LRRC31 Antibody

What is LRRC31 and what are its primary biological functions?

LRRC31 (Leucine-Rich Repeat-Containing Protein 31) is a protein with multiple leucine-rich repeat (LRR) domains that functions primarily at the protein level rather than the transcriptional level. It has two major identified biological functions. First, LRRC31 acts as a DNA repair suppressor by interacting with key repair proteins, including Ku70/Ku80 and ATR, resulting in inhibition of DNA-PKcs recruitment and activation, and disruption of the MSH2-ATR signaling module . Second, LRRC31 modulates epithelial barrier function, potentially through regulation of kallikrein (KLK) serine proteases . The protein shows basal expression in colonic and airway mucosal epithelium, but its expression can be significantly upregulated in certain disease states or in response to specific cytokines such as IL-13 .

Where is LRRC31 expressed in normal tissues?

LRRC31 exhibits basal mRNA expression in colonic and airway mucosal epithelium under normal conditions . Subcellular localization studies using confocal microscopy have revealed that LRRC31 is predominantly located in the nucleus, similar to ATR, suggesting its role in nuclear processes such as DNA repair regulation . Analysis of the TCGA database using Gene Expression Profiling Interactive Analysis (GEPIA) indicates that LRRC31 is expressed in most tumor types, with prostate adenocarcinoma (PRAD) showing the highest expression levels .

How does LRRC31 expression correlate with disease states?

Research has demonstrated significant correlations between LRRC31 expression and certain disease states. In eosinophilic esophagitis (EoE), LRRC31 mRNA expression increases dramatically (up to 137-fold) in patients with active disease compared to normal controls . This increased expression strongly correlates with esophageal eosinophilia (Pearson r = 0.60, P < 0.01) and with esophageal IL13 mRNA expression (Pearson r = 0.60, P < 0.0001) . In cancer research, analysis of TCGA data shows that patients with higher LRRC31 expression tend to have better survival outcomes, particularly in prostate adenocarcinoma, suggesting its potential tumor suppressor role .

How does LRRC31 regulate DNA double-strand break repair?

LRRC31 specifically inhibits DNA double-strand break (DSB) repair primarily through disruption of the non-homologous end joining (NHEJ) pathway. Mechanistically, LRRC31 interacts with Ku70/Ku80 heterodimers at the protein level but does not affect their binding to each other. Instead, it prevents the recruitment of DNA-PKcs to the Ku70/Ku80 complex at DSB sites . This inhibition disrupts the formation of the functional DNA-PK holoenzyme required for NHEJ-mediated repair. Studies using pEJ5-GFP (NHEJ) and DR-GFP (homologous recombination) reporter systems demonstrated that LRRC31 overexpression reduced NHEJ-mediated DSB repair by 52% and 40% in conditions without and with irradiation, respectively, while showing limited inhibitory effects on the homologous recombination pathway .

What experimental methods are optimal for studying LRRC31's effects on DNA repair?

To effectively study LRRC31's effects on DNA repair, researchers should employ multiple complementary approaches:

• DNA Damage Detection Assays: Neutral comet assay for detecting both single-strand breaks and double-strand breaks at the individual cell level. LRRC31-overexpressing cells show significantly extended comet tails (3.1 times greater average tail moment) compared to control cells following irradiation .

• γ-H2AX Immunofluorescence: Tracking formation and resolution of γ-H2AX foci over time (30 minutes to 48 hours post-irradiation) to assess DSB repair efficiency. LRRC31-overexpressing cells retain significantly higher levels of γ-H2AX foci, indicating impaired repair capacity .

• Repair Pathway Reporter Assays: Using pEJ5-GFP and DR-GFP reporters to quantify NHEJ and HR pathway activities, respectively, in cells with varied LRRC31 expression .

• Biochemical Fractionation: Isolating whole cell extracts (WCE) and chromatin binding proteins (CBP) to assess protein recruitment to DSB sites following irradiation .

• Immunoprecipitation-Western Blot (IP-WB): Detecting protein-protein interactions (e.g., LRRC31 with Ku70/Ku80) and complex formation or disruption .

