Recombinant Rat Bombesin receptor-activated protein C6orf89 homolog

What is the Bombesin Receptor-Activated Protein (BRAP) and how is it identified in different species?

BRAP, also known as C6orf89, was initially discovered as a potential interacting partner of the Bombesin Receptor Subtype-3 (BRS-3) through bacteria two-hybrid screening methods . In humans, this protein is encoded by the C6orf89 gene, while in mice, the homolog is encoded by the BC004004 gene . The rat BRAP shares significant structural similarities with its human and mouse counterparts. BRAP is classified as a type II membrane protein with a putative N-terminal transmembrane domain, featuring a luminal C-domain and a cytoplasmic N-domain . Sequence analysis using tools such as Swiss Model and AlphaFold structure prediction confirms this transmembrane architecture across species .

What are the primary biological functions of BRAP?

BRAP demonstrates several important biological functions that have been characterized in research settings. It exhibits histone deacetylase (HDAC) enhancer properties according to studies cited in PubMed:23460338 . Additionally, BRAP appears to play significant roles in cell cycle progression and wound repair processes, particularly in bronchial epithelial cells (PubMed:21857995) . Recent research has also revealed its involvement in inflammatory responses, especially in skin conditions. BRAP is abundantly expressed in keratinocytes, and its deficiency alters inflammatory processes in experimental models of psoriasis-like skin conditions . This suggests BRAP's importance in mediating crosstalk between epidermal cells and immune cells, potentially through cytokine regulation pathways .

What are the optimal conditions for expressing and purifying Recombinant Rat BRAP?

When working with Recombinant Rat BRAP, researchers should consider using expression systems that accommodate the protein's transmembrane nature. Based on the amino acid sequence (MDLAANEISIYDKLSETVDLVRQTGHQCGMSEKAIEKFIRQLLEKNEPQRGPPQYPLLIAVYKVLLTLGLILFTAYFVIQPFSSLAPEPVLSGANSWRSLVHHIRLVSLPITKKYMPENKGVPLQGSQEDKPFPDFDPWSSYNCEQNESEPIPANCTGCAQILPLKVTLPEDTPKNFERLRPLVIKTGQPLSSAEIQSFSCQYPEVTEGFTEGFFTKWWRCFPERWFPFPYPWRRPLNRSQILRELFPVFTQLPFPKDSSLNKCFLIQPEPVVGSKMHKVHDLFTLGSGEAMLQLIPPFQCRTHCQSVAMPIESGDIGYADAAHWKVYIVARGVQPLVICDGTTLSDL) , detergent-based extraction methods are recommended to solubilize the membrane-associated domains. For purification, affinity chromatography approaches using histidine or GST tags offer good yields while maintaining protein functionality. Western blot verification can be performed using validated antibodies such as the rabbit recombinant monoclonal antibody ab181073, which has been successfully used with human samples at 1/1000 dilution .

How can researchers effectively detect and measure BRAP expression in tissue samples?

For detecting BRAP expression in tissue samples, researchers can employ multiple complementary approaches. Western blotting using validated antibodies such as ab181073 has been shown to effectively detect the protein at the predicted band size of 40 kDa . This antibody has been successfully tested on human cell lines including A549, 293, and HepG2 at 20 μg lysate concentration . Immunohistochemistry techniques can localize BRAP expression within tissue sections, which is particularly valuable when studying its distribution in skin samples or other tissues of interest. For quantitative assessment, qPCR assays targeting the rat BRAP gene can measure mRNA expression levels. When designing such experiments, inclusion of appropriate controls is essential, such as using BRAP knockout tissues as negative controls and tissues known to highly express BRAP (such as keratinocytes) as positive controls .

What experimental models are suitable for studying BRAP function in inflammatory conditions?

