RBP5 Human

What is the distinction between RBP5 and RBBP5 in human cells?

RBP5 (Retinol-binding protein 5) and RBBP5 (Retinoblastoma binding protein 5) are different proteins with similar acronyms, which can lead to confusion in scientific literature. RBP5 is a 135-amino acid protein primarily involved in the intracellular transport of retinol (vitamin A) . In contrast, RBBP5 is described as a binding protein of retinoblastoma, which is one of the best-studied tumor suppressors .

RBBP5 functions within multiprotein complexes that modify chromatin structure through histone modifications, particularly as part of the Set1/Ash2 histone methyltransferase complex . When conducting research, it's critical to clearly specify which protein is being investigated and use appropriate specific antibodies and detection methods.

What are the primary biological functions of RBP5 in normal cellular processes?

RBP5 primarily functions in the intracellular transport of retinol (vitamin A), serving as a carrier protein that facilitates the movement of retinol within cells . This function is essential for maintaining proper retinoid homeostasis, which affects numerous cellular processes including:

• Cell differentiation and tissue development

• Gene expression regulation through retinoid signaling pathways

• Maintenance of epithelial cell integrity

• Visual function support

The protein shares approximately 51% sequence identity with zebrafish retinol binding proteins, suggesting evolutionary conservation of its fundamental structure and function . Methodologically, researchers studying RBP5 function should consider using retinol-binding assays, subcellular localization studies, and gene expression analyses following RBP5 modulation to comprehensively characterize its biological roles.

How does RBBP5 contribute to epigenetic regulation mechanisms?

RBBP5 plays a significant role in epigenetic regulation through its participation in histone methyltransferase complexes. According to the STRING database, RBBP5 works in association with other proteins like ASH2L and WDR5 to stimulate the histone methyltransferase activities of KMT2A, KMT2B, KMT2C, KMT2D, SETD1A, and SETD1B .

These complexes specifically methylate 'Lys-4' of histone H3, a modification that represents a specific tag for epigenetic transcriptional activation. Through these interactions, RBBP5 contributes to the regulation of gene expression patterns that control cell differentiation, development, and potentially oncogenic processes.

Methodologically, researchers investigating RBBP5's epigenetic functions should consider chromatin immunoprecipitation (ChIP) assays, histone methyltransferase activity assays, and genome-wide expression analyses following RBBP5 modulation to map its epigenetic influence across the genome.

What methodological approaches are most effective for measuring RBP5/RBBP5 expression in human tissue samples?

When measuring RBP5/RBBP5 expression in human tissue samples, researchers should employ multiple complementary techniques to ensure robust results:

• Immunohistochemistry (IHC): This technique was successfully used to examine RBBP5 expression levels in hepatocellular carcinoma tissue samples . IHC provides valuable information about protein localization and expression patterns within the tissue architecture. For optimal results:

  • Use antigen retrieval methods appropriate for formalin-fixed paraffin-embedded samples

  • Include positive and negative controls in each experimental run

  • Employ standardized scoring systems (H-score, Allred score) for semi-quantitative assessment

• Western blot analysis: This technique provides quantitative information about protein expression levels . Methodological considerations include:

  • Use of appropriate protein extraction buffers to maintain protein integrity

  • Loading equal amounts of protein across samples (verified by housekeeping proteins)

  • Optimization of antibody concentrations and incubation conditions

• Quantitative PCR (qPCR): While not explicitly mentioned in the search results, qPCR provides a complementary approach to assess mRNA expression levels.

For maximum validity, researchers should validate findings across multiple methodologies and include appropriate controls to account for technical and biological variability.

What experimental designs are optimal for studying the functional effects of RBBP5 knockdown in cancer models?

Based on research examining RBBP5 in hepatocellular carcinoma , the following experimental design recommendations can be made for studying RBBP5 knockdown effects:

• Cell model selection:

  • Use multiple cell lines representing the cancer type of interest

  • Include both cancer and non-malignant control cell lines

  • Consider patient-derived primary cells when available

• Knockdown approach:

  • Employ both transient (siRNA) and stable (shRNA) knockdown systems

  • Include at least 2-3 different knockdown constructs targeting different regions of RBBP5

  • Use appropriate non-targeting controls (scrambled sequences)

  • Verify knockdown efficiency at both mRNA and protein levels

• Functional assays:

  • Cell proliferation: Multiple time points and methodologies (MTT/MTS, BrdU incorporation)

