VBP1 Human

What is VBP1 and what is its primary function in human cells?

VBP1 (von Hippel-Lindau-binding protein 1) is a protein that physically interacts with the von Hippel-Lindau protein (pVHL), an E3-ubiquitin ligase. Its primary function involves enhancing pVHL stability and facilitating pVHL-mediated ubiquitination of hypoxia-inducible factor-1α (HIF-1α) . This process is critical for oxygen-dependent degradation of HIF-1α, which regulates cellular responses to hypoxia. Research approaches to study this function typically involve co-immunoprecipitation assays to detect protein-protein interactions, ubiquitination assays to measure HIF-1α degradation, and oxygen-dependent expression studies using controlled environmental chambers to simulate varying oxygen levels.

Where is VBP1 expressed in human tissues and how does expression vary across development?

Based on comparative studies with murine models, VBP1 expression appears to be tissue-specific during development but becomes more ubiquitous in adult tissues. In fetal stages, VBP1 is predominantly expressed in the central nervous system, retina, and liver . In adult tissues, VBP1 is ubiquitously expressed, though expression levels show tissue-specific patterns with particular concentration in brain, eye, kidney, and intestine .

Methodologically, researchers typically employ in situ hybridization and Northern blot analysis to map expression patterns. For human tissue studies, immunohistochemistry with anti-VBP1 antibodies on tissue microarrays provides comprehensive mapping of expression across multiple tissues simultaneously. RNA-seq and single-cell transcriptomics have become increasingly valuable for detecting subtle variations in expression across different cell types within tissues.

What is the genomic location of the VBP1 gene and are there known variants?

The human VBP1 gene is located in a region homologous to mouse chromosome Xq28 . Mapping studies typically employ fluorescence in situ hybridization (FISH) and comparative genomic hybridization to confirm localization. When investigating variants, next-generation sequencing approaches, including whole exome sequencing and targeted resequencing, are recommended for comprehensive detection. Analysis of potential variants should include assessment of conservation across species, prediction of functional impact using tools like SIFT and PolyPhen, and correlation with phenotypic data from clinical databases.

What are the most effective approaches for studying VBP1 function in vitro?

For in vitro studies of VBP1, several complementary approaches are recommended:

• Protein interaction studies: Co-immunoprecipitation followed by mass spectrometry to identify interaction partners beyond pVHL. Proximity ligation assays provide spatial resolution of interactions within cells.

• Gene expression modulation: CRISPR-Cas9 knockout, siRNA knockdown, or overexpression systems using lentiviral vectors allow for functional assessment under varied VBP1 levels.

• Functional assays: Ubiquitination assays, protein stability measurements using cycloheximide chase experiments, and reporter assays for HIF-1 activity provide mechanistic insights.

• Cellular phenotype assessment: Analysis of epithelial-mesenchymal transition markers, migration/invasion assays, and 3D culture systems reveal VBP1's role in complex cellular behaviors .

When designing these experiments, single-subject research designs with multiple condition implementations may provide more rigorous evidence than traditional group designs in some contexts, particularly when studying highly variable cellular responses .

How should researchers design experiments to investigate VBP1's role in hypoxia response pathways?

When investigating VBP1's role in hypoxia response pathways, a multi-level experimental approach is optimal:

• Controlled oxygen environments: Use hypoxia chambers with precise O₂ control (1-5%) and monitor cellular responses across different time points (acute vs. chronic hypoxia).

• HIF-1α stability measurements: Western blotting with time-course analysis following hypoxia induction, with and without VBP1 manipulation.

• Transcriptional activity assessment: Reporter assays using HIF-1-responsive elements (HRE) linked to luciferase to quantify downstream effects of VBP1 on HIF-1 activity .

• Target gene expression analysis: qRT-PCR arrays targeting known HIF-1α responsive genes to assess physiological relevance.