Based on recent research, several experimental models have proven effective for studying BRAP function in inflammatory conditions. The imiquimod (IMQ)-induced psoriasis-like skin inflammation model in mice has been particularly informative . In this approach, knockout mice (BC004004-/-) and wild-type controls are topically treated with IMQ daily for 7 days, followed by assessment of skin lesion development, histopathological changes, and inflammatory marker profiles . This model revealed that BRAP-deficient mice exhibit an altered pattern of inflammation with earlier onset and quicker remission of skin lesions .

For in vitro studies, siRNA silencing of BRAP in human keratinocyte-derived HaCaT cells has been successful in demonstrating the protein's role in cytokine release, particularly thymic stromal lymphopoietin (TSLP) . This approach allows researchers to investigate the molecular mechanisms through which BRAP influences inflammatory responses in a controlled environment.

How does BRAP deficiency alter cytokine expression patterns in inflammatory skin conditions?

BRAP deficiency introduces significant changes to cytokine expression patterns in inflammatory skin conditions, as demonstrated in the IMQ-induced psoriasis-like model. BRAP knockout mice (BC004004-/-) exhibited distinct cytokine profiles compared to wild-type controls when treated with IMQ . By day 4 of treatment, the knockout mice displayed elevated expression levels of IL-17A, suggesting a more robust activation of Th17 cells, which are key players in psoriasis pathogenesis .

Additionally, the serum levels of thymic stromal lymphopoietin (TSLP), a cytokine derived from keratinocytes, showed a distinctive pattern in BRAP-deficient conditions. TSLP levels increased in BC004004+/+ mice and reached peak concentration on day 4 of IMQ treatment . This finding was further supported by in vitro experiments where knockdown of BRAP in HaCaT keratinocyte cells led to increased TSLP release . These observations suggest that BRAP plays a regulatory role in cytokine production, particularly in the crosstalk between epidermal keratinocytes and immune cells during inflammatory responses.

What potential roles might BRAP play in cancer research given its connection to bombesin receptors?

While the direct functional relationship between BRAP and bombesin receptors remains under investigation, several avenues for cancer research emerge from their association. Bombesin receptors (BBRs) are overexpressed in numerous solid tumors and serve as autocrine growth factors when activated . Studies have shown that BRAP exhibits histone deacetylase (HDAC) enhancer properties and may influence cell cycle progression , suggesting potential epigenetic regulatory mechanisms relevant to cancer development.

Researchers investigating BRAP in cancer contexts should consider examining its expression in tumor tissues that overexpress bombesin receptors, such as prostate, breast, small cell lung, and gastrointestinal cancers . Analysis of BRAP expression levels in correlation with cancer progression and patient outcomes could reveal whether this protein functions as a tumor suppressor or promoter. Additionally, exploring how BRAP influences cellular responses to bombesin receptor antagonists, which are being developed as potential therapeutics , could yield insights into resistance mechanisms or enhanced efficacy.

How can chimeric receptor studies inform our understanding of species-specific differences in bombesin receptor pharmacology?

Chimeric receptor studies have provided valuable insights into species-specific differences in bombesin receptor pharmacology, particularly between rat and human BRS-3. Despite sharing 80% sequence identity, rat and human BRS-3 exhibit markedly different pharmacological properties . Research using chimeric receptors, where individual extracellular loops of rat BRS-3 were replaced with corresponding human sequences, revealed that the third extracellular loop (E3) is primarily responsible for these species differences .

This methodological approach can be applied to investigate whether BRAP interactions with bombesin receptors also display species specificity. Researchers could design experiments using chimeric constructs of rat and human BRAP to identify regions critical for potential receptor interactions or downstream signaling pathways. Such studies might explain why findings in rat models sometimes fail to translate to human systems, and could guide the development of more translatable research models for studying BRAP function.

What are common challenges in detecting BRAP protein expression and how can they be addressed?

Detecting BRAP protein expression can present several challenges for researchers. As a type II membrane protein with transmembrane domains, BRAP may require specialized extraction protocols to ensure complete solubilization from cellular membranes . Standard lysis buffers may not efficiently extract membrane-associated proteins, leading to false negative results.

