Recombinant Mouse Protein strawberry notch homolog 2 (Sbno2), partial

What is SBNO2 and what are its fundamental structural features?

SBNO2 belongs to the strawberry notch homolog protein family and functions as a transcriptional coregulator with dual coactivator and corepressor capabilities. In humans, the canonical SBNO2 protein consists of 1366 amino acid residues with a molecular mass of approximately 150.3 kDa, and up to two different isoforms have been reported . Mouse SBNO2 shares high homology with its human counterpart, reflecting evolutionary conservation of this protein across species. SBNO2 is particularly expressed in macrophages and contains multiple functional domains that enable its diverse regulatory activities . The protein is also known by synonyms including KIAA0963 and SNO, and orthologs have been identified in numerous species including mouse, rat, bovine, frog, chimpanzee, and chicken . This high degree of conservation across species underscores SBNO2's fundamental biological importance and makes mouse models particularly relevant for studying its functions with potential translational applications to human biology.

How is SBNO2 expression regulated in different cell types?

SBNO2 expression is tightly regulated in a cell type-specific manner, with notable expression patterns in macrophages and osteoclast precursors. Research has demonstrated that SBNO2 expression significantly increases following RANKL (Receptor Activator of Nuclear Factor κB Ligand) stimulation in monocyte-derived macrophages (MDMs) . This transcriptional induction depends on c-Fos and its downstream target Jdp2, but interestingly remains independent of NFATc1, as shown through experiments using the NFATc1 inhibitor FK506 . Additionally, SBNO2 has been identified as a direct target of BACH2 (BTB Domain and CNC Homolog 2), which functions as a transcriptional repressor. ChIP (Chromatin Immunoprecipitation) assays have revealed that BACH2 binds to specific regulatory elements of the SBNO2 gene, with stronger binding observed at certain regulatory sites . In gastric cancer, SBNO2 expression shows distinct patterns based on clinical parameters, with higher expression in both men and women with gastric cancer compared to healthy individuals, and elevated expression in patients older than 40 years . These findings indicate complex regulatory mechanisms controlling SBNO2 expression across different cellular contexts and disease states.

What are the primary functions of SBNO2 in mouse models?

Studies using mouse models have revealed several critical functions of SBNO2, particularly in bone biology and potentially in immune regulation. Most notably, SBNO2 plays an essential role in regulating osteoclast fusion. SBNO2-deficient mice exhibit osteopetrosis characterized by increased trabecular bone volume and number compared to wild-type mice . Histomorphometric analysis has demonstrated a significant reduction in the osteoclast surface/bone surface and eroded surface/bone surface ratios in SBNO2-deficient mice, although the osteoclast number/bone surface ratio remains normal . This indicates that osteoclast fusion, rather than differentiation, is specifically impaired in the absence of SBNO2. Additionally, the nuclei/osteoclast ratio is decreased in SBNO2-deficient mice, further confirming the fusion defect .

SBNO2-deficient mice also show lower serum levels of bone resorption markers, including TRAP 5b (TRACP5b) and serum type 1 collagen cross-linked C-terminal telopeptide (CTX), compared to wild-type mice . This confirms SBNO2's important role in normal bone homeostasis. Mechanistically, SBNO2 regulates the expression of DC-STAMP (Dendritic Cell-Specific Transmembrane Protein), which is essential for osteoclast fusion .

Interestingly, despite its expression in macrophages, SBNO2 deficiency does not significantly alter NF-κB activation or proinflammatory cytokine production in response to various TLR ligands in macrophages or dendritic cells . SBNO2-deficient mice showed no difference in survival or cytokine production during acute Staphylococcus aureus infection compared to wild-type mice, suggesting SBNO2 may be dispensable for certain aspects of inflammatory responses .

How does SBNO2 regulate osteoclast fusion at the molecular level?

