CYB5R2 Human

What is CYB5R2 and what are its primary functions in human cells?

CYB5R2 is a 276-amino-acid NADH-dependent flavin reductase that belongs to the flavoprotein pyridine nucleotide cytochrome reductase family. It contains one ferredoxin reductase-type flavin adenine dinucleotide-binding domain and participates in multiple cellular processes . Its primary functions include:

• Involvement in desaturation and elongation of fatty acids

• Participation in cholesterol biosynthesis pathways

• Contribution to drug metabolism mechanisms

• Role in methemoglobin reduction in erythrocytes

• Detoxification of environmental carcinogens, including polycyclic aromatic hydrocarbons and arylhydroxylamine carcinogens commonly found in cigarette smoke and fried foods

CYB5R2 is also responsible for NADH-dependent lucigenin chemiluminescence in spermatozoa by reducing both lucigenin and 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) . The diverse functions of this enzyme highlight its significance in cellular homeostasis and metabolism.

What protein interactions are critical for CYB5R2 function?

CYB5R2 engages in several crucial protein-protein interactions that facilitate its biological functions. Based on STRING interaction network analysis, its primary functional partners include:

These interactions are particularly important for electron transfer processes and enzymatic reactions where CYB5R2 serves as an electron donor . The high interaction scores with CYB5B and CYB5A (0.994 and 0.993, respectively) suggest these associations are especially critical for CYB5R2 function.

What evidence supports CYB5R2 as a tumor suppressor gene?

Multiple lines of evidence support CYB5R2's role as a tumor suppressor, particularly in nasopharyngeal carcinoma (NPC):

• Expression patterns: CYB5R2 transcript levels are decreased in NPC cell lines and tumor biopsies compared to normal nasopharyngeal epithelium .

• Epigenetic regulation: Promoter hypermethylation of CYB5R2 was detected in all six tested NPC cell lines and in 84% of primary NPC tumor biopsies but not in normal nasopharyngeal epithelium, suggesting epigenetic silencing is a key mechanism of inactivation .

• Clinical correlation: CYB5R2 methylation was significantly associated with lymph node metastasis in NPC patients (P < 0.05), indicating a relationship with disease progression .

• Functional studies: Ectopic expression of CYB5R2 in NPC cells:

  • Inhibited cell proliferation

  • Reduced colony formation capacity

  • Decreased cell migration

  • Suppressed in vivo tumor formation in nude mice models

These findings collectively establish CYB5R2 as a functional tumor suppressor gene that is frequently inactivated by promoter hypermethylation in NPC, with potential implications for its role in other cancers as well.

How does CYB5R2 regulate angiogenesis in tumors?

CYB5R2 exhibits significant anti-angiogenic effects through multiple mechanisms:

• Downregulation of VEGF: CYB5R2 transfection in NPC cells led to decreased expression of vascular endothelial growth factor (VEGF), a critical pro-angiogenic factor .

• Modulation of angiogenesis-related genes: PCR assays revealed that CYB5R2 overexpression alters the transcription of several genes involved in angiogenesis:

  • Upregulated genes: FAS, FOS, PIK3R1, ITGB3, MTSS1, IFNB1, and CDKN2A - many of which negatively regulate angiogenesis

  • Downregulated genes: ITGB5, IGF1, TEK, TGFBR1, and VEGF - several of which promote angiogenesis

• In vivo validation: Using a chick chorioallantoic membrane (CAM) embryo model, researchers demonstrated that CYB5R2-transfected NPC cells formed smaller tumors with significantly reduced neovascularization compared to control cells:

  • Neovascularization in CYB5R2-transfected CNE2 cells: 5.3%

  • Neovascularization in empty vector-transfected CNE2 cells: 16.6%

  • Neovascularization in CYB5R2-transfected HONE1 cells: 5.1%

  • Neovascularization in empty vector-transfected HONE1 cells: 13.5%

  • Both comparisons showed statistical significance (P < 0.001)

This multi-level regulation of angiogenesis represents a key mechanism through which CYB5R2 exerts its tumor suppressive effects, controlling both the tumor microenvironment and tumor growth.

