Recombinant Human Bombesin receptor-activated protein C6orf89 (C6orf89)

What is the structural and functional characterization of BRAP?

Recombinant Human Bombesin Receptor-Activated Protein (BRAP) is encoded by the C6ORF89 gene and contains 354 amino acids. It has been identified as a putative type II membrane protein with an N-terminal transmembrane (TM) domain and a potential catalytic region within its C-terminus. The protein is widely expressed in multiple cell types including bronchial epithelial cells, macrophages, and neurons, suggesting diverse physiological roles across tissues. Functionally, BRAP has been demonstrated to regulate immune and inflammatory responses, particularly in the human airway epithelium where it modulates NF-κB signaling pathways . Studies have shown that the overexpression of BRAP in cultured immortalized human bronchial epithelial cells promotes cellular proliferation while also regulating NF-κB transcriptional activity .

How does BRAP modulate inflammatory signaling in epithelial cells?

BRAP exhibits significant regulatory effects on NF-κB transcriptional activity in human bronchial epithelial cells (HBECs). Research has demonstrated that overexpression of BRAP through gene transfer inhibits both basal and inducible NF-κB transcriptional activity in these cells, while BRAP knockdown produces the opposite effect, enhancing NF-κB activity . The mechanism underlying this regulation involves BRAP's ability to enhance histone deacetylase (HDAC) activity, which subsequently affects gene expression patterns related to inflammation. More specifically, BRAP might increase HDAC activity leading to NF-κB regulation through its putative C-terminal domain . This modulation of NF-κB signaling positions BRAP as a significant regulator of immune and inflammatory responses, particularly in the context of airway epithelium functions and associated pathologies.

What expression patterns does BRAP exhibit in normal and diseased tissues?

BRAP demonstrates differential expression patterns across normal and diseased tissues, providing insights into its potential pathophysiological roles. In healthy tissues, BRAP is expressed in several cell types including bronchial epithelial cells, macrophages, and neurons, suggesting broad physiological functions . The protein contains a putative N-terminal transmembrane domain, indicating possible membrane localization that may be important for its cellular functions. In pathological contexts, immunohistochemistry analyses have revealed significant BRAP protein presence in many interstitial cells within fibrotic lung tissues, suggesting a potential role in fibrotic processes . Additionally, BRAP signals have been detected in neurons of brain tissue samples, indicating potential neurological functions that remain to be fully characterized. While comprehensive expression profiling across all human tissues remains incomplete, the available evidence suggests tissue-specific and context-dependent expression patterns that may change during disease progression.

What experimental approaches are optimal for studying BRAP-mediated regulation of NF-κB activity?

Investigating BRAP's regulatory effects on NF-κB activity requires sophisticated experimental approaches that capture the complexity of this signaling relationship. Researchers have successfully employed gene transfer techniques to achieve BRAP overexpression in human bronchial epithelial cells, allowing for the assessment of its inhibitory effects on both basal and inducible NF-κB transcriptional activity . Complementary knockdown experiments using RNA interference technologies provide opposite manipulations that further validate functional relationships. For mechanistic investigations, researchers have focused on BRAP's relationship with histone deacetylase (HDAC) activity, demonstrating that BRAP enhances HDAC function, which subsequently affects NF-κB signaling . This approach requires HDAC activity assays coupled with selective inhibitors to establish causality. Additionally, domain-mapping experiments utilizing truncated BRAP constructs have implicated the C-terminal region in mediating these effects. For in vivo relevance, researchers have developed knockout mouse models (BC004004−/−) lacking the homologous protein of BRAP to study systemic effects in complex biological contexts . The mouse homologous protein shares 83% identity with human BRAP, making this a valuable but imperfect model system.

How does the knockout of BRAP affect pulmonary fibrosis development, and what are the underlying mechanisms?