SBNO2 plays a crucial role in osteoclast fusion through several molecular mechanisms. Primarily, SBNO2 regulates the expression of DC-STAMP (Dendritic Cell-Specific Transmembrane Protein), which is essential for the cell-cell fusion of osteoclast precursors . In SBNO2-deficient mice, the reduced expression of DC-STAMP impairs the ability of osteoclast precursors to fuse, resulting in osteoclasts with fewer nuclei . This is evidenced by microcomputed tomographic (μ-CT) analysis of femurs, which shows that SBNO2-deficient mice develop osteopetrosis with a marked increase in trabecular bone volume and number compared to wild-type mice .

The expression of SBNO2 itself is significantly increased after RANKL stimulation in monocyte-derived macrophages (MDMs) . This transcriptional induction depends on c-Fos and its downstream target Jdp2, but is independent of NFATc1 (as shown by NFATc1 inhibition through FK506 treatment) . This places SBNO2 within a specific branch of the RANKL signaling cascade that regulates osteoclast fusion rather than differentiation.

Histomorphometric analysis reveals that while the osteoclast number/bone surface ratio remains normal in SBNO2-deficient mice, there is a significant reduction in the osteoclast surface/bone surface and eroded surface/bone surface ratios . The nuclei/osteoclast ratio is also decreased in these mice . Importantly, ruffled border formation (essential for bone resorption) appears normal in SBNO2-deficient osteoclasts, indicating that SBNO2 specifically regulates the fusion process rather than other aspects of osteoclast function or differentiation .

What protein interactions are critical for SBNO2 function?

SBNO2 engages in several key protein interactions that mediate its diverse functions in different cellular contexts. Recent research has revealed that SBNO2's intrinsically disordered region (IDR) directly interacts with TBK1 (TANK-binding kinase 1) and DNMT1 (DNA methyltransferase 1) . These interactions are enhanced in the presence of heme, suggesting a potential regulatory mechanism involving cellular heme levels . Interestingly, even smaller fragments of SBNO2 (spanning amino acids 381-481) that lack CP motifs (potential heme-binding sites) can still bind to TBK1 in a heme-dependent manner, indicating multiple interaction interfaces within the protein .

TBK1 and SBNO2 appear to form a double-negative feedback loop in which they repress each other's activity . Research has shown that BACH2 (which represses SBNO2) binds to the Tbk1 gene, and Tbk1 mRNA induction upon LPS stimulation is higher in Bach2-deficient B cells than in wild-type B cells . The TBK1 protein is also expressed at higher levels in Bach2-deficient splenic B cells . Furthermore, TBK1 can inhibit the repressor activity of BACH2, creating a regulatory circuit that influences the expression of targets like Slc48a1 (which encodes the heme transporter HRG1) .

Additional protein interaction studies have identified SBNO2 as potentially interacting with several other proteins, including CPSF1 (Cleavage and polyadenylation specificity factor subunit 1), CNOT1 (CCR4-NOT transcription complex subunit 1), MAP1S (Microtubule-associated protein 1S), ARID1A (AT-rich interactive domain-containing protein 1A), IDH3B (Isocitrate dehydrogenase 3 beta), and GTF2I (General transcription factor II-I) . These diverse interaction partners suggest broad regulatory roles for SBNO2 across multiple cellular processes.

What is the relationship between SBNO2 and cancer development?

SBNO2 has emerging roles in cancer biology, particularly in gastric cancer where it has been identified as a hub gene. Using both differentially expressed gene (DEG) analysis and weighted gene coexpression network analysis (WGCNA), researchers have identified SBNO2 as significantly upregulated in gastric cancer tissues compared to normal tissues . Analysis through multiple databases including TIMER, GEPIA2, UALCAN, and Human Cancer Metastasis Database has consistently confirmed the higher expression of SBNO2 in gastric cancer .

SBNO2 upregulation is not limited to gastric cancer. Analysis through the TIMER database has shown that SBNO2 expression is significantly elevated in several other cancer types, including bladder urothelial carcinoma, cholangiocarcinoma, esophageal carcinoma, and head and neck squamous cell carcinoma . This broad pattern of upregulation across multiple cancer types suggests a potentially conserved role in tumorigenesis.