  • Cell cycle analysis: Flow cytometry with propidium iodide staining

  • Apoptosis assays: Annexin V/PI staining, caspase activation

  • Drug sensitivity testing: Dose-response curves with relevant therapeutic agents

  • Migration/invasion assays for metastatic potential

• Molecular mechanism investigation:

  • Expression analysis of cell cycle regulators

  • Chromatin immunoprecipitation to assess changes in histone modifications

  • RNA-seq to identify global transcriptional changes

This comprehensive approach would enable robust characterization of RBBP5's role in cancer progression, similar to the findings that "Knockdown of RBBP5 significantly inhibited proliferation of HCC cells through cell cycle arrest" and "inhibition of RBBP5 increased the sensitivity of HCC cells to doxorubicin" .

What experimental controls are essential when investigating RBP5/RBBP5 protein interactions?

When investigating RBP5/RBBP5 protein interactions, the following experimental controls are essential for generating reliable and interpretable results:

• Input controls:

  • Total protein lysate samples to verify protein expression

  • Size markers to confirm expected molecular weights

  • Loading controls to ensure equal protein input across conditions

• Specificity controls:

  • IgG or other appropriate negative controls for immunoprecipitation experiments

  • Competitive binding assays with purified proteins

  • Reverse immunoprecipitation to confirm interactions

  • Pre-absorption controls for antibodies

• Functional validation controls:

  • Domain deletion/mutation constructs to map interaction regions

  • Knockdown/knockout of interaction partners to confirm specificity

  • Physiologically relevant stimuli that might affect interactions

• Technical controls:

  • Multiple antibody sources/epitopes when possible

  • Different protein interaction detection methods (co-IP, proximity ligation, FRET)

  • In vitro versus in vivo interaction confirmation

Given RBP5's potential interactions with proteins like URI1, ASH2L, and DPY30 , researchers should carefully validate each interaction using multiple approaches and appropriate controls to distinguish genuine interactions from artifacts.

How does RBBP5 contribute to hepatocellular carcinoma progression, and what methodologies best elucidate these mechanisms?

RBBP5 appears to play a significant role in hepatocellular carcinoma (HCC) progression through multiple mechanisms. According to research findings, RBBP5 was significantly upregulated in HCC tissues and cells compared to normal controls . This elevated expression correlated with several clinicopathological features, including:

• Elevated AFP levels

• Advanced TNM stage

• High Ki-67 expression (a proliferation marker)

• Larger tumor size

• Poor prognosis

Functionally, knockdown studies demonstrated that RBBP5 inhibition significantly reduced HCC cell proliferation through cell cycle arrest mechanisms. Additionally, RBBP5 inhibition increased the sensitivity of HCC cells to doxorubicin, suggesting a role in chemoresistance .

To optimally investigate these mechanisms, researchers should employ:

• Multi-omics approaches:

  • Transcriptomics (RNA-seq) following RBBP5 modulation

  • ChIP-seq to identify genome-wide binding sites and histone modification changes

  • Proteomics to identify altered signaling pathways

• Mechanistic studies:

  • Cell cycle phase analysis using flow cytometry

  • Expression analysis of cell cycle regulators

  • Detailed apoptotic pathway investigation

• In vivo validation:

  • Patient-derived xenograft models with RBBP5 modulation

  • Correlation of findings with patient samples and outcomes

  • Therapeutic intervention studies combining RBBP5 inhibition with chemotherapy

These methodological approaches would provide comprehensive insights into RBBP5's role in HCC progression and potential therapeutic targeting strategies.

What are the molecular mechanisms through which RBP5/RBBP5 interacts with epigenetic machinery, and how can these be experimentally validated?

Based on the search results, RBBP5 appears to be intimately connected with epigenetic regulation through interactions with histone methyltransferase complexes. The molecular mechanisms include:

• Complex formation with histone modifiers:

  • RBBP5 associates with ASH2L and WDR5 to stimulate the histone methyltransferase activities of KMT2A, KMT2B, KMT2C, KMT2D, SETD1A, and SETD1B

  • These complexes specifically methylate 'Lys-4' of histone H3, creating an epigenetic mark associated with transcriptional activation

• Connection to retinoid signaling:

  • DPY30, a potential interaction partner, plays "a crucial role in retinoic acid-induced differentiation along the neural lineage, regulating gene induction and H3 'Lys-4' methylation at key developmental loci"