Studies utilizing BRAP-deficient mouse models have revealed significant insights into its role in pulmonary fibrosis development. Researchers have constructed gene knockout mice (BC004004−/−) lacking the homologous protein of BRAP (which shares 83% identity with human BRAP) to investigate its function in vivo . While these knockout mice show no significant histological changes in major organs including heart, lung, liver, kidney, and spleen under normal conditions, they display remarkable phenotypic differences when challenged with fibrotic stimuli. When treated with bleomycin to establish a fibrotic lung injury model, BRAP-deficient mice demonstrated attenuated pulmonary fibrosis compared to wild-type controls . This suggests that BRAP normally contributes to fibrotic processes, and its absence provides protection against fibrotic development. The underlying mechanisms appear to involve fibrillar collagen metabolism and extracellular matrix regulation, as researchers observed that the fibrous connective tissue surrounding bones and joints in knockout mice was not as tough as in wild-type mice, indicating a possible defect in fibrillar collagens in the extracellular matrix due to lack of the BRAP homologous protein .

What are the optimal conditions for expressing and purifying recombinant human BRAP protein?

The expression and purification of recombinant human BRAP protein requires careful optimization due to its structural characteristics, particularly its putative transmembrane domain. Based on current research approaches, successful expression strategies typically employ mammalian expression systems such as HEK293 cells, which provide appropriate post-translational modifications and folding machinery for this complex human protein. For bacterial expression, specialized strains designed for membrane proteins coupled with lower induction temperatures (16-20°C) may improve solubility and folding. The purification process should begin with affinity chromatography, typically utilizing a histidine or GST tag fused to the recombinant protein. Critical buffer components should include: (1) mild detergents such as DDM or CHAPS (0.1-0.5%) to solubilize the transmembrane domain, (2) appropriate protease inhibitors to prevent degradation, and (3) reducing agents such as DTT to maintain protein stability. A multi-step purification approach incorporating size exclusion chromatography as a polishing step generally yields the highest purity. Quality control should include SDS-PAGE analysis, Western blotting with specific antibodies, and functional verification through HDAC activity enhancement assays to confirm that the purified protein retains its biological activity .

What experimental design is most effective for analyzing BRAP's enhancement of histone deacetylase activity?

An effective experimental design for analyzing BRAP's enhancement of histone deacetylase (HDAC) activity requires a multi-faceted approach that combines biochemical, cellular, and molecular techniques. The optimal design should include:

This experimental design should incorporate dose-dependent analyses with varying BRAP concentrations and time-course experiments to capture the dynamic nature of HDAC regulation. Additionally, the use of specific HDAC isoform inhibitors can help determine which HDAC family members are primarily affected by BRAP enhancement . The putative C-terminal domain of BRAP should receive particular attention, as previous research has implicated this region in mediating HDAC activity enhancement.

How can researchers effectively study the comparative biology of BRAP across different species models?

Studying the comparative biology of BRAP across different species requires a systematic approach that accounts for evolutionary conservation while identifying species-specific adaptations. The mouse homologous protein shares 83% identity with human BRAP, providing a foundation for comparative studies while necessitating careful interpretation of results . An effective research strategy should include:

• Sequence-structure-function analysis: Comprehensive alignment of BRAP sequences from multiple species to identify conserved domains versus variable regions. This should be coupled with homology modeling to predict three-dimensional structural conservation, particularly of the putative N-terminal transmembrane domain and C-terminal catalytic region.

• Cross-species expression studies: Expression of human BRAP in mouse cells and vice versa, with functional readouts including NF-κB activity and HDAC enhancement. This approach can identify whether functional differences arise from the protein itself or the cellular context.

• Domain-swapping experiments: Creation of chimeric proteins containing domains from different species' BRAP to localize regions responsible for species-specific functions.

• Equivalent knockout models: Development of comparable knockout models across species using CRISPR/Cas9 engineering with standardized phenotypic analysis protocols.

• Antibody validation: Rigorous validation of antibodies for cross-reactivity between species, which is essential for accurate protein detection. Current antibodies generated against human BRAP (fragment 100-250 aa) can detect both human and mouse proteins in Western blotting but show limitations in mouse immunohistochemistry applications .