Subtype analysis has revealed that SBNO2 expression in gastric cancer varies based on clinical parameters. Its expression is higher in both men and women with gastric cancer compared to healthy individuals and is particularly elevated in patients older than 40 years . SBNO2 expression is increased across all cancer stages (1-4) compared to normal tissues, with the highest levels observed in stage 2 lesions . Similarly, expression is elevated in grade 1-3 gastric cancer compared to normal tissues .

What experimental techniques are most effective for studying SBNO2?

Several experimental techniques have proven particularly effective for studying different aspects of SBNO2 biology. For protein detection and localization, immunofluorescence stands as the most common application for SBNO2 antibodies . Other immunodetection methods including ELISA, immunocytochemistry, and immunohistochemistry are also commonly employed . These techniques allow researchers to examine SBNO2 expression patterns across different tissues and cell types, as well as its subcellular localization.

For investigating SBNO2's role in bone biology, microcomputed tomographic (μ-CT) analysis has been instrumental in characterizing the osteopetrotic phenotype in SBNO2-deficient mice . This technique allows precise measurement of bone parameters such as trabecular bone volume and number. Complementary to μ-CT, histological examination of bone sections and histomorphometric analysis provide detailed insights into osteoclast parameters, including osteoclast surface/bone surface ratio and nuclei/osteoclast ratio .

To study SBNO2's transcriptional regulatory functions, chromatin immunoprecipitation (ChIP) assays have been employed to identify SBNO2 binding sites on DNA . Electrophoretic mobility shift assay (EMSA) has also been used to confirm direct binding of SBNO2 to specific DNA elements . For gene expression analysis, quantitative PCR (qPCR) has been valuable in measuring SBNO2 mRNA levels and the expression of potential target genes in different experimental conditions .

Protein-protein interaction studies have utilized various approaches including GST pull-down assays with recombinant proteins. For example, GST-SBNO2-IDR has been successfully used in pull-down experiments with FLAG-His-TBK1 and FLAG-His-DNMT1, demonstrating direct interactions that are enhanced in the presence of heme . Co-immunoprecipitation has also been employed to confirm these interactions using cell extracts .

For functional studies, the generation of SBNO2-deficient mice has been crucial in revealing the protein's role in osteoclast fusion and bone homeostasis . Analysis of serum bone resorption markers (TRAP 5b and CTX) in these mice has provided further insights into SBNO2's physiological functions .

How should SBNO2 knockout models be designed and validated?

Designing and validating SBNO2 knockout models requires careful consideration of several methodological aspects. For complete knockout models, the CRISPR/Cas9 system offers the most efficient approach. When designing guide RNAs, targeting early exons of the SBNO2 gene is recommended to ensure complete loss of function. Based on studies with existing SBNO2-deficient mice, targeting regions critical for DC-STAMP regulation would be particularly relevant for investigating osteoclast fusion defects .

Validation of SBNO2 knockout models should employ multiple complementary approaches. At the molecular level, confirmation of successful gene deletion should include genomic PCR to verify the targeted mutation, RT-PCR and qPCR to demonstrate absence of SBNO2 mRNA, and Western blotting to confirm the lack of SBNO2 protein . Particularly for conditional or tissue-specific knockouts, verification of gene deletion in the relevant tissue or cell type is essential.

Phenotypic validation should focus on the established roles of SBNO2. Since SBNO2-deficient mice develop osteopetrosis, μ-CT analysis of bone parameters is a critical validation step . Key measurements should include trabecular bone volume and number, which are significantly increased in SBNO2-deficient mice . Histological sections of proximal tibias should be examined for increased trabecular bone volume and number . Histomorphometric analysis should assess osteoclast surface/bone surface ratio, eroded surface/bone surface ratio, and nuclei/osteoclast ratio, all of which are affected in SBNO2 knockout mice .

Functional validation should include measurement of serum bone resorption markers, particularly TRAP 5b (TRACP5b) and serum type 1 collagen cross-linked C-terminal telopeptide (CTX), which are lower in SBNO2-deficient mice than in wild-type mice . In vitro osteoclast differentiation assays using bone marrow cells from knockout mice can confirm fusion defects while demonstrating normal differentiation capacity.