To experimentally validate these mechanisms, researchers should employ:

• Biochemical approaches:

  • Reconstitution of histone methyltransferase complexes with purified components

  • Structural studies of RBBP5 within these complexes (X-ray crystallography, cryo-EM)

  • In vitro histone methyltransferase assays with reconstituted complexes

• Cellular approaches:

  • ChIP-seq for RBBP5 and associated histone modifications

  • Sequential ChIP (Re-ChIP) to identify co-occupancy with other complex components

  • CRISPR-based approaches to modify specific domains of RBBP5

  • Proximity labeling techniques (BioID, APEX) to identify context-specific interaction partners

• Functional validation:

  • Gene expression analysis following RBBP5 modulation

  • Correlation of RBBP5 binding sites with histone modification patterns

  • Analysis of chromatin accessibility changes (ATAC-seq)

These methodological approaches would provide comprehensive insights into the molecular mechanisms through which RBBP5 influences epigenetic regulation and gene expression.

How do post-translational modifications affect RBP5/RBBP5 function, and what techniques can detect these modifications?

While the search results don't specifically address post-translational modifications (PTMs) of RBP5/RBBP5, this is a critical research question for understanding protein regulation. Given RBBP5's role in epigenetic regulation complexes , PTMs likely play important roles in modulating its function.

Potential PTMs that might regulate RBP5/RBBP5 function include:

• Phosphorylation (affecting protein-protein interactions or nuclear localization)

• Ubiquitination (regulating protein stability)

• Methylation or acetylation (influencing chromatin association)

• SUMOylation (altering protein interactions or localization)

Methodological approaches to study these modifications include:

• Identification techniques:

  • Mass spectrometry-based proteomics (phosphoproteomics, ubiquitinomics)

  • Western blotting with modification-specific antibodies

  • 2D gel electrophoresis to separate modified forms

  • Phos-tag gels for phosphorylation analysis

• Functional analysis methods:

  • Site-directed mutagenesis of modified residues

  • Expression of phosphomimetic or phospho-deficient mutants

  • Treatment with kinase/phosphatase inhibitors

  • Ubiquitination analysis with proteasome inhibitors

• Dynamics and regulation:

  • Time-course analysis following stimulation

  • Identification of responsible enzymes (kinases, E3 ligases)

  • Context-specific modification patterns in different cell types or conditions

• Structural impacts:

  • Conformational changes assessed by limited proteolysis

  • Protein interaction changes following modification

  • Subcellular localization shifts

These methodological approaches would provide insights into how PTMs regulate RBP5/RBBP5 function in different cellular contexts and how these modifications might be altered in disease states like cancer.

What are the best research designs to study RBP5/RBBP5 involvement in different cancer types?

To effectively study RBP5/RBBP5 involvement across different cancer types, researchers should implement a multi-faceted research design that combines clinical, cellular, and molecular approaches:

• Clinical investigation design:

  • Tissue microarray analysis across multiple cancer types

  • Correlation of expression with clinicopathological features

  • Survival analysis stratified by expression levels

  • Multi-cancer dataset mining (TCGA, ICGC) for expression patterns

• Comparative cellular models:

  • Panel of cell lines representing multiple cancer types

  • Matched normal-tumor cell models when available

  • Patient-derived primary cultures

  • 3D organoid models for physiological relevance

• Functional assessment design:

  • Standardized knockdown/overexpression approaches across cell types

  • Unified functional assay panel (proliferation, migration, drug sensitivity)

  • Context-specific assays relevant to each cancer type

  • Isogenic cell line pairs differing only in RBP5/RBBP5 status

• Molecular mechanism investigation:

  • ChIP-seq across cancer types to identify common and unique targets

  • Comparative transcriptomics to identify cancer-specific gene signatures

  • Protein complex analysis in different cellular contexts

  • Pathway analysis to identify cancer-type-specific dependencies

This comprehensive approach would build upon the hepatocellular carcinoma findings while enabling identification of both common and cancer-type-specific roles of RBP5/RBBP5, potentially revealing new therapeutic opportunities.

What statistical methodologies are most appropriate for analyzing correlations between RBP5/RBBP5 expression and clinical outcomes?

