins to both the immunoprecipitated bait protein and the input levels to account for variations in expression and IP efficiency.

• Comparison across multiple mutations: When analyzing multiple point mutations, use ANOVA with appropriate post-hoc tests rather than multiple t-tests to control for family-wise error rate.

• Dose-response relationships: When testing concentration-dependent effects, use regression analysis to determine EC50 values and Hill coefficients that can provide insights into cooperativity of interactions.

• Time-course experiments: Apply appropriate time-series analysis methods for experiments monitoring the kinetics of complex formation or downstream signaling events.

• Reproducibility assessment: Perform experiments with biological replicates (different protein preparations, different days, etc.) rather than just technical replicates to ensure robustness of findings.