To address this issue, researchers should employ detergent-based extraction methods using buffers containing appropriate non-ionic detergents such as Triton X-100 or NP-40. For Western blot applications, transfer conditions should be optimized for membrane proteins, potentially using longer transfer times or specialized transfer buffers containing SDS.

Another common challenge is antibody specificity. When using antibodies like ab181073, researchers should verify their specificity through positive controls (cell lines known to express BRAP) and negative controls (BRAP knockout samples or cells with siRNA knockdown) . If background issues occur, optimization of blocking conditions (using alternative blocking agents like 5% BSA instead of milk) and stringent washing steps can improve signal-to-noise ratio.

How can researchers address variability in BRAP knockout phenotypes in animal models?

Variability in BRAP knockout phenotypes has been observed in animal models, particularly in inflammatory response patterns . To address this challenge, researchers should implement several methodological approaches:

• Ensure genetic background consistency by using isogenic controls and sufficient backcrossing generations when working with knockout mice.

• Increase sample sizes to account for biological variability, conducting power calculations to determine appropriate numbers.

• Standardize experimental conditions including animal housing, handling, and treatment protocols to minimize environmental variables.

• Implement time-course studies rather than single timepoint analyses, as BRAP knockout mice have shown temporal differences in inflammatory responses compared to wild-type mice .

• Include comprehensive phenotyping beyond the primary outcome measures, as BRAP may influence multiple physiological systems simultaneously.

• Consider using conditional knockout models (tissue-specific or inducible) to distinguish developmental effects from acute functional roles of BRAP.

By addressing these factors systematically, researchers can reduce variability and generate more consistent and interpretable data from BRAP knockout models.

What are promising research avenues for elucidating BRAP's role in cellular signaling networks?

Several promising research avenues exist for further elucidating BRAP's role in cellular signaling networks. Given its histone deacetylase enhancer properties , investigating BRAP's influence on epigenetic regulation through chromatin immunoprecipitation sequencing (ChIP-seq) would identify affected gene regulatory regions. Phosphoproteomics analysis of cells with modulated BRAP expression could reveal downstream signaling pathways influenced by this protein.

The observed role of BRAP in keratinocyte-immune cell crosstalk warrants exploration of its involvement in other epithelial-immune interactions, such as in lung and intestinal tissues where epithelial barrier function is crucial. Co-immunoprecipitation studies coupled with mass spectrometry could identify novel BRAP interacting partners beyond bombesin receptors, potentially uncovering unexpected signaling connections.

Single-cell transcriptomics of tissues from BRAP knockout models during inflammatory challenges would provide high-resolution mapping of cellular responses, potentially identifying cell populations most affected by BRAP deficiency. Lastly, CRISPR-Cas9 screens targeting BRAP regulatory elements could identify factors controlling its expression in different physiological and pathological contexts.

How might translational research leverage BRAP as a potential therapeutic target?

Translational research could leverage BRAP as a potential therapeutic target in several disease contexts based on its emerging biological functions. Given BRAP's role in inflammatory skin conditions, developing targeted modulators of BRAP activity might offer novel approaches for treating psoriasis and other inflammatory dermatoses . Small molecule inhibitors or activators of BRAP could be designed to influence its histone deacetylase enhancer activity , potentially affecting epigenetic regulation in disease states.

For cancer applications, analyzing BRAP expression patterns in tumors that overexpress bombesin receptors could reveal correlations with disease progression or treatment response . If BRAP influences cellular responses to bombesin receptor antagonists, combination therapies targeting both pathways might enhance anti-tumor efficacy. Additionally, since BRAP affects TSLP release from keratinocytes , therapies modulating this interaction could impact allergic and inflammatory conditions where TSLP plays a central role.

Development of biomarkers based on BRAP expression or activity could aid in patient stratification for personalized treatment approaches. Importantly, any therapeutic development should consider potential differences between rat and human BRAP function, using humanized models when appropriate to ensure translational relevance.