LRRC59 Human

What is the molecular structure and cellular localization of LRRC59?

LRRC59 is a tail-anchored membrane protein consisting of a 244-amino acid cytoplasmic/nucleoplasmic region, a transmembrane domain (TMD) close to the C-terminus, and a 40-amino acid region that resides in the ER lumen. The cytoplasmic domain contains leucine-rich repeats (LRR) and a coiling domain facing the cytoplasm, with the wild-type cytoplasmic portion measuring approximately 27.8 kDa .

Methodologically, LRRC59 localization can be confirmed through subcellular fractionation and imaging techniques. The protein primarily localizes to the endoplasmic reticulum (ER) membrane and the nuclear envelope, showing prominent ER signals and a characteristic rim around the nucleus. Using rapamycin-dependent dimerization assays, researchers have demonstrated that LRRC59 can reach the inner nuclear membrane (INM), similar to established nuclear envelope proteins like emerin .

How is LRRC59 expression detected and quantified in research settings?

Multiple methodological approaches are employed to detect and quantify LRRC59:

• Immunohistochemistry (IHC): Tissue samples are processed through dewaxing, rehydration, and antigen retrieval before incubation with LRRC59 primary antibody (typically at 1:500 concentration) and subsequent visualization with HRP-labeled secondary antibodies. Positive expression appears as brownish-yellow staining, primarily in the cytoplasm with some nuclear presence .

• Quantitative real-time PCR (qRT-PCR): Used to measure LRRC59 mRNA expression across cell lines and tissues, providing relative expression levels.

• Western blot: Enables protein-level detection and semi-quantitative analysis of LRRC59 expression in cell and tissue lysates.

• Bioinformatic analysis: LRRC59 expression can be analyzed using databases like The Cancer Genome Atlas (TCGA), with techniques such as TPM (Transcripts Per Million) normalization for cross-sample comparisons .

• ROC curve analysis: Used to evaluate LRRC59's discriminatory power between cancer and normal tissues, with an area under the curve (AUC) of 0.808 (95% CI = 0.737–0.879) reported for bladder cancer, indicating good diagnostic potential .

What role does LRRC59 play in normal cellular processes?

LRRC59 was originally identified as a ribosome-binding protein that interacts with fibroblast growth factors. Research indicates it promotes importin α/β-dependent nuclear import of fibroblast growth factor 1 and the cancerous inhibitor of PP2A (CIP2A) .

Its strategic localization at both the ER and nuclear envelope suggests multifunctional roles in protein synthesis, membrane organization, and nuclear transport processes. The protein's ability to reach the inner nuclear membrane indicates potential functions in nuclear architecture or gene regulation .

Methodologically, protein interaction studies using co-immunoprecipitation, proximity ligation assays, and mass spectrometry have helped identify LRRC59's binding partners, though complete protein interaction networks remain to be fully characterized based on the available research data.

What mechanisms govern LRRC59's insertion into cellular membranes?

LRRC59 exhibits unusual membrane insertion properties that challenge conventional models. Research using purified microsomes demonstrates that LRRC59 can be post-translationally inserted into ER-derived membranes, even with artificial C-terminal extensions .

Surprisingly, the TRC-pathway (Transmembrane Recognition Complex), a major route for post-translational membrane insertion of tail-anchored proteins, is not required for LRRC59 membrane integration. Unlike established tail-anchored proteins such as emerin or Sec61β:

• Dominant negative WRB- or CAML-fragments do not inhibit LRRC59 membrane insertion

• TRC40-depletion from reticulocyte lysates does not prevent LRRC59 integration

These findings suggest LRRC59 utilizes alternative insertion mechanisms. Under certain conditions, the Signal Recognition Particle (SRP) or Hsc70 proteins could mediate post-translational events, bypassing the requirement for TRC40, WRB, or CAML. Recent research suggests the existence of multiple redundant pathways for membrane protein insertion that LRRC59 might utilize .

How does LRRC59 target and reach the inner nuclear membrane?

LRRC59 targeting to the inner nuclear membrane (INM) follows mechanisms distinct from canonical nuclear import pathways. Unlike soluble nucleoplasmic proteins, LRRC59 does not primarily depend on the importin α/β-mediated transport system .