• Parallel challenge models: Exposure of different species to identical stressors (e.g., bleomycin for pulmonary fibrosis) with standardized analysis methods to compare physiological responses.

This multi-faceted approach enables reliable cross-species comparisons while identifying evolutionarily conserved versus species-specific aspects of BRAP biology.

How should researchers interpret contradictory findings regarding BRAP's role in proliferation versus fibrosis?

Interpreting contradictory findings regarding BRAP's role in proliferation versus fibrosis requires nuanced analysis that considers cellular context, temporal dynamics, and pathway-specific effects. In cultured immortalized human bronchial epithelial cells, BRAP overexpression promotes proliferation while simultaneously inhibiting NF-κB activity . Conversely, in bleomycin-induced lung injury models, BRAP deficiency (through knockout of its mouse homolog) attenuates pulmonary fibrosis, suggesting that BRAP normally contributes to fibrotic processes . This apparent contradiction can be reconciled through several interpretative frameworks:

• Cell-type specificity: BRAP may exert different effects in epithelial cells (proliferative) versus mesenchymal or immune cells (pro-fibrotic), with the net outcome in complex tissues reflecting the dominant cell population in a given context.

• Temporal dynamics: BRAP's growth-promoting effects may be beneficial during normal epithelial renewal but become maladaptive during chronic injury when sustained proliferative signals contribute to fibrotic remodeling.

• Pathway interactions: While BRAP inhibits NF-κB in epithelial cells, it may simultaneously enhance other pathways relevant to fibrosis (e.g., TGF-β signaling) that were not evaluated in the original studies.

• Compensatory mechanisms: Acute BRAP manipulations in cell culture may trigger different responses than germline knockout in animals, where developmental compensation might occur.

• Disease stage-specific effects: BRAP might play distinct roles during initiation versus progression of fibrosis, with different experimental models capturing different temporal windows.

Researchers should carefully consider these possibilities when designing experiments and interpreting results, ideally using multiple complementary models and measuring multiple outcome parameters to build a more comprehensive understanding of BRAP's complex biology.

What methodological considerations are important when analyzing BRAP's interaction with the NF-κB pathway?

Analyzing BRAP's interaction with the NF-κB pathway requires careful methodological considerations to ensure accurate and reproducible results. When conducting such studies, researchers should address several critical factors:

• Stimulus specificity: Different NF-κB activators (TNF-α, IL-1β, LPS) may reveal distinct aspects of BRAP's regulatory function. Studies have shown that BRAP inhibits both basal and inducible NF-κB transcriptional activity in human bronchial epithelial cells, but the effect size may vary with different stimuli .

• Temporal dynamics: The timing of measurements is crucial, as BRAP may affect early signaling events (IκB phosphorylation, p65 nuclear translocation) differently than sustained transcriptional responses. Time-course experiments are essential to capture these dynamics.

• Readout selection: Multiple complementary assays should be employed, including:

  • Reporter gene assays (e.g., NF-κB-luciferase)

  • Western blotting for pathway components (p-IκB, nuclear p65)

  • ChIP assays to measure NF-κB binding to target gene promoters

  • RT-qPCR of NF-κB target genes

  • Protein-protein interaction studies (co-IP, PLA)

• HDAC involvement: Since BRAP enhances histone deacetylase (HDAC) activity, which subsequently affects NF-κB signaling, HDAC activity measurements should be incorporated into experimental designs . HDAC inhibitors can be used to determine whether BRAP's effects on NF-κB are HDAC-dependent.

• Cell type considerations: BRAP's effects may vary across cell types, necessitating validation in multiple relevant cell systems. Primary cells should be included alongside cell lines when possible.

• Genetic manipulation approaches: Both overexpression and knockdown/knockout approaches should be employed, with appropriate controls (empty vector, non-targeting siRNA) and verification of manipulation efficiency.

• Domain analysis: Structure-function studies using truncated or mutated BRAP constructs are essential to map which regions (particularly the C-terminal domain) mediate NF-κB regulation .