For cell-specific studies, bone marrow-derived macrophages (BMDMs) from SBNO2 knockout mice provide a valuable model system. These cells can be stimulated with RANKL to study osteoclast differentiation and fusion, with quantification of multinucleated TRAP-positive cells and nuclei per osteoclast to confirm the fusion defect .

What are the critical factors in purifying recombinant SBNO2 protein?

Purification of recombinant mouse SBNO2 presents several challenges that researchers must address to obtain functional protein for in vitro studies. Given that full-length SBNO2 is a large protein (~150 kDa) with multiple domains, a domain-based approach may be more practical than attempting to express the complete protein . The intrinsically disordered region (IDR) has been successfully expressed and used in interaction studies, making it a good starting point .

For expression systems, insect cells (using baculovirus) often provide a good compromise between proper folding and reasonable yield for large mammalian proteins like SBNO2. This system allows for some post-translational modifications while typically yielding more protein than mammalian expression systems. For smaller domains or fragments (such as the 381-481 amino acid region that retains TBK1 binding capacity), bacterial expression in E. coli may be sufficient .

Fusion tags can significantly improve both expression and purification efficiency. GST-tagged SBNO2-IDR has been successfully used in pull-down assays, indicating that GST fusion is compatible with proper folding of at least this domain . For full-length protein or larger fragments, solubility-enhancing tags like MBP (maltose-binding protein) or SUMO may be beneficial. His-tagging can facilitate purification through nickel affinity chromatography, as demonstrated with FLAG-His-TBK1 and FLAG-His-DNMT1 proteins used in interaction studies with SBNO2 .

An important consideration specific to SBNO2 is the role of heme in regulating its interactions. Research has shown that interactions between SBNO2's IDR and proteins like TBK1 and DNMT1 are enhanced in the presence of heme . Therefore, for functional studies, it may be necessary to incorporate heme during purification or to reconstitute purified protein with heme before use. This would be particularly important for interaction studies or assays designed to measure SBNO2's regulatory activities.

For quality control, purified SBNO2 should be validated through multiple approaches. SDS-PAGE and Western blotting can confirm size and immunoreactivity, while mass spectrometry can provide accurate mass determination. Functional validation should include binding assays with known interaction partners such as TBK1 or DNMT1, preferably in both the presence and absence of heme to confirm heme-dependent regulation .

How can researchers evaluate SBNO2's role in transcriptional regulation?

Evaluating SBNO2's role in transcriptional regulation requires a multi-faceted approach that combines molecular, cellular, and genomic techniques. As a transcriptional coregulator with both coactivator and corepressor functions, SBNO2's effects must be studied in the context of specific target genes and regulatory partners .

Chromatin immunoprecipitation (ChIP) assays represent a fundamental approach for identifying SBNO2 binding sites on DNA. This technique has been successfully applied to detect BACH2 binding to the Tbk1 gene, and similar approaches could identify direct SBNO2 targets . For genome-wide binding profiles, ChIP-sequencing (ChIP-seq) would provide comprehensive mapping of SBNO2 occupancy across the genome. This should be combined with transcriptomic analyses (RNA-seq) comparing wild-type and SBNO2-deficient cells to correlate binding with gene expression changes.

For studying SBNO2's impact on specific genes, quantitative PCR (qPCR) has proven effective. For example, qPCR has been used to demonstrate that Tbk1 mRNA induction upon LPS stimulation is higher in Bach2-deficient B cells than in wild-type cells . Similar approaches can examine how SBNO2 deficiency affects the expression of potential target genes. DC-STAMP represents a particularly relevant target for investigation given SBNO2's role in regulating its expression in the context of osteoclast fusion .

