Recombinant Mouse Bombesin receptor-activated protein C6orf89 homolog

What is the Bombesin receptor-activated protein (BRAP) and how is it related to C6orf89?

Bombesin receptor-activated protein (BRAP) is a protein encoded by the C6orf89 gene in humans. It was initially identified as a potential binding partner for the Bombesin Receptor Subtype-3 (BRS-3) using bacteria two-hybrid screening methods . In mice, the homolog of BRAP is encoded by the BC004004 gene, which shares approximately 83% sequence similarity with the human version . BRAP is abundantly expressed in keratinocytes, which are the predominant cell type in the epidermis, suggesting its importance in skin biology and potentially in inflammatory skin conditions .

What are the known functional domains of mouse BRAP homolog?

The mouse BRAP homolog, while sharing significant structural similarity with human BRAP, has distinct functional domains that contribute to its biological activities. Research indicates that BRAP deficiency alters inflammatory responses, particularly in skin conditions like psoriasis. The protein appears to play a regulatory role in cytokine production, especially thymic stromal lymphopoietin (TSLP), which mediates communication between epidermal cells and immune cells . Studies with knockout mice (BC004004-/-) have revealed that BRAP deficiency leads to altered inflammatory patterns, with evidence suggesting it regulates keratinocyte-immune cell crosstalk through TSLP-dependent mechanisms .

How is BRAP expression regulated during inflammatory responses?

BRAP expression demonstrates dynamic regulation during inflammatory responses. Research using imiquimod (IMQ)-induced psoriasis models has shown that the first application of IMQ triggers an increase in BRAP homolog expression in the skin of wild-type mice . This suggests that BRAP may be upregulated as part of the early inflammatory response. The precise molecular mechanisms controlling this upregulation remain under investigation, but the rapid response indicates that BRAP likely plays a role in the initial stages of inflammation regulation . Understanding these regulatory mechanisms is crucial for researchers examining BRAP's role in inflammatory conditions.

What chemical compounds are known to alter C6orf89 homolog expression?

Several chemical compounds have been documented to influence the expression of C20h6orf89 (the rat homolog of C6orf89). Research data indicates differential effects on expression depending on the compound:

These data provide important considerations for experimental design when studying BRAP expression regulation .

What are the essential controls when studying recombinant mouse BRAP in inflammatory models?

When studying recombinant mouse BRAP in inflammatory models, researchers should implement several critical controls:

• Genetic Controls: Include both wild-type (BC004004+/+) and knockout (BC004004-/-) mice in parallel experiments to accurately assess BRAP-dependent effects .

• Temporal Controls: Monitor inflammatory responses at multiple time points (e.g., days 1, 4, and 7 after inflammatory induction) to capture the dynamic nature of inflammation, as BRAP deficiency alters both the onset and resolution of inflammation .

• Cytokine Profile Controls: Measure multiple cytokines simultaneously (IL-17A, IL-1β, TGF-β1, IL-23, and TSLP) to comprehensively assess the inflammatory landscape, as BRAP deficiency differentially affects various inflammatory mediators .

• Cell-Specific Controls: When conducting in vitro experiments, include both BRAP-silenced and control cells to distinguish cell-autonomous effects from systemic responses .

These controls are essential because BRAP deficiency has been shown to cause altered inflammation kinetics rather than simply exacerbating or attenuating inflammation .

What are the most effective methods for silencing BRAP expression in experimental models?

For effective BRAP silencing in experimental models, researchers have successfully utilized several approaches:

• siRNA Transfection: Human BRAP expression can be efficiently downregulated using Stealth RNAi™ siRNAs targeting the C6orf89 gene (e.g., siRNA IDs: HSS137527, HSS137528, and HSS137529) transfected with Lipofectamine™ 3000 according to manufacturer protocols . This method is particularly effective for in vitro studies using keratinocyte cell lines like HaCaT.