By addressing these methodological considerations, researchers can generate more reliable and comprehensive insights into BRAP's complex role in NF-κB pathway regulation.

How can bioinformatic approaches enhance our understanding of BRAP functions across tissues?

Bioinformatic approaches offer powerful tools to expand our understanding of BRAP functions across diverse tissues, particularly when experimental data is limited. These computational methods can integrate multiple data types to generate new hypotheses and guide experimental design:

• Expression correlation networks: By analyzing RNA-seq data across tissues, researchers can identify genes whose expression patterns correlate with BRAP, suggesting functional relationships or shared regulatory mechanisms. This approach can reveal tissue-specific co-expression patterns that might explain the variable effects of BRAP in different contexts, such as its role in bronchial epithelial cells versus interstitial cells in fibrotic lungs .

• Protein-protein interaction prediction: Computational methods can predict potential BRAP binding partners based on sequence, structure, and co-expression data. These predictions can guide experimental validation of interactions, particularly with components of the NF-κB pathway and histone deacetylase complexes.

• Pathway enrichment analysis: Integration of BRAP-associated genes into pathway databases can identify biological processes beyond the currently known NF-κB and HDAC connections, potentially explaining its diverse roles in proliferation and fibrosis.

• Structural modeling and docking: Homology modeling of BRAP's structure, particularly its putative N-terminal transmembrane domain and potential catalytic C-terminal region , can predict functional sites and guide mutagenesis studies.

• Single-cell transcriptomic analysis: Mining single-cell RNA-seq datasets can reveal cell type-specific expression patterns of BRAP across tissues, potentially explaining contextual differences in function.

• Cross-species conservation analysis: Evolutionary analysis of BRAP across species can identify highly conserved regions likely to be functionally critical, distinguishing them from more variable regions that might confer species-specific functions.

• Disease association studies: Integration of GWAS and eQTL data can connect BRAP genetic variants with disease phenotypes, potentially uncovering new roles beyond the currently studied pulmonary fibrosis context.

These bioinformatic approaches can provide a more comprehensive understanding of BRAP biology while guiding focused experimental studies in the most promising directions.

What are the most promising therapeutic applications targeting BRAP in inflammatory and fibrotic diseases?

Based on current understanding of BRAP's biological functions, several promising therapeutic applications targeting this protein in inflammatory and fibrotic diseases can be envisioned:

• Small molecule inhibitors: Development of compounds targeting BRAP's C-terminal domain could disrupt its enhancement of histone deacetylase activity, potentially reducing inflammatory and fibrotic responses. This approach is supported by evidence that BRAP enhances HDAC activity leading to NF-κB regulation .

• Anti-fibrotic applications: Given that BRAP knockout mice show attenuated bleomycin-induced pulmonary fibrosis , strategies to downregulate BRAP expression or activity specifically in pulmonary interstitial cells could represent a novel approach for treating idiopathic pulmonary fibrosis and other fibrotic lung diseases.

• Combined HDAC/BRAP targeting: Synergistic therapeutic approaches combining partial HDAC inhibition with BRAP modulation might allow for more selective anti-inflammatory effects with reduced side effects compared to conventional HDAC inhibitors.

• Cell-specific delivery strategies: Targeted delivery of BRAP-modulating agents to specific cell populations (e.g., airway epithelial cells for asthma/COPD or interstitial fibroblasts for fibrosis) could improve therapeutic index by concentrating effects in disease-relevant cells.

• Biomarker-guided therapy: Development of BRAP expression or activity assays as companion diagnostics could identify patients most likely to benefit from BRAP-targeted therapies, enabling precision medicine approaches.

• Combination with standard therapies: BRAP modulators could potentially enhance the efficacy of current anti-inflammatory or anti-fibrotic treatments by targeting complementary pathways.

While promising, these therapeutic applications require further validation through additional preclinical studies and careful translation to human disease contexts, particularly given some apparent contradictions between in vitro cellular effects and in vivo disease models .

