ITGB1BP3 Human

What is ITGB1BP3 and what are its primary functions?

ITGB1BP3, also known as Nicotinamide riboside kinase 2, is a member of the uridine kinase family and NRK subfamily. It serves multiple critical functions in cellular metabolism and regulation. Primarily, ITGB1BP3 catalyzes the phosphorylation of nicotinic acid riboside and nicotinamide riboside to create nicotinic acid mononucleotide and nicotinamide mononucleotide, functioning as an essential enzyme in nicotinamide metabolism pathways . Additionally, ITGB1BP3 plays a significant role in cellular adhesion by reducing laminin matrix deposition and cell adhesion to laminin specifically, while not affecting adhesion to fibronectin . The protein is also involved in the regulation of paxillin (PXN) at both the protein level and in terms of tyrosine phosphorylation, suggesting its involvement in cellular signaling pathways . Furthermore, ITGB1BP3 has been implicated in the regulation of terminal myogenesis, indicating its importance in muscle cell development and differentiation .

What are the common synonyms and identifiers for ITGB1BP3?

When conducting literature searches or database queries, researchers should be aware of the various nomenclature used for this protein:

Understanding these alternative names is crucial when conducting comprehensive literature reviews, as different research groups may use various nomenclature in their publications . When searching protein and genomic databases, including all synonyms will ensure complete retrieval of relevant information and prevent overlooking important research findings.

How should I design experiments to study ITGB1BP3's role in cell adhesion?

When designing experiments to study ITGB1BP3's role in cell adhesion, follow a systematic approach that addresses both gain-of-function and loss-of-function scenarios. Begin by clearly defining your dependent and independent variables according to experimental design principles .

Step 1: Define variables

• Independent variable: ITGB1BP3 expression levels (overexpression, normal expression, knockdown)

• Dependent variable: Cell adhesion to different matrices (laminin, fibronectin, collagen)

• Control variables: Cell type, passage number, culture conditions, matrix concentration

Step 2: Develop hypotheses
Formulate specific, testable hypotheses based on known functions of ITGB1BP3:

• H1: Overexpression of ITGB1BP3 will decrease cell adhesion to laminin but not to fibronectin

• H2: Knockdown of ITGB1BP3 will increase cell adhesion to laminin but not affect adhesion to fibronectin

Step 3: Experimental treatments

• Generate stable cell lines with ITGB1BP3 overexpression using appropriate expression vectors

• Create ITGB1BP3 knockdown cell lines using siRNA or CRISPR-Cas9

• Include vector-only and scrambled siRNA controls

Step 4: Quantitative adhesion assays

• Plate cells on wells coated with different matrices (laminin, fibronectin, collagen)

• Allow adhesion for standardized time periods (30 min, 1 hour, 2 hours)

• Wash, fix, and quantify adhered cells using crystal violet staining or fluorescent labeling

• Measure adhesion strength using detachment assays with graduated shear stress

Step 5: Molecular mechanism analysis

• Assess matrix deposition using immunofluorescence

• Evaluate focal adhesion formation by staining for paxillin and phospho-paxillin

• Quantify integrin expression and activation states using flow cytometry

This comprehensive experimental design will enable thorough investigation of how ITGB1BP3 specifically affects laminin-dependent adhesion processes while controlling for other variables that might influence the results .

What are the optimal conditions for using recombinant ITGB1BP3 in in vitro studies?

When working with recombinant ITGB1BP3 protein in vitro, optimization of storage, handling, and reaction conditions is critical for maintaining enzyme activity and experimental reproducibility.

Storage and Stability Considerations:

• Store the recombinant ITGB1BP3 at 4°C if the entire vial will be used within 2-4 weeks

• For longer storage periods, keep the protein frozen at -20°C

• For long-term storage, add a carrier protein (0.1% Human Serum Albumin or Bovine Serum Albumin)

• Avoid multiple freeze-thaw cycles to prevent protein degradation and activity loss

Buffer and Reaction Conditions:
The standard formulation of recombinant ITGB1BP3 (0.25mg/ml) contains:

• 20mM Tris-HCl buffer (pH 8.0)

