NUCB2 Human, His

What is NUCB2 and how does it relate to nesfatin-1?

NUCB2 is a protein encoded by the NUCB2 gene in humans, acting as a calcium-binding EF-hand protein . It functions as a precursor protein that undergoes cleavage by prohormone convertase, resulting in the formation of three peptide fragments: nesfatin-1, nesfatin-2, and nesfatin-3 . Of these fragments, nesfatin-1 has been extensively characterized as an anorexigenic factor first identified in 2006, while the roles of nesfatin-2 and nesfatin-3 remain largely undiscovered .

Methodologically, researchers must carefully distinguish between targeting the full-length NUCB2 protein versus its processed fragments in experimental designs. When studying the protein's role in appetite regulation, energy homeostasis, or cancer progression, this distinction becomes crucial for accurate interpretation of results.

What experimental approaches should be used to study NUCB2 expression patterns in human tissues?

To effectively study NUCB2 expression in human tissues, researchers should implement a multi-modal approach:

• Quantitative PCR (qPCR): For measuring NUCB2 mRNA levels across various tissues

• Western blotting: To quantify protein expression with specific antibodies against NUCB2

• Immunohistochemistry: For spatial localization within tissue sections

• RNA sequencing: To examine transcriptional regulation and alternative splicing

When comparing expression across experimental conditions, researchers should note that NUCB2 is broadly distributed in both the central nervous system and peripheral tissues, including adipose tissue, pancreas, cardiomyocytes, and reproductive systems . Expression levels vary significantly between tissue types, with particularly high expression observed in specific cancer types, including glioblastoma .

What are optimal strategies for expressing recombinant His-tagged NUCB2?

For efficient expression of His-tagged NUCB2, researchers should optimize several parameters:

• Expression system selection:

  • E. coli BL21(DE3) for high yield but potential refolding requirements

  • Mammalian cell lines (HEK293 or CHO) for proper post-translational modifications

  • Insect cell systems (Sf9, Hi5) for intermediate yield with eukaryotic processing

• Tag position considerations:

  • N-terminal His-tag may interfere with signal peptide processing

  • C-terminal His-tag may affect calcium-binding properties of EF-hand domains

  • Internal His-tag placement requires careful domain structure analysis

• Expression conditions:

  • Lower temperature induction (16-20°C) to improve folding

  • Reduced IPTG concentration (0.1-0.5 mM) for bacterial systems

  • Supplementation with calcium (1-2 mM) in culture media to stabilize EF-hand domains

Since NUCB2 contains calcium-binding domains, researchers must carefully consider buffer compositions throughout the expression and purification process to maintain structural integrity.

What are the critical quality control measures for purified His-tagged NUCB2?

Following purification of His-tagged NUCB2, researchers should implement rigorous quality control protocols:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (target >95% purity)

  • Size exclusion chromatography to confirm monodispersity

  • Mass spectrometry to verify molecular weight and detect modifications

• Structural validation:

  • Circular dichroism spectroscopy to assess secondary structure

  • Thermal shift assays to evaluate protein stability

  • Dynamic light scattering to detect aggregation

• Functional verification:

  • Calcium-binding assays using isothermal titration calorimetry

  • Interaction studies with known binding partners (e.g., NDN)

  • Activity assays relevant to downstream applications

These quality control measures ensure that the recombinant His-tagged NUCB2 maintains native-like properties for subsequent experimental applications.

How does NUCB2 expression correlate with glioblastoma progression and prognosis?

NUCB2 expression shows significant correlation with glioblastoma (GBM) progression and patient outcomes. Analysis of clinical data reveals:

This data demonstrates that high-grade GBM (WHO Grade III/IV) is significantly associated with high NUCB2 expression compared to lower-grade tumors . Furthermore, multivariate analysis confirms NUCB2 expression as an independent prognostic factor in GBM:

High NUCB2 expression is associated with poor prognosis in patients with GBM . When conducting clinical studies on NUCB2 in GBM, researchers should stratify patients based on NUCB2 expression levels and correlate with standard clinicopathological features and treatment outcomes.