Reporter gene assays provide another valuable tool for studying SBNO2's transcriptional regulatory function. Luciferase reporters containing promoter regions of putative SBNO2 target genes can be constructed and transfected into cells with or without SBNO2 expression. For example, the Slc48a1 reporter carrying the Slc48a1 MARE sequence has been used to demonstrate the interplay between BACH2, MAFK, and TBK1 in transcriptional regulation . Similar approaches could elucidate how SBNO2 regulates its target genes.

Protein-protein interaction studies should complement transcriptional analyses, as SBNO2's coregulator function likely involves interactions with various transcription factors and chromatin modifiers. Pull-down assays, co-immunoprecipitation, and mass spectrometry-based interactome analyses can identify the key interaction partners involved in different regulatory contexts .

What techniques are most effective for investigating SBNO2 in cancer models?

Investigating SBNO2 in cancer models requires a combination of bioinformatic, molecular, cellular, and in vivo approaches. Given SBNO2's identification as a hub gene in gastric cancer and its association with poor prognosis, effective techniques for studying its role in cancer are particularly important .

Bioinformatic analyses have proven valuable for establishing SBNO2's relevance in cancer. The combination of differentially expressed gene (DEG) analysis and weighted gene coexpression network analysis (WGCNA) successfully identified SBNO2 as a hub gene in gastric cancer . These approaches can be applied to other cancer types to determine if SBNO2 plays similar roles. Database mining using resources like TIMER, GEPIA2, UALCAN, Human Cancer Metastasis Database, and Kaplan-Meier plotter has validated SBNO2's expression patterns and prognostic significance in gastric cancer . These resources can be leveraged for preliminary analysis of SBNO2 in other cancer types.

For molecular and cellular studies, modulating SBNO2 expression in cancer cell lines provides critical insights. CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown in relevant cancer cell lines (such as gastric cancer lines for studies related to findings in that cancer type) can reveal how SBNO2 influences cancer cell proliferation, migration, invasion, and response to therapies. Conversely, overexpression studies can determine if increased SBNO2 promotes oncogenic phenotypes.

Immunohistochemical analysis of SBNO2 expression in tumor tissue microarrays allows correlation of expression levels with clinical parameters and outcomes. This approach has shown that SBNO2 expression varies based on gender, age, cancer stage, and tumor grade in gastric cancer patients . Similar analyses can be conducted for other cancer types to establish clinical relevance.

Given SBNO2's suggested role in immune cell infiltration in gastric cancer, techniques for studying the tumor immune microenvironment are particularly relevant . Flow cytometry and immunohistochemistry can assess immune cell composition in relation to SBNO2 expression. Single-cell RNA sequencing of tumor samples can provide detailed insights into how SBNO2 expression in cancer cells correlates with immune cell populations and their activation states.

What are the emerging areas of SBNO2 research in immunology?

While SBNO2 is notably expressed in macrophages, its role in immune function appears more nuanced than initially expected. Research has shown that SBNO2 deficiency does not alter NF-κB activation or proinflammatory cytokine production in response to various TLR ligands in monocyte-derived macrophages . Additionally, SBNO2-deficient mice challenged with Staphylococcus aureus acute infection exhibit normal survival and cytokine production . These findings suggest that SBNO2 may be dispensable for certain canonical inflammatory responses.

The interaction between SBNO2's intrinsically disordered region (IDR) and TBK1 (TANK-binding kinase 1) is another area worth exploring . Given TBK1's established role in innate immune signaling, this interaction suggests potential involvement of SBNO2 in specific immune pathways. The finding that this interaction is enhanced by heme adds another layer of complexity, suggesting possible integration of heme sensing with immune regulation .

The relationship between SBNO2 and BACH2 also merits further investigation in immune contexts. BACH2 is a known regulator of B cell differentiation and T cell function, and the finding that it represses SBNO2 expression suggests potential roles for SBNO2 in adaptive immunity . The double-negative feedback loop between TBK1 and BACH2, in which TBK1 inhibits BACH2's repressor activity while BACH2 represses Tbk1 expression, creates a regulatory circuit that may influence immune cell differentiation and function .