How can LRRC31 be manipulated to sensitize cancer cells to radiation therapy?

LRRC31 manipulation presents a promising approach for sensitizing cancer cells, particularly breast cancer brain metastases (BCBMs), to radiation therapy. Experimental evidence demonstrates that overexpression of LRRC31 significantly increases radiation sensitivity with a dose enhancement factor (DEF) of 1.7 at surviving fraction 0.4 . This radiosensitizing effect has been validated in multiple breast cancer cell lines, including 231BR, MCF7, and 4T1-BR5.

For effective manipulation in research settings, the following methods have demonstrated success:

• Gene Overexpression Systems: Stable transfection of LRRC31 expression constructs in cancer cell lines has shown consistent radiosensitizing effects across multiple models .

• Nanoparticle-Mediated Gene Delivery: Targeted delivery of LRRC31 gene via nanoparticles has demonstrated efficacy in vivo, significantly improving the survival of tumor-bearing mice after irradiation .

• Combination with DNA-PK Inhibitors: Since LRRC31 inhibits DNA-PKcs recruitment and activation, combining LRRC31 overexpression with DNA-PK inhibitors such as NU7441 may provide synergistic effects .

The radiosensitizing mechanism involves both enhanced DNA damage accumulation and increased apoptosis. LRRC31 overexpression promotes small but statistically significant increases in G2-M phase cell populations and enhances cellular apoptosis following irradiation .

How does IL-13 regulate LRRC31 expression in epithelial cells?

IL-13 significantly upregulates LRRC31 expression in epithelial cells, as demonstrated in multiple experimental systems. In air-liquid interface (ALI) differentiated esophageal epithelial cells (EPC2s), IL-13 treatment increased LRRC31 mRNA by 18-fold (P < 0.01) compared to untreated controls . At the protein level, IL-13 stimulation resulted in a 14-fold increase in LRRC31 expression relative to HSP90 loading control .

This induction appears to be part of a broader IL-13-mediated gene expression program in epithelial cells. In EPC2 cells engineered to overexpress LRRC31, IL-13 treatment further amplified LRRC31 expression, indicating a potential positive feedback loop. Importantly, IL-13-induced CCL26 (eotaxin-3, an eosinophil chemoattractant) expression was also enhanced in LRRC31-overexpressing cells compared to controls, suggesting that LRRC31 may modulate other IL-13-responsive genes .

What is the relationship between LRRC31 and epithelial barrier function?

LRRC31 plays a significant role in enhancing epithelial barrier function. Differentiated EPC2 cells overexpressing LRRC31 demonstrate a 1.9-fold increase in transepithelial electrical resistance (TEER, P < 0.05) and a 2.8-fold decrease in paracellular flux (P < 0.05) compared to control cells . These parameters are standard measures of barrier integrity in epithelial models.

The molecular mechanism underlying this effect appears to involve regulation of kallikrein (KLK) serine proteases. RNA sequencing analysis of differentiated LRRC31-overexpressing EPC2 cells identified 38 dysregulated genes (P < 0.05), including 5 kallikrein serine proteases . LRRC31 overexpression decreased KLK expression and activity, while IL-13 treatment, which induces LRRC31, also affected KLK expression patterns .

This relationship suggests a potential protective role for LRRC31 in maintaining epithelial barrier integrity, particularly in inflammatory conditions where IL-13 levels are elevated, such as eosinophilic esophagitis.

What experimental models are best suited for studying LRRC31's role in barrier function?

To effectively study LRRC31's role in epithelial barrier function, the following experimental models and approaches are recommended:

• Air-Liquid Interface (ALI) Culture Systems: ALI-differentiated esophageal epithelial cells (EPC2s) have proven effective for studying LRRC31's impact on barrier function. These cultures recapitulate the polarized epithelium and allow for manipulation of LRRC31 expression via stable transfection or induction with IL-13 .