What molecular mechanisms underlie NUCB2's effect on tumor cell migration and invasion?

NUCB2 promotes tumor cell migration and invasion through several interrelated molecular mechanisms:

• Epithelial-Mesenchymal Transition (EMT) regulation:

  • Knockdown of NUCB2 leads to increased E-cadherin levels (epithelial marker)

  • NUCB2 knockdown results in decreased N-cadherin levels (mesenchymal marker)

  • These changes indicate NUCB2's role in promoting EMT, a critical process for cancer cell invasion

• Angiogenesis and proliferation pathways:

  • NUCB2 knockdown decreases VEGF expression, suggesting a role in promoting angiogenesis

  • Reduced cyclin D1 levels following NUCB2 knockdown indicates its involvement in cell cycle regulation

• Functional impact on cellular behavior:

  • Wound healing assays demonstrate decreased migration ability following NUCB2 knockdown

  • Matrigel invasion assays show substantial reduction in invasive potential with NUCB2 suppression

These findings were verified through both siRNA (#1 and #2) approaches in multiple GBM cell lines (GBM8401 and U87-MG), confirming the consistency of NUCB2's role in promoting aggressive phenotypes in GBM .

How does NUCB2 contribute to chemotherapy and radiotherapy resistance in glioblastoma?

NUCB2 confers resistance to both chemotherapy and radiotherapy in GBM through distinct molecular mechanisms:

• Chemotherapy resistance (Temozolomide/TMZ):

  • NUCB2 plasmid-transfected GBM cells exhibit significantly higher cell viability than control groups after TMZ treatment

  • Western blot analysis reveals decreased levels of PARP and cleaved caspase-3 in NUCB2-overexpressing cells, indicating reduced apoptosis

  • This suggests NUCB2 overexpression protects GBM cells from TMZ-induced cell death

• Radiotherapy resistance:

  • Colony formation assays demonstrate that NUCB2-overexpressing cells form more colonies after radiation treatment compared to control cells

  • NUCB2 overexpression increases expression of DNA repair proteins (Ku70, Ku80, Rad51, and Rad52)

  • Enhanced DNA repair capacity enables GBM cells to survive radiation-induced DNA damage

These findings indicate that targeting NUCB2 could potentially sensitize GBM cells to standard therapies. Researchers investigating this approach should employ combination treatment protocols testing NUCB2 inhibition alongside standard chemotherapy and radiotherapy regimens.

How does NUCB2 knockout affect hepatic glucose metabolism in animal models?

NUCB2 knockout significantly impacts hepatic glucose metabolism, as demonstrated in NUCB2/nesfatin-1 global knockout (NUCB2-KO) rat models:

• Experimental approach:

  • Pancreatic-euglycemic clamping (PEC) procedure on wild-type and NUCB2-KO rats

  • Duodenal infusion of either glucose or saline

  • Monitoring of blood glucose, insulin, free fatty acid (FFA), and triglyceride (TG) levels

• Key findings:

  • Wild-type rats showed increased intravenous glucose infusion rate (GIR) and decreased hepatic glucose production (HGP) with glucose infusion compared to saline infusion

  • NUCB2-KO rats demonstrated impaired response to intestinal glucose sensing

  • This suggests that NUCB2/nesfatin-1 is essential for proper regulation of hepatic glucose production in response to intestinal glucose signals

These findings establish NUCB2's role in the gut-brain-liver axis, regulating hepatic glucose production through intestinal nutrient sensing mechanisms .

What experimental approaches are most effective for studying NUCB2's role in insulin sensitivity?