What novel experimental techniques could advance our understanding of BRAP's membrane topology and subcellular localization?

Advanced experimental techniques could significantly enhance our understanding of BRAP's membrane topology and subcellular localization, which remain incompletely characterized despite its identification as a putative type II membrane protein with an N-terminal transmembrane domain . The following innovative approaches could provide critical insights:

• Cryo-electron microscopy (cryo-EM): This technique could resolve BRAP's structure at near-atomic resolution while preserving its native membrane environment, particularly when combined with lipid nanodisc reconstitution to maintain the transmembrane domain in a native-like state.

• Super-resolution microscopy techniques: Methods such as STORM, PALM, or STED microscopy would enable visualization of BRAP's subcellular distribution with nanometer precision, potentially revealing distinct localization patterns in different cell types or under various stimulation conditions.

• Split-fluorescent protein complementation assays: These approaches could map BRAP's topology by determining which domains reside on which side of the membrane, while simultaneously identifying interaction partners in specific subcellular compartments.

• Proximity labeling techniques: Methods such as BioID or APEX2 fused to BRAP could identify neighboring proteins in living cells, providing insights into its microenvironment within membrane compartments.

• Live-cell imaging with optogenetic tools: Combining fluorescently tagged BRAP with optogenetic control of its activity could reveal dynamic changes in localization coupled with function.

• Mass spectrometry-based spatial proteomics: Techniques like LOPIT (localization of organelle proteins by isotope tagging) could systematically map BRAP's distribution across subcellular compartments.

• Single-molecule tracking: This approach could measure the diffusional behavior of BRAP molecules in the membrane, potentially revealing functional interactions or confinement in specific membrane domains.

• Correlative light and electron microscopy (CLEM): This technique could connect fluorescence imaging of tagged BRAP with ultrastructural details of its cellular context.

These advanced techniques would provide crucial information about how BRAP's localization relates to its diverse functions in regulating NF-κB activity, enhancing HDAC function, and contributing to processes like cell proliferation and fibrotic responses.

How might single-cell multi-omics approaches reveal new insights about BRAP's role in heterogeneous tissues?

Single-cell multi-omics approaches offer unprecedented opportunities to unravel BRAP's complex roles in heterogeneous tissues, potentially resolving apparent contradictions in existing research findings. These advanced technologies can provide several key insights:

• Cell type-specific expression patterns: Single-cell RNA sequencing (scRNA-seq) can identify which specific cell populations within complex tissues express BRAP at the highest levels. This is particularly relevant given BRAP's known expression in bronchial epithelial cells and interstitial cells of fibrotic lungs , but likely extends to other cell types that remain uncharacterized.

• Regulatory network reconstruction: By integrating single-cell transcriptomics with epigenomic data (scATAC-seq), researchers can map the regulatory networks controlling BRAP expression in different cellular contexts, potentially explaining tissue-specific functions.

• Protein-protein interaction landscapes: Single-cell proteomics and proximity labeling approaches can reveal cell type-specific BRAP interaction partners, potentially explaining why it promotes proliferation in some contexts while contributing to fibrosis in others.

• Dynamic responses to perturbations: Single-cell technologies applied to stimulated or injured tissues can track how BRAP expression and activity change during disease progression in specific cell populations.

• Spatial context integration: Spatial transcriptomics methods can map BRAP expression patterns within intact tissue architecture, revealing potential relationships between BRAP-expressing cells and their microenvironment.

• Trajectory analysis of cellular transitions: During processes like fibrotic transformation, single-cell approaches can track how BRAP expression and associated pathways change as cells transition between phenotypic states.

• Cell-cell communication analysis: Single-cell data can identify potential paracrine signaling relationships between BRAP-expressing cells and neighboring populations, revealing non-cell-autonomous mechanisms of action.

These approaches could explain the seemingly contradictory findings that BRAP promotes proliferation in cultured bronchial epithelial cells while contributing to fibrosis in vivo , potentially by revealing distinct functions in different cell populations within the same tissue or identifying sequential roles during disease progression.