• Barrier Function Measurements:

  • Transepithelial electrical resistance (TEER) measurements to assess tight junction integrity

  • Paracellular flux assays using fluorescent markers to measure barrier permeability

  • Both parameters should be measured in LRRC31-overexpressing and control cells, with and without IL-13 stimulation

• Kallikrein Activity Assays: Since LRRC31 appears to regulate kallikrein expression and activity, incorporating assays to measure KLK activity provides insight into the molecular mechanisms of LRRC31's barrier-enhancing effects .

• RNA Sequencing: For comprehensive assessment of gene expression changes induced by LRRC31 overexpression, RNA-seq has successfully identified dysregulated genes, including multiple KLK family members .

• Patient-Derived Samples: For clinical relevance, correlating LRRC31 expression in patient biopsies with disease parameters (e.g., eosinophil counts, IL-13 levels) can validate findings from in vitro models .

What are the critical considerations for optimizing LRRC31 antibody-based detection in different experimental contexts?

When optimizing LRRC31 antibody-based detection across various experimental contexts, researchers should consider these critical factors:

• Antibody Validation: Given LRRC31's varied expression levels across tissues, comprehensive validation is essential. Western blot analysis can confirm specificity, as demonstrated in studies where LRRC31 protein was readily detectable in active EoE patient esophageal tissue with a 6-fold increase compared to normal tissue . For cancer research applications, validation in breast cancer cell lines with manipulated LRRC31 expression is recommended .

• Subcellular Localization: Confocal microscopy analysis has shown that LRRC31 is primarily localized in the nucleus, similar to ATR . Therefore, nuclear extraction protocols may be necessary for efficient detection in some applications.

• Protein Interaction Studies: For co-immunoprecipitation experiments studying LRRC31's interactions with DNA repair proteins (Ku70/Ku80, ATR) or other binding partners, optimizing lysis conditions is crucial. Successful IP-WB protocols have been established for detecting LRRC31's interactions with these proteins .

• Cross-Reactivity Assessment: When studying LRRC31 in multiple species (human vs. mouse models), antibody cross-reactivity should be carefully evaluated, as expression patterns may differ between species.

• Fixation Methods: For immunohistochemistry or immunofluorescence applications, appropriate fixation methods must be optimized to preserve LRRC31 epitopes while maintaining cellular architecture.

How can researchers effectively study LRRC31 protein-protein interactions?

To effectively study LRRC31 protein-protein interactions, researchers should implement the following methodological approaches:

• Co-Immunoprecipitation (Co-IP) with Western Blot Analysis: This approach has successfully identified LRRC31's interactions with key DNA repair proteins. Specifically:

  • Transfect cells with constructs expressing tagged versions of LRRC31 (e.g., Myc-tagged) and potential interacting partners (e.g., Flag-tagged)

  • Immunoprecipitate with anti-Myc antibody

  • Analyze lysates by Western blot using anti-Flag antibody to detect interactions

• Mass Spectrometry-Based Interactome Analysis:

  • Immunoprecipitate LRRC31 from cell lysates

  • Separate co-IP proteins using SDS-PAGE

  • Recover protein bands and subject them to mass spectrometry for identification of novel interacting partners

• Proximity Ligation Assays (PLA): For detecting protein-protein interactions in situ with high specificity and sensitivity, particularly useful for visualizing where in the cell these interactions occur.

• Chromatin Fractionation Assays: For studying interactions at DNA damage sites:

  • Prepare whole cell extracts (WCE) and chromatin binding protein (CBP) fractions after inducing DNA damage

  • Analyze LRRC31 and interacting partners in these fractions by Western blot

• Functional Validation of Interactions: Beyond identifying interactions, validating their functional significance is critical:

  • Assess if LRRC31 alters the activity of interacting proteins (e.g., inhibition of DNA-PKcs phosphorylation at Serine 2056)

  • Use structure-function analysis with LRRC31 domain mutants to map interaction regions

What technical challenges exist in detecting low levels of endogenous LRRC31 expression?