• 100mM Sodium Chloride

• 1mM Dithiothreitol

• 40% glycerol

For enzymatic activity assays, the following conditions are typically optimal:

• Temperature: 37°C (physiological) or 30°C (compromise between activity and stability)

• pH range: 7.5-8.5 (with optimal activity around pH 8.0)

• Required cofactors: ATP or other phosphate donors

• Divalent cations: Magnesium chloride (1-5mM)

Activity Measurement:
When assessing the kinase activity of ITGB1BP3:

• Use nicotinamide riboside or nicotinic acid riboside as substrates

• Include ATP as a phosphate donor

• Measure product formation (nicotinamide mononucleotide or nicotinic acid mononucleotide) using HPLC, mass spectrometry, or coupled enzyme assays

• Determine enzyme kinetics parameters (Km, Vmax) under varying substrate concentrations

Following these guidelines will ensure optimal activity and stability of recombinant ITGB1BP3 in your experimental protocols.

What expression systems are available for studying ITGB1BP3?

Several expression systems are available for ITGB1BP3 research, each with distinct advantages depending on your experimental goals:

Bacterial Expression Systems:
Recombinant ITGB1BP3 can be efficiently produced in E. coli with an N-terminal His-tag for purification . This system allows for:

• High protein yield

• Cost-effective production

• Simple purification via affinity chromatography

• Suitability for structural and biochemical studies

Mammalian Expression Systems:
For functional studies requiring proper post-translational modifications:

• Plasmid #51920 allows expression of full-length Integrin beta-1 precursor ectodomain in mammalian cells

• Features include His tag, enzymatic biotinylation sequence, and CMV promoter

• Stop codon before C-terminal rat Cd4d3+4 tag allows controlled expression

Selection Criteria for Expression System:

When designing expression constructs, consider including:

• Appropriate affinity tags for detection and purification

• Promoters suitable for your target cell type

• Regulatory elements for controlled expression

• Fusion partners that may enhance solubility or detection

These expression systems provide versatile tools for investigating different aspects of ITGB1BP3 biology in appropriate experimental contexts.

How is ITGB1BP3 implicated in cancer chemoresistance?

ITGB1BP3 has emerged as a significant factor in cancer chemoresistance, particularly in ovarian cancer. Microarray analysis has identified ITGB1BP3 among 10 genes that were significantly upregulated in chemo-resistant ovarian cancer sublines compared to chemosensitive cells . This finding suggests that ITGB1BP3 may play a role in modulating cancer cell response to chemotherapeutic agents.

Mechanisms of ITGB1BP3-Mediated Chemoresistance:

Several potential mechanisms may explain how ITGB1BP3 contributes to chemoresistance:

• Extracellular Matrix Remodeling: As ITGB1BP3 reduces laminin matrix deposition and affects cell adhesion , it may alter the tumor microenvironment in ways that protect cancer cells from drug exposure or efficacy.

• Cell Signaling Modulation: ITGB1BP3's involvement in the regulation of paxillin (PXN) suggests it may influence signaling pathways that promote cell survival under chemotherapeutic stress.

• Metabolic Reprogramming: As a nicotinamide riboside kinase, ITGB1BP3 participates in NAD+ metabolism , which is critical for cellular energetics and DNA repair mechanisms that can contribute to drug resistance.

Research Approaches to Study ITGB1BP3 in Chemoresistance:

To investigate ITGB1BP3's role in chemoresistance, researchers should consider:

• Gene expression profiling of matched sensitive/resistant cell lines

• CRISPR-Cas9 knockout or knockdown of ITGB1BP3 followed by drug sensitivity testing

• Overexpression studies to determine if ITGB1BP3 alone can confer resistance

• Pathway analysis to identify interaction partners in resistance mechanisms

• In vivo xenograft studies comparing tumors with varying ITGB1BP3 expression levels

This research direction may yield valuable insights for developing strategies to overcome chemoresistance in ovarian cancer and potentially other malignancies where ITGB1BP3 is dysregulated.

What are the relationships between ITGB1BP3 and other ECM-associated proteins?

ITGB1BP3 functions within a complex network of extracellular matrix (ECM) proteins and signaling molecules. Understanding these relationships provides insight into its broader biological roles and potential therapeutic targeting.