• Gene Knockout Models: For in vivo studies, BC004004-/- knockout mice provide a comprehensive model for studying BRAP deficiency effects at the organismal level . These models are particularly valuable for investigating complex phenotypes like altered inflammatory responses in skin conditions.

• Verification Methods: Regardless of the silencing approach, researchers should verify BRAP suppression through:

  • Western blot analysis using specific antibodies (e.g., BRAP antibody, Abcam, cat: ab181073; 1:2,000 dilution)

  • RT-PCR for mRNA expression levels

  • Functional assays to confirm phenotypic changes (e.g., TSLP release measurement in culture media or serum)

The choice between these methods depends on the specific research questions, with siRNA approaches offering transient, cell-specific silencing, while knockout models provide insights into systemic effects of constitutive BRAP deficiency .

What are the key phenotypic differences between BC004004-/- knockout mice and wild-type controls in inflammatory models?

BC004004-/- knockout mice display several distinct phenotypic differences when compared to wild-type controls in inflammatory models, particularly in imiquimod (IMQ)-induced psoriasis-like inflammation:

• Altered Inflammatory Kinetics: Knockout mice develop skin lesions with earlier and more acute onset, followed by quicker remission compared to wild-type mice, which exhibit milder but more sustained inflammation .

• Temporal Cytokine Profile Differences:

  • On day 1 of IMQ treatment: Increased TGF-β1 and IL-23 in knockout mice

  • On day 4: Significantly higher levels of IL-1β, TGF-β1, and IL-17A in knockout mice, corresponding with peak inflammation severity

  • By day 7: Inflammation in knockout mice subsides more rapidly than in wild-type mice

• TSLP Expression: Substantially higher levels of thymic stromal lymphopoietin (TSLP) in both skin tissue and serum of knockout mice, with peak TSLP levels observed on day 4 of treatment .

• Th17 Cell Activation: Enhanced activation of Th17 cells in knockout mice, as evidenced by elevated IL-17A expression, suggesting a more robust but transient immune response .

These phenotypic differences suggest that BRAP functions as a regulator of inflammatory kinetics rather than simply suppressing inflammation, highlighting its potential role in modulating the temporal aspects of inflammatory responses .

Beyond skin inflammation, what other phenotypes have been observed in BC004004 knockout mice?

BC004004 knockout mice exhibit several phenotypes beyond skin inflammation, demonstrating the multifaceted role of BRAP in various physiological processes:

• Altered Fibrotic Responses: BC004004-/- mice show attenuation of injury-induced fibrosis in both lungs and kidneys, suggesting that BRAP may play a role in promoting fibrotic responses after tissue injury .

• Behavioral Changes: Exacerbation of stress-induced behavioral changes has been observed in knockout mice, indicating a potential role for BRAP in neurological responses to stress .

• Immune System Modulation: The knockout mice demonstrate altered cytokine profiles not only in skin but in systemic circulation, suggesting broader immune system effects beyond localized inflammation .

• Keratinocyte Function: BRAP deficiency alters keratinocyte cytokine production, particularly thymic stromal lymphopoietin (TSLP), which may influence various epithelial-immune cell interactions throughout the body .

These diverse phenotypes suggest that BRAP homologs serve as important modulators in multiple organ systems, potentially through common mechanisms involving cytokine regulation and cell-cell communication pathways . Researchers investigating BRAP should consider these broader systemic effects when designing experiments and interpreting results.

How does BRAP deficiency contribute to altered inflammatory responses in psoriasis-like models?

BRAP deficiency contributes to altered inflammatory responses in psoriasis-like models through several interconnected mechanisms:

• Enhanced TSLP Production: BC004004-/- mice exhibit significantly elevated TSLP levels in both skin tissue and serum during imiquimod (IMQ) treatment. This increase in TSLP was confirmed through both in vivo studies and in vitro experiments where BRAP silencing in human keratinocyte-derived HaCaT cells led to increased TSLP release .