Detecting low levels of endogenous LRRC31 expression presents several technical challenges that researchers should address through specific methodological approaches:

• Tissue-Specific Expression Variability: LRRC31 shows variable basal expression across tissues, with detectable levels in colonic and airway mucosal epithelium but potentially lower expression in other tissues . This necessitates:

  • Establishing tissue-specific baseline expression levels

  • Using positive control samples (e.g., IL-13-stimulated epithelial cells) alongside experimental samples

• Signal Amplification Strategies: For immunohistochemistry or Western blot applications with low abundance proteins:

  • Consider tyramide signal amplification (TSA) methods

  • Use high-sensitivity chemiluminescence substrates for Western blot

  • Optimize protein loading quantities (may require larger amounts of starting material)

• RNA Detection Alternatives: When protein detection is challenging, qPCR provides a sensitive alternative:

  • Studies have successfully detected LRRC31 mRNA expression in normal tissue samples and demonstrated dramatic upregulation (137-fold) in disease states

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Consider droplet digital PCR for absolute quantification of low-abundance transcripts

• Enrichment Techniques: For protein detection:

  • Employ subcellular fractionation to concentrate LRRC31 in nuclear extracts

  • Consider immunoprecipitation prior to Western blot to enrich target protein

• Antibody Selection and Validation: Critical for low-abundance proteins:

  • Validate antibody specificity using overexpression and knockout controls

  • Compare multiple antibodies targeting different epitopes

  • Optimize antibody concentration and incubation conditions

How can researchers address experimental variability in LRRC31 functional assays?

Addressing experimental variability in LRRC31 functional assays requires systematic control measures and standardized protocols:

• DNA Repair Assays Standardization:

  • For neutral comet assays measuring DNA damage, establish consistent electrophoresis conditions and objective quantification methods for comet tail moment analysis

  • When assessing γ-H2AX foci formation and resolution, implement automated imaging and analysis platforms to quantify foci numbers across multiple time points

  • For reporter assays measuring NHEJ and HR pathway activities, normalize to transfection efficiency controls and include positive control treatments (e.g., DNA-PK inhibitors like NU7441)

• Cell Type Considerations:

  • Account for intrinsic differences in DNA repair capacity between cell lines

  • Establish baseline repair kinetics for each model system

  • When comparing different cell lines (e.g., 231BR, MCF7, 4T1-BR5), standardize radiation doses and assessment timepoints

• Epithelial Barrier Function Assays:

  • For transepithelial electrical resistance (TEER) measurements, ensure consistent electrode placement and temperature control

  • In paracellular flux assays, standardize molecular tracer concentration and exposure time

  • Allow complete epithelial differentiation in ALI cultures (typically 7-14 days) before functional testing

• Expression Level Control:

  • Validate LRRC31 expression levels by both qPCR and Western blot

  • Use inducible expression systems to achieve consistent expression across experiments

  • Include multiple LRRC31-expressing clones to account for clonal variation effects

• Statistical Approach:

  • Implement robust statistical methods appropriate for the specific assay

  • Increase biological replicates (n≥3) to account for intrinsic variability

  • Report effect sizes and confidence intervals alongside p-values

What are the most effective controls for validating LRRC31 antibody specificity?

To rigorously validate LRRC31 antibody specificity, researchers should implement the following control strategies:

• Genetic Manipulation Controls:

  • LRRC31 Overexpression: Use cells with confirmed LRRC31 overexpression as positive controls. Studies have successfully employed this approach, demonstrating increased LRRC31 signal by Western blot in overexpressing cells .

  • LRRC31 Knockdown: CRISPR-Cas9 with specific sgRNAs targeting LRRC31 (e.g., sgLRRC-2) has been shown to effectively reduce LRRC31 expression and can serve as negative controls .

  • Dose-dependent expression: When possible, use systems with varying LRRC31 expression levels to confirm signal proportionality.

• Peptide Competition Assays:

  • Pre-incubate the LRRC31 antibody with excess purified LRRC31 peptide (corresponding to the epitope)

  • This should abolish or significantly reduce specific binding in Western blot or immunohistochemistry applications

• Cross-Validation with Multiple Antibodies:

  • Compare results using antibodies targeting different LRRC31 epitopes

  • Consistent detection patterns across different antibodies strengthen specificity confidence

• Physiological Induction Controls:

  • Use IL-13 treatment of epithelial cells as a positive control for LRRC31 induction

  • This approach has consistently demonstrated significant upregulation of both LRRC31 mRNA (18-fold) and protein (14-fold)

• Tissue Specificity Controls:

  • Include tissues with known LRRC31 expression (colonic and airway mucosal epithelium) as positive controls

  • Include tissues known to have minimal expression as negative controls

  • Use patient samples with active EoE as high-expression positive controls

How should researchers interpret conflicting results between LRRC31 mRNA and protein expression data?