When analyzing correlations between RBP5/RBBP5 expression and clinical outcomes, researchers should employ robust statistical methodologies that account for the complexity of clinical data:

• Expression categorization approaches:

  • Receiver Operating Characteristic (ROC) curve analysis to determine optimal cut-off values

  • Quartile or percentile-based stratification

  • Continuous variable analysis to avoid information loss from dichotomization

• Survival analysis methods:

  • Kaplan-Meier survival curves with log-rank tests for univariate analysis

  • Cox proportional hazards models for multivariate analysis

  • Time-dependent ROC analysis for prognostic performance

  • Restricted mean survival time (RMST) for non-proportional hazards

• Association with clinical parameters:

  • Chi-square or Fisher's exact tests for categorical variables

  • Mann-Whitney U or t-tests for continuous variables

  • Correlation coefficients (Spearman's, Pearson's) for continuous associations

  • Logistic regression for binary outcomes

• Multiple testing considerations:

  • Bonferroni correction for conservative multiple testing adjustment

  • False Discovery Rate (FDR) methods for less stringent correction

  • Bootstrapping for robust confidence interval estimation

• Predictive modeling approaches:

  • Machine learning algorithms to identify complex patterns

  • Cross-validation to assess generalizability

  • Model performance metrics (AUC, C-index, Brier score)

  • Nomogram development for clinical application

These statistical approaches would provide comprehensive assessment of RBP5/RBBP5's clinical significance, similar to the approach that identified associations between RBBP5 expression and "elevated level of AFP, advanced TNM stage, high Ki-67 expression, larger tumor size, and poor prognosis" .

What experimental approaches can distinguish between direct and indirect effects of RBP5/RBBP5 on gene expression?

Distinguishing between direct and indirect effects of RBP5/RBBP5 on gene expression requires sophisticated experimental designs that capture different levels of molecular regulation:

• Chromatin association mapping:

  • ChIP-seq to identify direct binding sites of RBP5/RBBP5

  • CUT&RUN or CUT&Tag for higher resolution binding profiles

  • ChIP-exo for base-pair resolution of binding sites

  • Re-ChIP to identify co-occupancy with other complex members

• Temporal dynamics analysis:

  • Time-course expression analysis following RBP5/RBBP5 modulation

  • Nascent RNA sequencing (GRO-seq, PRO-seq) to capture immediate transcriptional changes

  • Pulse-chase labeling of newly synthesized RNA

  • Conditional/inducible systems for precise temporal control

• Direct interaction verification:

  • DNA binding assays (EMSA, DNA footprinting) if direct DNA binding is suspected

  • Chromatin tethering experiments (artificial recruitment to specific loci)

  • Proximity labeling to identify interactions in native context

  • In vitro transcription assays with purified components

• Dissection of molecular intermediates:

  • Sequential knockdown experiments (RBP5/RBBP5 + potential mediators)

  • Epistasis analysis with genetic approaches

  • Inhibitor studies targeting specific pathways

  • Reconstitution experiments in knockout backgrounds

• Integrative approaches:

  • Integration of binding data with expression changes

  • Network analysis to identify direct vs. indirect nodes

  • Mathematical modeling of gene regulatory networks

  • Multi-omics integration (epigenome, transcriptome, proteome)

These methodological approaches would enable researchers to distinguish genes directly regulated by RBP5/RBBP5-containing complexes from those affected through secondary mechanisms, providing clearer insights into their fundamental roles in transcriptional regulation.

How should researchers interpret contradictory findings about RBP5/RBBP5 function across different experimental systems?

When faced with contradictory findings about RBP5/RBBP5 function across different experimental systems, researchers should apply a systematic approach to interpretation:

• Contextual framework analysis:

  • Evaluate cellular context differences (cell type, tissue origin, species)

  • Consider experimental conditions (growth factors, confluency, oxygen levels)

  • Assess developmental or differentiation stage variations

  • Examine disease state contexts (normal vs. transformed cells)

• Methodological assessment:

  • Compare protein modulation approaches (knockdown vs. knockout vs. overexpression)

  • Evaluate timing differences (acute vs. chronic modulation)

  • Assess experimental readouts (direct vs. surrogate measures)

  • Consider technical limitations of each methodology

• Molecular complexity considerations:

  • Investigate protein isoform differences across systems

  • Evaluate post-translational modification status

  • Assess complex composition variations (ASH2L, WDR5 availability)

  • Consider feedback mechanisms and compensatory responses

• Integrative interpretation strategies:

  • Develop unifying models that accommodate seemingly contradictory findings

  • Identify boundary conditions where function transitions occur

  • Design bridging experiments to directly test contradictions

  • Consider context-dependent dual functions (common in epigenetic regulators)

• Validation approaches:

  • Replicate key experiments under identical conditions

  • Employ orthogonal methodologies to address the same question

  • Validate findings in more physiologically relevant models

  • Consider multi-laboratory collaborations for independent verification

This systematic approach acknowledges that proteins like RBBP5, which function within complex epigenetic regulatory networks , may indeed have context-dependent and sometimes opposing functions depending on cellular context, available interaction partners, and specific experimental conditions.