• Modified Cytokine Cascade: BRAP deficiency alters the expression pattern of multiple psoriasis-related cytokines:

  • Initial increase in TGF-β1 and IL-23 upon IMQ application

  • Elevated IL-17A levels on day 4 of treatment, suggesting enhanced Th17 cell activation

  • Higher IL-1β expression corresponding with peak inflammation

• Altered Inflammatory Kinetics: Rather than simply exacerbating inflammation, BRAP deficiency modifies the temporal pattern of inflammatory responses, leading to earlier onset but faster resolution of inflammation .

• Keratinocyte-Immune Cell Crosstalk: The elevated level of TSLP released from keratinocytes due to BRAP deficiency appears to mediate communication between epidermal cells and immune cells, contributing to the altered pathological changes observed in psoriasis-like skin lesions .

This evidence suggests that BRAP functions as a regulator of inflammatory dynamics rather than a simple inhibitor or promoter of inflammation, making it a potential target for therapies aimed at modulating the course rather than simply suppressing inflammatory responses .

What is the relationship between BRAP, TSLP, and inflammatory responses in keratinocytes?

The relationship between BRAP, TSLP (thymic stromal lymphopoietin), and inflammatory responses in keratinocytes represents a critical regulatory axis in skin inflammation:

• BRAP as a TSLP Regulator: Research demonstrates that BRAP functions as a negative regulator of TSLP production in keratinocytes. When BRAP expression is reduced or eliminated through knockout (BC004004-/-) or siRNA silencing, TSLP production and release significantly increase .

• Temporal Dynamics: During IMQ-induced inflammation:

  • TSLP levels in BC004004-/- mice gradually increased during treatment

  • Peak TSLP levels occurred on day 4, coinciding with peak inflammation

  • TSLP levels in knockout mice were significantly higher than in wild-type controls throughout the treatment period

• In Vitro Confirmation: Studies using HaCaT cells (human keratinocyte-derived cells) with C6orf89 silencing showed increased TSLP release into culture media, confirming that this relationship is cell-autonomous and not solely dependent on complex in vivo interactions .

• Immunohistochemical Evidence: IHC analysis showed that IMQ treatment increased TSLP content within the epidermis in both knockout and wild-type mice, but TSLP was consistently more abundant in BC004004-/- mice on days 1, 4, and 7 of treatment .

This regulatory relationship suggests that BRAP serves as a checkpoint in keratinocyte-derived inflammatory signaling, with important implications for understanding and potentially treating inflammatory skin conditions like psoriasis . The BRAP-TSLP axis represents a potential therapeutic target for modulating skin inflammation at the level of keratinocyte-immune cell communication.

What are the potential molecular mechanisms by which BRAP regulates TSLP production?

The molecular mechanisms by which BRAP regulates TSLP production remain under investigation, though current research suggests several potential pathways:

• Protein-Protein Interactions: BRAP was initially identified as a binding partner for Bombesin Receptor Subtype-3 (BRS-3) . This suggests that BRAP may regulate TSLP production through interactions with receptor-mediated signaling pathways that ultimately control TSLP gene expression or protein processing.

• Inflammatory Signaling Regulation: The altered expression of multiple inflammatory cytokines (IL-1β, TGF-β1, IL-17A, IL-23) in BRAP-deficient models suggests that BRAP may regulate TSLP through broader effects on inflammatory signaling networks . These cytokines form interconnected networks that could converge on TSLP regulation.

• Epigenetic Regulation: Some chemical compounds that interact with the C20h6orf89 gene (rat homolog) influence methylation patterns. For example, benzo[a]pyrene increases methylation of the C6orf89 promoter . This suggests BRAP may be involved in epigenetic regulatory mechanisms that control TSLP expression.

• Keratinocyte-Specific Signaling: The cell-autonomous increase in TSLP production following BRAP silencing in keratinocytes indicates that BRAP likely acts through keratinocyte-specific signaling pathways rather than solely through interactions with immune cells .