ITGB1BP3 and Collagen Interactions:
Interestingly, ITGB1BP3 is upregulated alongside several collagen genes (COL3A1, COL5A2, COL15A1) in chemoresistant ovarian cancer cells . This co-regulation pattern suggests a coordinated role in ECM remodeling that may contribute to drug resistance and altered cell behavior. The relationship may involve:

• Direct or indirect interaction between ITGB1BP3 and collagen molecules

• Co-regulation through shared transcriptional control mechanisms

• Functional cooperation in modifying cellular adhesion properties

ITGB1BP3 and Laminin Pathway:
ITGB1BP3 has been specifically shown to reduce laminin matrix deposition and cell adhesion to laminin, but not to fibronectin . This selective effect suggests a specific regulatory role in laminin-dependent processes. Laminin subunit alpha 1 (LAMA2) is downregulated in chemoresistant cells , possibly indicating a compensatory relationship with ITGB1BP3 upregulation.

ITGB1BP3 and Integrin Signaling:
Despite its name indicating a relationship with integrin beta 1, the specific molecular interactions between ITGB1BP3 and integrins require further investigation. The downregulation of integrin subunit alpha 1 (ITGA1) in chemoresistant cells alongside ITGB1BP3 upregulation suggests a potential regulatory relationship that could influence integrin-mediated adhesion and signaling.

Interaction with Growth Factor Pathways:
ITGB1BP3 may intersect with growth factor signaling networks:

• Transforming growth factor beta induced (TGFBI) is upregulated alongside ITGB1BP3 in chemoresistant cells

• This co-regulation suggests potential interaction between ITGB1BP3 and TGF-β pathway components

• Such interactions could influence cell differentiation, proliferation, and response to therapy

These relationships position ITGB1BP3 at an important intersection of ECM organization, cellular adhesion, and signal transduction pathways relevant to both normal physiology and disease states.

How can ITGB1BP3 genetic variants be analyzed in disease association studies?

Genetic variation in ITGB1BP3 may contribute to disease susceptibility or treatment response. To effectively analyze ITGB1BP3 genetic variants in disease association studies, researchers should implement a systematic approach:

Single Nucleotide Polymorphism (SNP) Identification Strategy:

• Comprehensive SNP Profiling:

  • Utilize next-generation sequencing techniques to identify both common and rare variants

  • Include promoter regions, exons, introns, and regulatory elements

  • Cross-reference findings with existing databases like dbSNP and gnomAD

• Functional Classification of Variants:

  • Coding variants: missense, nonsense, frameshift

  • Regulatory variants: promoter, enhancer, silencer regions

  • Splice site variants: potential impact on mRNA processing

  • Structural variants: insertions, deletions, copy number variations

Study Design Considerations:

When designing genetic association studies for ITGB1BP3:

• Population Selection:

  • Define appropriate case and control groups with careful phenotyping

  • Consider ancestry-specific variations and population stratification

  • Calculate required sample size based on expected effect sizes

• Analytical Approaches:

  • Perform both single-variant and haplotype-based analyses

  • Consider gene-environment interactions, particularly for treatment response

  • Implement appropriate statistical corrections for multiple testing

Example Application: Treatment Response Association:

The methodology used in statin response association studies provides a model for ITGB1BP3 variant analysis:

• Identify candidate SNPs in ITGB1BP3 that might affect enzyme function or expression

• Genotype these variants in well-characterized patient cohorts

• Analyze associations between variants and clinical outcomes or biomarkers

• Conduct functional validation of significant associations

This approach could be particularly valuable for investigating ITGB1BP3's role in cancer chemoresistance , where genetic variations might predict treatment response or suggest personalized therapeutic strategies.

How can I resolve inconsistent results in ITGB1BP3 expression studies?

Inconsistent results in ITGB1BP3 expression studies can arise from various methodological factors. Addressing these systematically can improve reproducibility and reliability of your findings.