When researchers encounter discrepancies between LRRC31 mRNA and protein expression data, systematic analytical approaches can help resolve these conflicts:

• Temporal Dynamics Assessment:

  • Consider time-course experiments to track both mRNA and protein expression following stimulation (e.g., IL-13 treatment)

  • LRRC31 mRNA increases may precede detectable protein changes

  • Studies have shown that while IL-13 increases LRRC31 mRNA by 18-fold, protein increases (14-fold) may follow different kinetics

• Post-Transcriptional Regulation Analysis:

  • Investigate microRNA-mediated regulation of LRRC31 mRNA

  • Assess mRNA stability through actinomycin D chase experiments

  • Consider polysome profiling to determine translation efficiency

• Protein Stability Considerations:

  • Evaluate LRRC31 protein half-life through cycloheximide chase experiments

  • Investigate potential post-translational modifications affecting detection

  • Assess proteasomal degradation pathways using inhibitors like MG132

• Detection Method Limitations:

  • For Western blot analysis, ensure extraction methods efficiently recover nuclear proteins, as LRRC31 is predominantly nuclear

  • Consider native versus denaturing conditions for protein detection

  • Evaluate antibody sensitivity thresholds compared to qPCR detection limits

• Biological Context Interpretation:

  • In research settings, whole-transcript expression analysis has shown that LRRC31 functions primarily at the protein rather than transcriptional level

  • In clinical samples, LRRC31 mRNA strongly correlates with protein expression (as demonstrated in EoE studies) , but this relationship may vary by tissue context

  • Consider cell-type specific factors that may influence the mRNA-protein correlation

What are the most promising therapeutic applications for LRRC31 manipulation in cancer treatment?

Based on current research findings, LRRC31 manipulation offers several promising therapeutic applications in cancer treatment:

• Radiation Sensitization for Brain Metastases:

  • LRRC31 overexpression sensitizes breast cancer brain metastases (BCBMs) to radiation therapy with a dose enhancement factor of 1.7

  • This effect has been validated across multiple breast cancer cell lines (231BR, MCF7, 4T1-BR5)

  • Targeted delivery of LRRC31 gene via nanoparticles significantly improved survival in mouse models after irradiation

  • This approach could potentially allow for dose reduction in radiation therapy, minimizing toxicity to normal brain tissue

• Combination Therapy Strategies:

  • Since LRRC31 inhibits both the NHEJ pathway and disrupts the ATR-MSH2 module, combining LRRC31 overexpression with:

    • PARP inhibitors could create synthetic lethality through simultaneous targeting of multiple DNA repair pathways

    • ATR inhibitors might provide synergistic effects through comprehensive disruption of ATR signaling

    • Traditional DNA-damaging chemotherapeutics could enhance therapeutic efficacy

• Tumor Suppression Activities:

  • Beyond radiation sensitization, LRRC31 demonstrates intrinsic tumor suppressor properties:

    • LRRC31 inhibits cell proliferation in vitro and tumor development in vivo

    • It regulates both cell cycle (increased G2-M phase) and apoptosis

    • TCGA database analysis shows patients with higher LRRC31 expression have better survival outcomes

  • These properties suggest potential for LRRC31 as a therapeutic gene in cancer treatment strategies

• Delivery System Development:

  • Ongoing research should focus on optimizing delivery systems:

    • Nanoparticle formulations for targeted LRRC31 gene delivery to tumors

    • Viral vector approaches for stable expression

    • Cell-penetrating peptide conjugates for LRRC31 protein delivery

How might LRRC31 function in other inflammatory or barrier dysfunction diseases beyond EoE?