What is the relationship between CYB5R2 methylation and cancer progression?

The relationship between CYB5R2 promoter methylation and cancer progression has been most extensively studied in nasopharyngeal carcinoma:

• Prevalence of methylation: Promoter hypermethylation of CYB5R2 was detected in 84% of primary NPC tumor biopsies compared to none in normal nasopharyngeal epithelium, indicating this is a tumor-specific epigenetic alteration .

• Association with metastasis: Clinical data analysis revealed that CYB5R2 methylation status was significantly associated with lymph node metastasis in NPC patients (P < 0.05), suggesting it may serve as a prognostic biomarker .

• Functional consequences: The methylation-induced silencing of CYB5R2 appears to contribute to:

  • Enhanced cell proliferation

  • Increased colony formation

  • Greater migration capacity

  • Higher tumorigenicity in vivo

• Reversibility: The endogenous expression of CYB5R2 could be restored in NPC cell lines by treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, demonstrating that this epigenetic silencing is reversible .

These findings suggest that CYB5R2 methylation is not merely a biomarker but actively contributes to cancer progression, particularly metastasis, by silencing an important tumor suppressor gene. The epigenetic mechanism of CYB5R2 inactivation offers potential therapeutic opportunities through epigenetic modifying agents.

What are the recommended models for studying CYB5R2 function in vivo?

Based on successful experimental approaches documented in the literature, several in vivo models are recommended for investigating CYB5R2 function:

• Chick Chorioallantoic Membrane (CAM) Embryo Model:

  • Particularly useful for studying angiogenesis and tumor formation

  • Allows implantation of CYB5R2-expressing or control cells

  • Enables quantification of tumor size and blood vessel formation

  • Provides a relatively rapid assessment (typically 7-10 days)

  • Allows for immunohistochemical analysis of protein expression patterns (e.g., VEGF)

• Nude Mice Xenograft Models:

  • Gold standard for assessing tumorigenicity

  • Permits longer-term studies of tumor growth and progression

  • Can be used to assess the effects of CYB5R2 expression on tumor size, invasion, and metastasis

  • Allows for tissue collection and molecular analysis

• Cell Line Models:

  • NPC cell lines (CNE2, HONE1) have been successfully used to study CYB5R2 function

  • Transfection with CYB5R2 expression vectors enables gain-of-function studies

  • These cells can then be used in both in vitro assays and the in vivo models described above

When designing experiments, researchers should consider combining multiple models to comprehensively assess the biological functions of CYB5R2, particularly its roles in tumor suppression, angiogenesis, and metabolism.

How can researchers effectively analyze CYB5R2 promoter methylation?

For comprehensive analysis of CYB5R2 promoter methylation, researchers should employ a multi-method approach:

• Methylation-Specific PCR (MSP):

  • Allows rapid screening of methylation status

  • Requires primer sets specifically designed to detect either methylated or unmethylated alleles

  • Samples should be run in duplicate for reliability

  • Results are typically visualized on agarose gels

  • Provides qualitative assessment of methylation status

• Bisulfite Genomic Sequencing (BGS):

  • Offers quantitative, base-resolution analysis of methylation

  • Process involves:

    • Sodium bisulfite modification of DNA

    • PCR amplification with BGS primers targeting the CYB5R2 promoter region

    • Subcloning of PCR products into competent cells

    • Sequencing of multiple isolated plasmid clones (8-10 recommended)

    • Analysis of CpG site methylation patterns

• Methylation Array Technologies:

  • For genome-wide contextual analysis

  • Can identify patterns of CYB5R2 methylation in relation to other genes

  • Useful for larger epidemiological or clinical correlation studies

• Functional Validation:

  • Treatment with demethylating agents (e.g., 5-aza-2′-deoxycytidine)

  • Quantification of CYB5R2 re-expression using real-time PCR

  • Assessment of phenotypic effects following re-expression

For clinical samples, combining MSP (for rapid screening) with BGS (for detailed analysis of positive samples) provides the most comprehensive assessment of CYB5R2 methylation status and its potential correlation with disease parameters.