Common Sources of Variability and Solutions:

1. Cell Culture Conditions:

• Problem: Variability in cell density, passage number, or growth phase

• Solution: Standardize seeding density, use cells within a defined passage range, and harvest at consistent confluence levels (70-80% recommended)

2. Reagent and Sample Quality:

• Problem: Degraded RNA or protein samples leading to inconsistent detection

• Solution: Implement rigorous quality control measures:

  • For RNA: Verify RIN values >8 before RT-PCR

  • For protein: Confirm integrity via Coomassie staining before Western blot

  • Store recombinant ITGB1BP3 at 4°C if using within 2-4 weeks, or at -20°C with carrier protein (0.1% HSA or BSA) for longer storage

3. Detection Method Limitations:

• Problem: Different antibodies or primer sets give conflicting results

• Solution: Validate reagents using:

  • Multiple antibody clones targeting different epitopes

  • Alternative primer pairs spanning different exon junctions

  • Positive and negative control samples with known ITGB1BP3 expression

4. Reference Gene Selection:

• Problem: Unstable reference genes leading to normalization errors

• Solution: Validate multiple reference genes for your specific experimental system using algorithms like geNorm or NormFinder

Systematic Troubleshooting Approach:

By systematically addressing these potential sources of variability, researchers can significantly improve the consistency and reliability of ITGB1BP3 expression data across experiments and between laboratories.

What experimental controls are essential when studying ITGB1BP3's effect on cellular processes?

Essential Experimental Controls:

1. Expression Controls:

• Positive Control: Cell line known to express high levels of ITGB1BP3 (e.g., certain muscle cells given its role in myogenesis )

• Negative Control: Cell line with minimal ITGB1BP3 expression or CRISPR-edited knockout

• Verification Method: Western blot or qRT-PCR to confirm expression levels in all experimental groups

2. Manipulation Controls:

For overexpression studies:

• Empty Vector Control: Cells transfected with the same vector lacking the ITGB1BP3 insert

• Irrelevant Protein Control: Overexpression of an unrelated protein of similar size

• Expression Level Verification: Titration of expression construct to achieve physiologically relevant levels

For knockdown/knockout studies:

• Scrambled siRNA/shRNA: Non-targeting sequence with similar chemical properties

• Rescue Control: Re-expression of ITGB1BP3 in knockout cells to confirm specificity

• Off-target Effect Assessment: Evaluation of multiple siRNA sequences targeting different regions

3. Functional Assay Controls:

For cell adhesion studies:

• Substrate Controls: Compare adhesion to laminin (affected by ITGB1BP3 ) vs. fibronectin (unaffected by ITGB1BP3 )

• Time Course Controls: Multiple time points to distinguish adhesion vs. proliferation effects

• Cell Viability Control: Ensure manipulations don't affect cell survival

For kinase activity studies:

• Enzyme-free Control: Reaction mixture without ITGB1BP3 to establish baseline

• Heat-inactivated Enzyme Control: Denatured ITGB1BP3 to control for non-enzymatic effects

• Substrate Specificity Control: Test multiple potential substrates including non-physiological candidates

4. Experimental Design Controls:

• Biological Replicates: Independent experiments from different cell preparations

• Technical Replicates: Multiple measurements from the same biological sample

• Blinding: Researcher analyzing results should be unaware of sample identity

Implementing these controls according to established experimental design principles will significantly enhance the reliability and interpretability of ITGB1BP3 research findings.

How might ITGB1BP3 contribute to metabolic regulation in disease contexts?

ITGB1BP3, functioning as Nicotinamide riboside kinase 2, may play significant roles in metabolic regulation that extend beyond its better-characterized functions in cell adhesion and myogenesis. Understanding these metabolic connections could reveal new therapeutic targets for various diseases.

NAD+ Metabolism and Energy Homeostasis:

As ITGB1BP3 catalyzes the phosphorylation of nicotinamide riboside to nicotinamide mononucleotide , it directly contributes to NAD+ biosynthesis. This positions ITGB1BP3 as a potential regulator of:

• Cellular Energetics: NAD+ is essential for mitochondrial function and ATP production

• Sirtuin Activity: NAD+-dependent deacetylases regulate numerous metabolic processes

• PARP Function: NAD+-consuming enzymes involved in DNA repair and cellular stress responses

These pathways are particularly relevant in diseases characterized by metabolic dysregulation and mitochondrial dysfunction.