Critically, researchers should include appropriate controls for each oxygen condition and validate with multiple cell lines to ensure findings are not cell-type specific. Single-subject research designs with repeated measurements under varied conditions can provide robust evidence of VBP1's specific effects .

What considerations should guide the selection of model systems for studying VBP1 in cancer contexts?

Selection of appropriate model systems for VBP1 cancer research should be guided by these considerations:

For cancer metastasis studies specifically, models that recapitulate epithelial-mesenchymal transition (EMT) are particularly valuable, as VBP1 has been shown to suppress HIF-1α-induced EMT in vitro and tumor metastasis in vivo . Xenograft models with tagged tumor cells allow for direct visualization of metastatic processes influenced by VBP1.

How does VBP1 interact with the VHL-Elongin BC-Cullin2-Rbx1 (VBC) complex, and what methodologies best capture this interaction?

VBP1 enhances the stability of pVHL and facilitates pVHL-mediated ubiquitination of HIF-1α through direct protein interaction . To effectively study this complex interaction:

• Structural biology approaches: Cryo-electron microscopy and X-ray crystallography can resolve the VBP1-VBC complex structure at atomic resolution, providing insights into binding interfaces.

• Interaction kinetics measurements: Surface plasmon resonance (SPR) and microscale thermophoresis (MST) provide quantitative binding kinetics under varied conditions.

• Domain mapping: Truncation and point mutation analysis followed by binding assays identifies critical interaction domains.

• In-cell interaction dynamics: FRET/BRET approaches with fluorescently tagged proteins reveal real-time interaction dynamics in living cells.

• Functional consequence assessment: Reconstituted ubiquitination assays using purified components determine how VBP1 enhances VBC complex activity.

A multi-method approach is recommended as each technique provides complementary information about different aspects of the interaction. When analyzing contradictory results between methods, researchers should consider differences in protein conformation, post-translational modifications, and cellular context.

What are the conflicting findings regarding VBP1 expression in tumors, and how can researchers resolve these contradictions?

Current literature presents seemingly contradictory findings regarding VBP1 expression in tumors. While some studies suggest VBP1 suppresses tumor metastasis, consistent differences in VBP1 expression levels between tumors and normal tissues have not been universally detected . To resolve these contradictions:

• Comprehensive tissue analysis: Using tissue microarrays across multiple tumor types with matched normal controls, stratified by stage and grade.

• Context-dependent analysis: Evaluating VBP1 expression in relation to hypoxic markers, pVHL status, and HIF-1α levels rather than in isolation.

• Functional stratification: Correlating VBP1 expression with EMT markers and metastatic potential.

• Single-cell approaches: Employing single-cell RNA-seq to detect cell population-specific expression patterns that might be masked in bulk analysis.

• Multi-omics integration: Combining transcriptomic, proteomic, and epigenetic data to identify regulatory mechanisms affecting VBP1 function beyond expression levels.

When designing studies to address these contradictions, researchers should employ single-subject research designs with multiple condition implementations to establish causal relationships more definitively than traditional group comparisons, which may mask important individual variations .

How does VBP1 function as a tumor suppressor, and what experimental designs best demonstrate this property?

VBP1 functions as a tumor suppressor primarily by enhancing pVHL stability and facilitating pVHL-mediated ubiquitination of HIF-1α, thereby suppressing HIF-1α-induced epithelial-mesenchymal transition (EMT) and tumor metastasis . To effectively demonstrate this tumor suppressor function:

• Gain/loss-of-function studies: Compare metastatic potential in isogenic cell lines with VBP1 overexpression or knockout, using both in vitro invasion assays and in vivo metastasis models.

• Rescue experiments: Determine whether VBP1 reintroduction in VBP1-deficient tumor cells reverses metastatic phenotypes.

• Pathway dissection: Use epistasis experiments with HIF-1α constitutively active mutants to determine whether VBP1's effects are HIF-1α-dependent.

• Correlation with clinical outcomes: Analyze patient survival data stratified by VBP1 expression levels, controlling for confounding factors.