What techniques are most effective for studying CYB5R2's role in gene expression regulation?

To comprehensively investigate CYB5R2's impact on gene expression regulation, researchers should employ the following techniques:

• PCR Arrays for Pathway Analysis:

  • Use specialized arrays like the PAHS-033A Human Cancer Pathway Finder Superarray

  • This approach allows simultaneous analysis of multiple genes (84 genes in 6 cancer-related signaling pathways)

  • Provides a broad overview of pathways affected by CYB5R2 expression

  • Can identify candidates for further investigation

• Quantitative Real-Time PCR:

  • Essential for validating changes identified in array experiments

  • Provides precise quantification of expression changes

  • Use the 2^(-ΔΔCT) method to determine relative expression levels

  • Include appropriate housekeeping genes (e.g., GAPDH) as internal controls

  • Perform experiments in triplicate for statistical reliability

• Protein Expression Analysis:

  • Western blotting to confirm translation of transcriptional changes

  • Immunohistochemistry to assess protein expression in tissue contexts (as demonstrated in the CAM model for VEGF)

• Functional Assays:

  • Luciferase reporter assays to assess effects on promoter activity

  • ChIP assays to identify direct binding targets of affected transcription factors

  • Pathway inhibitor studies to validate specific pathway involvement

The most effective approach combines initial broad screening (PCR arrays) followed by targeted validation (qRT-PCR) and functional characterization of the identified pathways. For CYB5R2 specifically, particular attention should be paid to genes involved in angiogenesis, apoptosis, cell cycle control, and DNA damage repair, as these have been shown to be significantly affected by CYB5R2 expression .

How do researchers reconcile varying expression patterns of CYB5R2 across different cancer types?

The literature indicates that CYB5R2 expression varies across different cancer types, presenting a complex picture that requires careful experimental design and interpretation:

• Expression patterns documented in literature:

  • Downregulated in nasopharyngeal carcinoma (NPC)

  • Abnormally inactivated in prostate and breast cancers

  • Upregulated in B cell acute lymphocytic leukemia

To reconcile these varying patterns, researchers should consider:

• Tissue-specific functions: CYB5R2 may have different roles depending on the cellular context. In epithelial cancers, it appears to function primarily as a tumor suppressor, while in hematological malignancies, its role may differ.

• Methodological approaches:

  • Multi-cancer analysis: Compare CYB5R2 expression, methylation, and mutation profiles across multiple cancer types using public databases (TCGA, ICGC)

  • Functional studies in different cell types: Perform knockdown and overexpression experiments in multiple cell lineages to determine if CYB5R2's effects are tissue-dependent

  • Context-dependent interaction analysis: Investigate if CYB5R2 interacts with different partner proteins in different tissues

  • Pathway analysis: Determine if CYB5R2 activates or inhibits different signaling pathways in different cancer types

  • Genetic background considerations: Assess if genetic or epigenetic alterations in other genes modify CYB5R2 function

• Potential reconciling hypotheses:

  • CYB5R2 may function as a "conditional tumor suppressor" depending on cellular context

  • Post-translational modifications may alter CYB5R2 function in different tissues

  • Alternative splicing may generate tissue-specific isoforms with different functions

  • The metabolic state of the cell may determine whether CYB5R2 promotes or inhibits cancer progression

Researchers should design experiments that directly test these hypotheses to develop a unified understanding of CYB5R2's role across different cancer types.

What are the challenges in targeting CYB5R2 for cancer therapy?