Potential Disease Connections:

Several disease contexts where ITGB1BP3's metabolic functions might be significant include:

• Cancer Metabolism:

  • The upregulation of ITGB1BP3 in chemoresistant ovarian cancer may reflect metabolic adaptation

  • Cancer cells often exhibit altered NAD+ metabolism to support rapid proliferation

  • Targeting ITGB1BP3 might sensitize resistant tumors by disrupting metabolic adaptations

• Cardiovascular Disorders:

  • NAD+ precursors have shown cardioprotective effects in various models

  • ITGB1BP3 genetic variants might influence cardiovascular risk or treatment response

  • The protein's dual roles in metabolism and adhesion could affect vascular integrity

• Neurodegenerative Diseases:

  • NAD+ depletion is implicated in neurodegeneration

  • ITGB1BP3 activity might influence neuronal energy homeostasis

  • The protein's effect on ECM interactions could also impact neuronal connectivity

Research Approaches:

To investigate ITGB1BP3's metabolic roles, researchers should consider:

• Metabolomic profiling of cells with modified ITGB1BP3 expression

• Assessment of NAD+ levels and consumption rates in disease models

• Analysis of mitochondrial function in response to ITGB1BP3 manipulation

• Investigation of interactions between ITGB1BP3 and other metabolic regulators

This emerging research direction could significantly expand our understanding of ITGB1BP3's biological significance beyond its currently established functions.

What techniques are most effective for identifying ITGB1BP3 interaction partners?

Identifying protein interaction partners is crucial for understanding ITGB1BP3's cellular functions. Multiple complementary approaches can be employed to comprehensively map its interactome.

Affinity-Based Approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use validated antibodies against endogenous ITGB1BP3 or epitope-tagged recombinant protein

  • Perform under various cellular conditions (growth factors, stress, differentiation)

  • Identify binding partners using mass spectrometry

  • Validate key interactions with reverse Co-IP and Western blotting

• Proximity Labeling Techniques:

  • BioID: Fusion of ITGB1BP3 with a biotin ligase to biotinylate proximal proteins

  • APEX2: Peroxidase-based labeling of neighboring proteins

  • Advantages: Captures transient interactions and works in native cellular compartments

  • Combine with quantitative proteomics for interaction strength assessment

Genetic and Functional Approaches:

• Yeast Two-Hybrid Screening:

  • Use ITGB1BP3 domains as bait to screen cDNA libraries

  • Particularly useful for identifying direct binary interactions

  • Follow with mammalian verification systems (mammalian two-hybrid)

• Genetic Interaction Mapping:

  • CRISPR-based screens to identify synthetic lethal or rescue interactions

  • Correlation of gene expression patterns across tissues and conditions

  • Analysis of co-regulated genes from transcriptomic studies

Structural and Biophysical Methods:

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Quantify binding kinetics using recombinant ITGB1BP3

  • Determine affinity constants for potential interaction partners

  • Map binding interfaces with mutational analysis

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Identify regions of ITGB1BP3 that undergo conformational changes upon binding

  • Map interaction surfaces with high resolution

Computational Prediction and Analysis:

• Protein-Protein Interaction Prediction:

  • Structure-based docking simulations

  • Co-expression network analysis across tissues and conditions

  • Evolutionary analysis of co-conserved protein domains

• Interactome Visualization and Analysis:

  • Network analysis to identify key nodes and potential functional clusters

  • Pathway enrichment to contextualize interaction partners

Integrating multiple complementary approaches provides the most comprehensive and reliable identification of physiologically relevant ITGB1BP3 interaction partners, forming the foundation for mechanistic studies of its cellular functions.

How might ITGB1BP3 be targeted for therapeutic development?

Given ITGB1BP3's multiple biological functions and disease associations, several therapeutic approaches targeting this protein could be developed. Strategic intervention points exist at the level of expression, enzymatic activity, and protein-protein interactions.