Further research employing techniques such as protein-protein interaction studies, chromatin immunoprecipitation, and signaling pathway inhibition will be necessary to fully elucidate the specific molecular mechanisms connecting BRAP to TSLP regulation .

How might the study of BRAP contribute to understanding of epithelial-immune cell crosstalk?

The study of BRAP offers significant insights into epithelial-immune cell crosstalk, particularly in inflammatory skin conditions:

• Keratinocyte-Derived Signaling: Research has demonstrated that BRAP deficiency in keratinocytes leads to increased production of TSLP, a crucial cytokine that acts as a bridge between epithelial cells and immune cells . This provides a mechanistic model for how epithelial-derived signals influence immune responses.

• Temporal Regulation of Inflammation: BC004004-/- mice exhibit altered inflammatory kinetics with earlier onset and faster resolution of inflammation . This suggests that BRAP influences not only the magnitude but also the timing of epithelial-immune interactions, potentially through regulated release of signaling molecules.

• Cytokine Network Modulation: The altered expression patterns of multiple cytokines (IL-17A, IL-1β, TGF-β1, IL-23) in BRAP-deficient models reveal how a single epithelial protein can reshape entire cytokine networks that mediate communication between tissue compartments .

• Translational Relevance: Understanding BRAP's role in epithelial-immune crosstalk could lead to novel therapeutic approaches for conditions characterized by dysregulated communication between these cell types, such as psoriasis, atopic dermatitis, and potentially other inflammatory conditions .

By elucidating how BRAP regulates this cellular crosstalk, researchers may identify new targets for therapeutic intervention that specifically modulate epithelial-immune communication rather than broadly suppressing immune function .

What are the most promising applications for recombinant mouse BRAP in current research?

Recombinant mouse BRAP offers several promising applications in current research:

• Functional Reconstitution Studies: Introducing recombinant BRAP into BC004004-/- systems allows researchers to establish causal relationships between BRAP function and observed phenotypes . This approach helps determine which aspects of altered inflammatory responses are directly attributable to BRAP deficiency versus secondary effects.

• Protein-Protein Interaction Mapping: Recombinant BRAP can be used to identify binding partners in various cellular contexts, expanding our understanding of its regulatory networks beyond the initially identified Bombesin Receptor Subtype-3 (BRS-3) . This approach could reveal novel mechanisms of BRAP function.

• Structure-Function Analysis: Using recombinant BRAP variants with specific domain mutations enables identification of critical regions required for its regulatory functions, particularly in TSLP production and inflammatory modulation . This information could guide future drug development targeting specific BRAP domains.

• Comparative Studies: Comparing the activities of recombinant mouse BRAP (encoded by BC004004) with human BRAP (encoded by C6orf89) provides insights into conserved functions and species-specific differences . Such comparisons are essential for translating findings from mouse models to human applications.

• Therapeutic Potential Assessment: Evaluating how recombinant BRAP administration affects inflammatory processes in various disease models could identify new therapeutic strategies for conditions involving dysregulated epithelial-immune cell communication .

These applications highlight how recombinant mouse BRAP serves as both an investigative tool and a potential therapeutic agent in research focused on inflammatory regulation .

What unexplored disease models might benefit from investigation of BRAP function?

Based on current understanding of BRAP function, several unexplored disease models may benefit from investigation:

• Autoimmune Disorders: Given BRAP's role in regulating inflammatory kinetics and cytokine production, autoimmune conditions beyond psoriasis—such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis—might be influenced by BRAP-dependent mechanisms .

• Fibrotic Diseases: BC004004-/- mice exhibit attenuation of injury-induced fibrosis in lungs and kidneys , suggesting that BRAP modulation might be relevant for conditions like idiopathic pulmonary fibrosis, liver cirrhosis, and renal fibrosis.