Developing therapeutic strategies targeting CYB5R2 presents several significant challenges that researchers must address:

• Restoring expression of a silenced gene:

  • CYB5R2 is frequently silenced through promoter hypermethylation in certain cancers

  • While demethylating agents (e.g., 5-aza-2′-deoxycytidine) can restore expression in vitro , clinical application faces challenges:

    • Lack of specificity (genome-wide demethylation)

    • Potential toxicity

    • Transient effect requiring repeat administration

    • Difficulty achieving sufficient local concentration

• Delivery mechanisms:

  • For gene therapy approaches, efficient delivery of CYB5R2 to target tissues remains challenging

  • Viral vectors may induce immune responses

  • Non-viral vectors typically show lower efficiency

  • Targeting specifically to tumor cells is difficult

  • Blood-brain barrier presents additional challenges for central nervous system tumors

• Context-dependent effects:

  • As CYB5R2 shows variable expression across cancer types , therapeutic approaches would need to be cancer-specific

  • Upregulation in some cancers (e.g., B cell acute lymphocytic leukemia) suggests potential negative effects of widespread CYB5R2 restoration

• Biomarker development and patient selection:

  • Need to develop reliable assays for CYB5R2 methylation in liquid biopsies

  • Identifying patients who would benefit most from CYB5R2-targeted therapy

  • Establishing cutoff values for methylation status that predict therapeutic response

• Downstream targeting alternatives:

  • Identifying and targeting the critical downstream effectors of CYB5R2 (e.g., VEGF pathway) may be more feasible than directly targeting CYB5R2 itself

  • This requires comprehensive understanding of CYB5R2-regulated pathways in specific cancers

Researchers developing CYB5R2-based therapeutic strategies should consider combination approaches that address the epigenetic silencing while also targeting key downstream pathways affected by CYB5R2 loss.

How do researchers design experiments to assess CYB5R2's role in detoxification of environmental carcinogens?

To thoroughly investigate CYB5R2's role in detoxifying environmental carcinogens, researchers should design comprehensive experimental approaches:

• In vitro enzyme activity assays:

  • Purify recombinant CYB5R2 protein

  • Assess its ability to metabolize known carcinogens (polycyclic aromatic hydrocarbons, arylhydroxylamine compounds)

  • Determine enzyme kinetics (Km, Vmax) for different substrates

  • Compare activity with other detoxification enzymes

• Cell-based xenobiotic metabolism studies:

  • Generate cell lines with controlled CYB5R2 expression (overexpression, knockdown, knockout)

  • Expose cells to environmental carcinogens at various concentrations

  • Measure:

    • Carcinogen metabolism rates

    • Formation of DNA adducts

    • Oxidative stress markers

    • Cell viability and apoptosis

    • Mutagenesis rates

• Mass spectrometry-based metabolomics:

  • Analyze metabolite profiles in CYB5R2-expressing versus control cells after carcinogen exposure

  • Identify specific metabolic pathways affected by CYB5R2 activity

  • Track the biotransformation of carcinogens through various metabolic intermediates

• Animal models for carcinogen susceptibility:

  • Develop CYB5R2 knockout or tissue-specific conditional knockout mice

  • Expose to environmental carcinogens (e.g., benzo[a]pyrene, cigarette smoke)

  • Assess:

    • Tumor incidence and progression

    • Tissue-specific DNA damage

    • Carcinogen metabolite profiles in various tissues

    • Long-term carcinogenesis outcomes

• Human population studies:

  • Analyze CYB5R2 expression and promoter methylation status in populations with varying carcinogen exposure

  • Correlate with cancer incidence and outcomes

  • Investigate gene-environment interactions through case-control studies

These experimental approaches should be integrated to build a comprehensive understanding of CYB5R2's role in carcinogen detoxification and its potential as a biomarker for carcinogen exposure risk. Special attention should be given to nasopharyngeal carcinoma, where environmental factors (like preserved foods containing nitrosamines) interact with CYB5R2 silencing.

What are the latest methodologies for studying CYB5R2 protein-protein interactions?