Future research should explore SBNO2's role in specialized immune cell functions, particularly those not addressed by the NF-κB and cytokine production assays in previous studies. This includes potential impacts on antigen presentation, pattern recognition receptor signaling beyond TLRs, lymphocyte development, and immune memory formation. Given SBNO2's established role in osteoclast fusion, its potential involvement in other cell fusion events relevant to immunity (such as multinucleated giant cell formation in granulomatous diseases) also warrants investigation.

What therapeutic potential does targeting SBNO2 hold for bone disorders?

The clear phenotype of osteopetrosis in SBNO2-deficient mice suggests significant therapeutic potential for targeting SBNO2 in bone disorders . By regulating osteoclast fusion through DC-STAMP expression, SBNO2 represents a specific intervention point that could be exploited to modulate bone resorption without completely blocking osteoclast differentiation or function.

For conditions characterized by excessive bone resorption, such as osteoporosis, Paget's disease, and bone metastases, inhibiting SBNO2 activity could potentially reduce osteoclast fusion and thereby decrease bone resorption. Unlike bisphosphonates or RANKL inhibitors that broadly target osteoclast survival or formation, SBNO2 inhibition would specifically impact fusion, potentially allowing for more precise modulation of osteoclast activity. This approach might preserve the single-nucleated osteoclasts necessary for bone remodeling while preventing the formation of highly resorptive multinucleated osteoclasts.

The development of SBNO2 inhibitors would require detailed structural understanding of the protein, particularly its interaction interfaces with DC-STAMP regulatory elements and protein partners involved in transcriptional control. Small molecule inhibitors targeting these interactions or siRNA-based approaches to reduce SBNO2 expression could be explored as therapeutic strategies.

For genetic bone disorders characterized by osteopetrosis, understanding the role of SBNO2 provides new insights into disease mechanisms and potential interventions. While activating SBNO2 to increase bone resorption would be the theoretical approach for treating these conditions, practical therapeutic development would face significant challenges.

A particularly promising direction is the exploration of SBNO2's relationship with RANKL signaling. Since SBNO2 expression is induced by RANKL through c-Fos and Jdp2 , this pathway could offer additional intervention points. Compounds that modulate this specific branch of RANKL signaling might achieve more targeted effects than current broad RANKL inhibitors.

The development of bone-targeted delivery systems would be crucial for any SBNO2-directed therapy to maximize efficacy while minimizing potential off-target effects in other tissues where SBNO2 is expressed. Nanoparticle formulations with bone-seeking properties or osteoclast-targeted delivery vehicles could enable localized modulation of SBNO2 activity.

How might SBNO2 be utilized as a prognostic or therapeutic target in cancer?

To develop SBNO2 as a prognostic tool, standardized assays for measuring its expression in tumor samples would be needed. Immunohistochemistry protocols could be optimized for use in clinical pathology laboratories, and qPCR-based tests could be developed for quantitative assessment. The finding that SBNO2 expression varies based on cancer stage and grade in gastric cancer suggests that these parameters should be considered when establishing reference ranges and cutoff values for prognostic applications .

Several approaches could be explored for targeting SBNO2 in cancer:

• Transcriptional inhibition: Developing small molecules that disrupt SBNO2's interactions with transcriptional machinery or target genes.

• RNA interference: siRNA or antisense oligonucleotides designed to reduce SBNO2 expression.

• Protein-protein interaction inhibitors: Compounds that block SBNO2's interactions with specific partners such as TBK1 or DNMT1, which have been shown to interact with SBNO2 in a heme-dependent manner .

• Exploiting heme sensitivity: Since SBNO2 interactions are modulated by heme, strategies that alter heme availability or binding could potentially modulate SBNO2 function .

The suggestion that SBNO2 plays a role in immune cell infiltration in gastric cancer adds another dimension to its therapeutic potential . Targeting SBNO2 might not only directly affect cancer cell behavior but also modulate the tumor immune microenvironment. This dual mechanism could enhance the efficacy of immunotherapies, potentially converting "cold" tumors (with low immune infiltration) to "hot" tumors that respond better to immune checkpoint inhibitors.