What bioinformatic approaches are most effective for integrating RBP5/RBBP5 genomic binding data with transcriptional outcomes?

To effectively integrate RBP5/RBBP5 genomic binding data with transcriptional outcomes, researchers should employ sophisticated bioinformatic approaches that capture the complexity of epigenetic regulation:

• Multi-omics data integration:

  • ChIP-seq for RBP5/RBBP5 and associated histone marks (H3K4me3, H3K4me1)

  • RNA-seq or nascent RNA sequencing for transcriptional effects

  • ATAC-seq or DNase-seq for chromatin accessibility correlation

  • Integration with existing datasets (ENCODE, Roadmap Epigenomics)

• Peak-to-gene assignment methodologies:

  • Proximity-based assignment with defined distance thresholds

  • Topologically associated domain (TAD) considerations

  • Chromatin interaction data (Hi-C, ChIA-PET) integration

  • Enhancer-promoter linking algorithms

• Statistical approaches for correlation:

  • Gene Set Enrichment Analysis (GSEA) for pathway connections

  • Regression models accounting for multiple factors

  • Machine learning approaches for pattern recognition

  • Bayesian network modeling of cause-effect relationships

• Functional classification tools:

  • Motif enrichment analysis for co-binding factors

  • Biological pathway enrichment (GO, KEGG, Reactome)

  • Gene regulatory network reconstruction

  • Comparative analysis across cell types/conditions

• Visualization and interpretation frameworks:

  • Genome browsers with multi-track visualization

  • Network visualization tools for interaction mapping

  • Heat maps and meta-gene plots for global patterns

  • Integrative dashboards for multi-dimensional data exploration

These bioinformatic approaches would enable researchers to move beyond correlation to establish causal relationships between RBP5/RBBP5 binding and transcriptional outcomes, particularly in the context of its role in histone methyltransferase complexes that specifically modify histone H3 at lysine 4 .

How can researchers apply findings about RBP5/RBBP5 function to develop potential therapeutic strategies for cancer?

Based on findings that RBBP5 plays significant roles in hepatocellular carcinoma progression , researchers can systematically develop therapeutic strategies through the following methodological approaches:

• Target validation approaches:

  • Genetic modulation in preclinical models (conditional knockouts, inducible systems)

  • Patient-derived xenograft response to RBBP5 inhibition

  • Synthetic lethality screening to identify context-specific vulnerabilities

  • Biomarker development to identify responsive patient populations

• Drug development strategies:

  • Protein-protein interaction (PPI) inhibitor design targeting RBBP5-complex interactions

  • Structure-based drug design if structural data is available

  • Allosteric modulator screening

  • Degradation-based approaches (PROTACs, molecular glues)

• Combination therapy rationales:

  • Synergy testing with standard chemotherapeutics (expanding on the finding that "inhibition of RBBP5 increased the sensitivity of HCC cells to doxorubicin")

  • Combinations with other epigenetic modifiers (HDAC inhibitors, DNA methyltransferase inhibitors)

  • Pathway-based combinations targeting complementary mechanisms

  • Immunotherapy combinations if epigenetic changes affect tumor immunogenicity

• Translational research approaches:

  • Pharmacodynamic biomarker development

  • Ex vivo drug sensitivity testing in patient samples

  • Early-phase clinical trial designs with molecular stratification

  • Resistance mechanism anticipation and mitigation strategies

• Precision medicine applications:

  • Patient stratification based on RBBP5 expression or activity

  • Development of companion diagnostics

  • Monitoring of treatment response through liquid biopsy approaches

  • Adaptive therapy approaches based on dynamic biomarkers

These methodological approaches leverage the finding that RBBP5 inhibition not only suppresses cancer cell proliferation but also enhances chemosensitivity , suggesting multiple therapeutic angles that could be clinically relevant across cancer types where RBBP5 dysregulation occurs.