To comprehensively characterize CYB5R2's interactome and understand its functional networks, researchers should employ these cutting-edge methodologies:

• Proximity-based labeling techniques:

  • BioID or TurboID: Fuse CYB5R2 with a biotin ligase to biotinylate proximal proteins

  • APEX2: Use ascorbate peroxidase fusion to label proximal proteins with biotin-phenol

  • Advantages:

    • Captures transient interactions

    • Works in native cellular environments

    • Can be targeted to specific cellular compartments

  • Follow with mass spectrometry to identify labeled proteins

• Co-immunoprecipitation with mass spectrometry (Co-IP-MS):

  • Use epitope-tagged CYB5R2 (avoiding tags that interfere with FAD binding domain)

  • Perform reciprocal Co-IPs with identified partners (CYB5A, CYB5B, etc.)

  • Quantitative approaches (SILAC, TMT labeling) can distinguish specific from non-specific interactions

• Crosslinking mass spectrometry (XL-MS):

  • Apply chemical crosslinkers to stabilize CYB5R2 protein complexes

  • Digest and analyze by MS to identify interaction interfaces

  • Particularly useful for mapping electron transfer pathways between CYB5R2 and partners

• Fluorescence-based interaction assays:

  • FRET (Förster Resonance Energy Transfer) to detect direct protein interactions

  • BiFC (Bimolecular Fluorescence Complementation) to visualize interactions in living cells

  • F2H (Fluorescent Two-Hybrid) assay to screen for novel interactors

• Structural biology approaches:

  • Cryo-EM of CYB5R2 complexes with interaction partners

  • X-ray crystallography of co-crystals with binding partners

  • NMR spectroscopy to map interaction surfaces

• Computational prediction and validation:

  • Molecular docking simulations based on protein structures

  • Network analysis using existing databases (STRING as shown in search results)

  • Machine learning approaches to predict novel interactions

Each methodology has strengths and limitations, so a multi-method approach is recommended. For CYB5R2 specifically, attention should be paid to:

• Interactions that relate to electron transfer (CYB5A, CYB5B)

• Complexes involved in fatty acid metabolism (SCD)

• Associations relevant to its tumor suppressor function (VEGF pathway components)

• Interactions that may be affected by promoter methylation or altered in cancer cells

Integration of these approaches will provide a systems-level understanding of how CYB5R2 functions within cellular networks and how its dysregulation contributes to pathological states.

How can CYB5R2 methylation status be developed as a diagnostic or prognostic biomarker?

Developing CYB5R2 methylation as a clinically useful biomarker requires systematic research and validation approaches:

• Biomarker development pipeline:

  • Discovery phase:

    • The existing data showing 84% methylation in NPC tumors and correlation with lymph node metastasis provides strong preliminary evidence

    • Expand analysis to larger, diverse patient cohorts

    • Correlate with comprehensive clinical parameters

  • Analytical validation:

    • Develop standardized methylation detection assays:

      • Quantitative MSP (qMSP)

      • Digital droplet PCR for methylation analysis

      • Next-generation sequencing-based methylation profiling

    • Establish analytical performance characteristics:

      • Sensitivity and specificity

      • Reproducibility (intra- and inter-laboratory)

      • Sample stability requirements

  • Clinical validation:

    • Prospective studies in target populations

    • Define clear clinical endpoints:

      • For diagnosis: sensitivity, specificity, positive/negative predictive values

      • For prognosis: hazard ratios for survival, time to progression

    • Determine optimal specimen types (tissue, circulating tumor DNA, etc.)

    • Establish clinically meaningful cutoff values

• Potential clinical applications:

  • Early detection of nasopharyngeal carcinoma in high-risk populations

  • Prediction of lymph node metastasis risk to guide treatment decisions

  • Monitoring of treatment response and minimal residual disease

  • Selection of patients for epigenetic therapy approaches

• Integration with other biomarkers:

  • Develop multi-marker panels including CYB5R2 methylation

  • Combine with EBV DNA detection for NPC

  • Incorporate into existing cancer biomarker algorithms

• Technical considerations:

  • Detection in minimally invasive samples (nasopharyngeal brushings, liquid biopsies)

  • Development of point-of-care testing where appropriate

  • Quality control measures for clinical implementation