• Allergic Conditions: The relationship between BRAP and TSLP (a key mediator in allergic responses) suggests potential relevance in allergic conditions such as asthma, atopic dermatitis, and allergic rhinitis .

• Neurological Disorders: The observation that BC004004-/- mice show exacerbation of stress-induced behavioral changes points to potential applications in studying neuroinflammatory and neurodegenerative conditions .

• Epithelial Barrier Disorders: BRAP's abundant expression in keratinocytes and its influence on epithelial-immune crosstalk suggest relevance to conditions characterized by epithelial barrier dysfunction, such as inflammatory bowel diseases and chronic obstructive pulmonary disease .

Future research exploring BRAP function in these disease contexts could reveal new pathophysiological mechanisms and therapeutic targets across a spectrum of inflammatory and immune-mediated conditions .

How should researchers interpret apparently contradictory findings in BRAP functional studies?

When confronted with apparently contradictory findings in BRAP functional studies, researchers should consider several interpretative frameworks:

• Temporal Dynamics Analysis: The complex kinetics of inflammation in BC004004-/- mice demonstrate that BRAP deficiency causes altered inflammatory patterns rather than simply enhancing or suppressing inflammation . Researchers should examine time-course data rather than single time points to avoid misinterpreting transient effects as contradictory findings.

• Context-Dependent Function: BRAP appears to have different effects depending on the tissue context—attenuating fibrosis in lungs and kidneys while modifying inflammatory patterns in skin . These seemingly contradictory functions may reflect tissue-specific signaling environments.

• Dose-Dependent Effects: Chemical interaction data shows that different compounds can both increase or decrease C6orf89 homolog expression , suggesting that BRAP may function within optimal concentration ranges rather than following simple more/less paradigms.

• Methodological Differences: When evaluating seemingly contradictory results, researchers should carefully examine:

  • Complete vs. partial knockdown approaches

  • Global vs. conditional knockout models

  • Acute vs. chronic experimental timeframes

  • In vitro vs. in vivo experimental systems

• Pleiotropy Consideration: BRAP likely participates in multiple signaling pathways simultaneously. Enhanced activity in one pathway coupled with reduced activity in another could lead to apparently contradictory phenotypes that actually reflect BRAP's multifunctional nature .

By applying these interpretative approaches, researchers can reconcile seemingly contradictory findings and develop more nuanced models of BRAP function that account for its complex regulatory roles across different contexts .

What are the key considerations when comparing results between human BRAP and mouse homolog studies?

When comparing results between human BRAP (C6orf89) and mouse homolog (BC004004) studies, researchers should consider several critical factors:

• Sequence Homology Context: While the mouse homolog shares 83% similarity with human BRAP , differences in the remaining sequence may affect:

  • Protein-protein interaction capabilities

  • Post-translational modifications

  • Subcellular localization

  • Regulatory responses

• Expression Pattern Differences: Although both human BRAP and mouse homolog are abundantly expressed in keratinocytes , potential differences in expression levels across other tissues may lead to varying phenotypic effects between species.

• Experimental Model Translation:

  • Mouse models offer systemic knockout approaches (BC004004-/-) that aren't possible in human studies

  • Human studies typically rely on cell culture with siRNA knockdown

  • These methodological differences may account for apparently discrepant results

• Disease Context Variations: Psoriasis in humans and IMQ-induced psoriasis-like inflammation in mice share features but aren't identical, potentially affecting how BRAP function manifests in disease models .

• Data Integration Approaches: When comparing across species, researchers should:

  • Prioritize conserved phenotypes as likely representing core BRAP functions

  • View species-specific findings as potential areas for investigation rather than contradictions

  • Use orthologous approaches (like using both human HaCaT cells and mouse primary keratinocytes) to bridge species differences

By carefully considering these factors, researchers can more accurately translate findings between mouse models and human studies, avoiding misinterpretation of species-specific phenomena as contradictory results .