• Therapeutic targeting validation: Test whether enhancing VBP1 function through small molecules inhibits tumor progression.

When interpreting results, researchers should be careful to distinguish between correlation and causation, particularly when analyzing clinical data. Appropriately designed research questions should focus on mechanisms rather than simply associations .

How does VBP1 expression correlate with clinical outcomes in VHL-associated cancers?

Current research suggests complex relationships between VBP1 expression and clinical outcomes in VHL-associated cancers. To properly investigate these correlations:

Research questions investigating clinical correlations should be specific enough to answer thoroughly but complex enough to develop meaningful answers over the space of a paper or thesis . Integration of molecular data with clinical outcomes requires careful statistical planning to avoid false correlations.

What methodological approaches are most effective for targeting the VBP1-pVHL-HIF-1α axis therapeutically?

When exploring therapeutic targeting of the VBP1-pVHL-HIF-1α axis, researchers should consider these methodological approaches:

• Small molecule screening: High-throughput screening of compound libraries to identify molecules that enhance VBP1-pVHL interaction or VBP1 stability.

• Peptide mimetics: Design of peptides that mimic key VBP1 interaction domains to enhance pVHL-mediated HIF-1α degradation.

• Targeted protein degradation: PROTAC (Proteolysis Targeting Chimera) approaches targeting HIF-1α in a VBP1-independent manner as a parallel strategy.

• RNA therapeutics: Antisense oligonucleotides or siRNA delivery systems to modulate VBP1 expression in tumors.

• Combination strategies: Testing VBP1-enhancing approaches with existing anti-angiogenic therapies, as both target HIF-dependent pathways.

Evaluation should include not only target engagement and pathway modulation metrics but also phenotypic outcomes like reduced EMT and metastasis. Pre-clinical testing should incorporate both in vitro and in vivo models with appropriate controls and sample sizes to ensure statistical power .

How might emerging single-cell technologies advance our understanding of VBP1 function in heterogeneous tissues?

Single-cell technologies offer unprecedented opportunities to understand VBP1 function in complex tissues:

• Single-cell RNA sequencing: Reveals cell type-specific expression patterns and co-expression networks involving VBP1, particularly important given the heterogeneous expression observed in tissues like brain, eye, kidney, and intestine .

• Single-cell proteomics: Emerging mass cytometry (CyTOF) and single-cell Western blot technologies allow protein-level quantification of VBP1 and interaction partners.

• Spatial transcriptomics: Methods like Visium and MERFISH provide spatial context to VBP1 expression patterns, particularly valuable in tumors with hypoxic gradients.

• Live-cell imaging: CRISPR-based tagging of endogenous VBP1 with fluorescent proteins enables real-time tracking of expression and localization dynamics.

These approaches can resolve contradictory findings from bulk analyses by revealing cell population-specific functions of VBP1. When designing single-cell studies, researchers should consider both technical variability and biological heterogeneity, incorporating appropriate controls and statistical methods for high-dimensional data analysis.

What are the understudied aspects of VBP1 biology that merit further investigation?

Several aspects of VBP1 biology remain understudied and represent valuable research opportunities:

• Post-translational modifications: Systematic characterization of how phosphorylation, ubiquitination, or other modifications regulate VBP1 function and interaction with pVHL.

• Non-HIF functions: Investigation of HIF-independent roles of VBP1, potentially involving other protein quality control pathways or transcriptional regulation.

• Tissue-specific functions: Exploration of why VBP1 shows specific expression patterns in brain, eye, kidney, and intestine, and whether these reflect tissue-specific roles .

• Developmental dynamics: Examination of why VBP1 expression changes during development, particularly in the placenta where high expression at day 12 decreases in later stages .

• Evolutionary conservation: Comparative analysis of VBP1 function across species to identify essential versus adaptable aspects of its biology.

When formulating research questions for these areas, researchers should ensure they are feasible to answer within practical constraints while remaining complex enough to merit detailed investigation .