To comprehensively investigate NUCB2's role in insulin sensitivity, researchers should employ a multi-level experimental approach:

• In vivo metabolic phenotyping:

  • Hyperinsulinemic-euglycemic clamp (gold standard for insulin sensitivity assessment)

  • Pancreatic-euglycemic clamping with tissue-specific infusions

  • Glucose tolerance tests (GTT) and insulin tolerance tests (ITT)

  • Measurement of relevant metabolic parameters (glucose, insulin, free fatty acids, triglycerides)

• Tissue-specific analyses:

  • Liver: hepatic glucose production assays, glycogen content measurement

  • Muscle: ex vivo glucose uptake, glycogen synthesis

  • Adipose tissue: lipolysis assays, glucose uptake

  • Duodenum: nutrient sensing mechanisms, signal transduction pathways

• Molecular signaling studies:

  • Insulin signaling pathway components (IRS, PI3K, AKT)

  • AMPK pathway activation

  • mTOR signaling

From the available research, the pancreatic-euglycemic clamping approach with duodenal infusions has proven particularly valuable for understanding NUCB2's role in gut-brain-liver signaling and glucose homeostasis .

What explains the contrasting roles of NUCB2 across different cancer types?

NUCB2 exhibits contrasting roles across cancer types, functioning as a negative prognostic indicator in most cancers (including GBM, breast, colon, bladder, prostate cancers) while playing a tumor-suppressive role in adrenocortical and ovarian epithelial carcinoma . Several hypotheses might explain these contradictory functions:

• Tissue-specific processing:

  • Differential processing of NUCB2 into nesfatin-1, nesfatin-2, and nesfatin-3 across tissues

  • Varying ratios of these peptides may exert opposite effects on cell growth and survival

• Signaling context:

  • Tissue-specific expression of downstream effectors

  • Interaction with different binding partners (e.g., NDN has been identified as an interactor)

  • Integration with tissue-specific signaling networks

• Concentration-dependent effects:

  • Biphasic dose-response relationships where low vs. high expression levels trigger distinct pathways

  • Threshold effects in signaling activation

To investigate these contradictions, researchers should employ tissue-specific conditional knockout models, detailed interactome analysis, and comprehensive signaling pathway mapping across different cancer types.

How might NUCB2 targeting overcome treatment resistance in glioblastoma?

Based on preclinical evidence, NUCB2 targeting shows significant potential for overcoming treatment resistance in GBM through multiple mechanisms:

• Chemosensitization potential:

  • NUCB2 knockdown enhanced sensitivity to temozolomide in GBM cells

  • Increased apoptosis markers (PARP and cleaved caspase-3) observed with NUCB2 suppression during TMZ treatment

  • This suggests NUCB2 inhibition could lower effective TMZ doses and improve treatment response

• Radiosensitization strategies:

  • NUCB2 knockdown reduced expression of DNA repair proteins (Ku70, Ku80, Rad51, and Rad52)

  • Decreased colony formation after radiation treatment with NUCB2 suppression

  • This indicates potential for enhanced radiotherapy efficacy with NUCB2 targeting

• In vivo evidence:

  • Mice implanted with NUCB2-knockdown GBM cells showed significantly prolonged survival

  • Reduced tumor progression was observed using IVIS imaging in the NUCB2 knockdown group

Researchers developing NUCB2-targeted therapeutic approaches should focus on combination therapy protocols, selective delivery systems (particularly for crossing the blood-brain barrier), and identification of patient subgroups most likely to benefit from NUCB2 inhibition.

What methodological approaches can resolve discrepancies in NUCB2 functional studies?

Resolving discrepancies in NUCB2 functional studies requires sophisticated methodological approaches:

• Context-specific experimental design:

  • Clear definition of experimental scope (full-length NUCB2 vs. nesfatin-1)

  • Standardized concentration ranges across studies

  • Consistent cell types and physiological conditions

• Comprehensive protein characterization:

  • Analysis of post-translational modifications

  • Identification of protein-protein interactions in tissue-specific contexts

  • Structural biology approaches to determine conformation in different cellular environments

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Network analysis to identify context-dependent signaling pathways

  • Single-cell approaches to capture heterogeneity in responses

• Cross-validation requirements:

  • Multiple model systems (cell lines, primary cultures, animal models)

  • Diverse technical approaches for key findings

  • Replication across independent laboratories

These approaches will help address contradictory findings regarding NUCB2's role in cancer progression, treatment resistance, and metabolic regulation, enabling more consistent and reliable research outcomes.