Given LRRC31's established roles in epithelial barrier function and inflammatory disease, its potential functions in other conditions deserve investigation:

• Inflammatory Bowel Disease (IBD):

  • LRRC31 shows basal expression in colonic epithelium , suggesting potential relevance in IBD

  • Its IL-13-responsive nature may be significant in ulcerative colitis, where IL-13 plays a pathogenic role

  • LRRC31's enhancement of barrier function may represent a protective mechanism in intestinal inflammation

  • Research should investigate LRRC31 expression patterns in IBD patient tissues and functional consequences of its modulation in colonic epithelial models

• Allergic Airway Diseases:

  • LRRC31 expression in airway mucosal epithelium and its IL-13 responsiveness suggest potential roles in:

    • Asthma, where IL-13 drives goblet cell metaplasia and airway hyperresponsiveness

    • Allergic rhinitis, where epithelial barrier dysfunction contributes to pathogenesis

  • Studies examining LRRC31 in bronchial or nasal epithelial cultures under Th2 cytokine stimulation could provide valuable insights

• Atopic Dermatitis:

  • Epithelial barrier dysfunction is central to atopic dermatitis pathogenesis

  • LRRC31's regulation of kallikreins is particularly relevant, as KLK dysregulation contributes to skin barrier impairment

  • The protein's IL-13 responsiveness aligns with the Th2-dominant inflammation in atopic dermatitis

  • Keratinocyte models with manipulated LRRC31 expression could help elucidate its role in maintaining skin barrier integrity

• Other Eosinophilic Gastrointestinal Disorders (EGID):

  • Beyond EoE, LRRC31 may function similarly in eosinophilic gastritis or eosinophilic colitis

  • Comparative studies across different EGID subtypes could reveal tissue-specific functions

What novel approaches could enhance detection of LRRC31 protein interactions and regulatory mechanisms?

Advancing understanding of LRRC31's protein interactions and regulatory mechanisms requires innovative methodological approaches:

• Proximity-Based Protein Interaction Mapping:

  • BioID or TurboID approaches: Fusion of biotin ligase to LRRC31 for proximity-dependent biotinylation of interacting partners, followed by streptavidin pulldown and mass spectrometry

  • APEX2 proximity labeling: Similar approach using peroxidase-catalyzed biotinylation

  • These methods could expand beyond known interactions with Ku70/Ku80 and ATR to identify novel binding partners in different cellular contexts

• Live-Cell Imaging of LRRC31 Dynamics:

  • CRISPR-mediated endogenous tagging of LRRC31 with fluorescent proteins

  • Tracking LRRC31 recruitment to DNA damage sites in real-time following laser microirradiation

  • FRET-based approaches to visualize interaction with known partners (Ku70/Ku80, ATR) in living cells

  • These approaches could provide insights into the temporal dynamics of LRRC31's inhibitory functions

• Domain-Specific Functional Analysis:

  • Structure-function studies using truncated or mutated LRRC31 constructs

  • Particular focus on leucine-rich repeat (LRR) domains implicated in protein-protein interactions

  • Identification of minimal functional domains required for DNA repair inhibition versus barrier function enhancement

  • X-ray crystallography or cryo-EM structural analysis of LRRC31 in complex with binding partners

• Transcriptional and Post-Transcriptional Regulation:

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors regulating LRRC31 expression

  • Investigation of epigenetic regulation through bisulfite sequencing and histone modification analysis

  • RNA-binding protein immunoprecipitation (RIP) to identify factors controlling LRRC31 mRNA stability or translation

  • miRNA target prediction and validation to characterize post-transcriptional regulation

• Single-Cell Analysis Approaches:

  • Single-cell RNA sequencing to characterize heterogeneity in LRRC31 expression across cell populations

  • Single-cell protein analysis through mass cytometry (CyTOF) incorporating LRRC31 antibodies

  • These approaches could reveal cell state-dependent regulation and function of LRRC31 that might be masked in bulk analyses

By implementing these advanced approaches, researchers can develop a more comprehensive understanding of LRRC31's complex regulatory networks and diverse biological functions.