Small Molecule Inhibitors:

The nicotinamide riboside kinase activity of ITGB1BP3 provides a clear target for small molecule inhibition:

• Competitive Inhibitors:

  • Design compounds that compete with nicotinamide riboside for the substrate binding site

  • Structure-based drug design using recombinant ITGB1BP3

  • High-throughput screening of chemical libraries against purified protein

• Allosteric Inhibitors:

  • Target regulatory sites that influence enzymatic activity

  • May offer greater selectivity between ITGB1BP3 (NRK2) and related kinases

  • Identify potential binding pockets through molecular dynamics simulations

Biologics and Peptide-Based Approaches:

• Blocking Peptides:

  • Design peptides that interfere with ITGB1BP3's interaction with laminin or other binding partners

  • Target specific protein-protein interaction domains

  • Conjugate to cell-penetrating sequences for intracellular delivery

• Monoclonal Antibodies:

  • Develop antibodies that neutralize extracellular functions

  • Potential for antibody-drug conjugates in cancer contexts where ITGB1BP3 is overexpressed

Genetic and RNA-Based Approaches:

• RNAi Therapeutics:

  • siRNA or shRNA delivery systems targeting ITGB1BP3 mRNA

  • Particularly relevant for cancer applications where ITGB1BP3 is upregulated

  • Requires effective delivery systems to target tissues of interest

• CRISPR-Based Therapeutics:

  • Gene editing to correct disease-associated variants

  • Targeted epigenetic modulation to alter expression levels

Potential Clinical Applications:

Biomarker Applications:

Beyond direct targeting, ITGB1BP3 may serve as a biomarker for:

• Predicting chemotherapy response in cancer patients

• Identifying patients who might benefit from specific therapeutic interventions

• Monitoring treatment efficacy through expression level changes

These diverse approaches to ITGB1BP3-directed therapeutics reflect the protein's multifaceted biological roles and disease associations, offering numerous avenues for translational research and drug development.

What evidence supports ITGB1BP3 as a biomarker in disease diagnosis or prognosis?

The potential of ITGB1BP3 as a biomarker is emerging through evidence from various disease contexts, particularly in cancer and cellular response pathways. Understanding this evidence is crucial for evaluating its clinical utility.

Cancer Biomarker Potential:

The upregulation of ITGB1BP3 in chemoresistant ovarian cancer cells represents the strongest evidence for its biomarker potential . This finding suggests several clinical applications:

• Prediction of Chemotherapy Response:

  • ITGB1BP3 expression levels might predict patient response to standard chemotherapy regimens

  • Could guide first-line treatment selection in ovarian cancer patients

  • May identify patients who would benefit from alternative treatment approaches

• Disease Monitoring:

  • Changes in ITGB1BP3 expression during treatment could indicate developing resistance

  • Potential liquid biopsy target if shed into circulation or detectable in extracellular vesicles

• Risk Stratification:

  • Expression patterns might correlate with disease aggressiveness or recurrence risk

  • Could be incorporated into multi-gene prognostic panels

Genetic Variation as Predictive Biomarkers:

Similar to approaches used in statin response studies , genetic variants in ITGB1BP3 could serve as predictive biomarkers:

• Pharmacogenomic Applications:

  • Specific polymorphisms might predict response to therapies affecting pathways where ITGB1BP3 functions

  • Could guide personalized treatment selection and dosing

  • Particularly relevant for metabolic and cardiovascular interventions

• Disease Risk Assessment:

  • Variants might contribute to genetic risk scores for conditions involving ECM dysregulation

  • Potential relevance to muscular disorders given role in myogenesis

Methodological Considerations for Biomarker Validation:

For ITGB1BP3 to transition from potential to validated biomarker, researchers should:

• Establish Analytical Validity:

  • Develop standardized assays for ITGB1BP3 detection in clinical specimens

  • Determine appropriate cutoff values for different clinical applications

  • Ensure reproducibility across laboratories and platforms

• Demonstrate Clinical Validity:

  • Conduct prospective studies in well-defined patient cohorts

  • Calculate sensitivity, specificity, and predictive values for specific clinical outcomes

  • Compare performance to existing biomarkers and standards of care

• Assess Clinical Utility:

  • Determine if ITGB1BP3 testing improves patient outcomes

  • Evaluate cost-effectiveness and implementation feasibility

  • Consider combination with other biomarkers for improved performance

While current evidence suggests promising biomarker potential, particularly in cancer contexts, comprehensive clinical validation studies are needed to establish ITGB1BP3's definitive value in disease diagnosis, prognosis, or treatment selection.