Experimental approaches using rapamycin-dependent dimerization assays reveal that the cytoplasmic domain size is the critical determinant of INM localization, suggesting a passive diffusion model. This size-dependency manifests in several ways:

• Constructs with small cytoplasmic domains (HA-FRB-LRRC59, mCherry-FRB-LRRC59) rapidly reach the INM

• Constructs with larger domains (mCherry-FRB-MBP-LRRC59 at 107.5 kDa) show significantly slower INM targeting

• Dimerization-promoting tags (GST-tagged LRRC59) dramatically reduce INM targeting

These observations support a model where LRRC59 reaches the INM through peripheral channels of the nuclear pore complex that impose size restrictions on transmembrane protein passage .

What is LRRC59's role in cancer progression and immune regulation?

LRRC59 has emerged as a significant factor in cancer biology, particularly in bladder cancer. Comprehensive analyses combining transcriptomics, proteomics, and clinical data reveal:

What clinicopathological factors correlate with LRRC59 expression?

LRRC59 expression shows significant associations with multiple clinicopathological parameters in bladder cancer:

Univariate analysis further identified several factors with significant odds ratios, including:

• Histologic grade (OR = 0.046, 95% CI = 0.003–0.333, p = 0.003)

• Cancer subtype (OR = 0.508, 95% CI = 0.331–0.774, p = 0.002)

• Race (OR = 2.373, 95% CI = 1.377–4.195, p = 0.002)

• Primary therapy outcome (OR = 0.516, 95% CI = 0.319–0.828, p = 0.003)

What methodological considerations are important when studying LRRC59's methylation status and epigenetic regulation?

LRRC59's methylation status appears to be clinically significant, with hypomethylation associated with poor prognosis in bladder cancer . Researchers studying this aspect should consider:

• Methylation analysis platforms: The UALCAN and MethSurv databases have been successfully employed to analyze LRRC59 methylation patterns. These platforms allow for correlation between methylation status and clinical outcomes.

• Integration with expression data: Combined analysis of methylation patterns and expression levels provides deeper insights into regulatory mechanisms. Hypomethylation often correlates with increased expression, but exceptions may exist.

• CpG island mapping: Identifying specific CpG islands within the LRRC59 gene locus that correlate most strongly with expression changes and clinical outcomes can provide mechanistic insights.

• Cell-type specificity: Methylation patterns may vary across different cell types within tumor tissues, requiring single-cell methodologies or microdissection approaches for precise analysis.

• Functional validation: Using demethylating agents (like 5-aza-2'-deoxycytidine) to experimentally manipulate methylation status can help establish causative relationships between methylation, expression, and functional outcomes.

• Temporal considerations: Methylation patterns may change during disease progression, necessitating longitudinal sampling approaches.

How can LRRC59's prognostic value be integrated into clinical decision-making models?

LRRC59 demonstrates independent prognostic value in bladder cancer, suggesting potential clinical utility . To integrate this biomarker into decision-making frameworks:

What experimental designs can resolve contradictions in LRRC59's membrane insertion mechanisms?

The unusual membrane insertion properties of LRRC59 present several experimental challenges and contradictions . To resolve these issues, researchers might consider:

• Comparative analyses using domain swapping: Exchanging transmembrane domains and C-terminal regions between LRRC59 and canonical TRC40-dependent tail-anchored proteins (like emerin) to identify critical sequence determinants.

• In vitro reconstitution systems: Creating purified component systems with defined membrane compositions to test insertion efficiency under strictly controlled conditions.

• Real-time tracking approaches:

  • Fluorescence pulse-chase experiments to monitor the timing of membrane insertion

  • Single-molecule tracking to observe individual insertion events

  • FRAP (Fluorescence Recovery After Photobleaching) to measure membrane integration kinetics

• Genetic screening in diverse systems:

  • CRISPR-based screens to identify novel factors involved in LRRC59 membrane insertion

  • Synthetic genetic array analysis in yeast models expressing human LRRC59

• Structural biology approaches:

  • Cryo-EM studies of LRRC59 during membrane insertion

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes during insertion

• Systematic inhibition strategies:

  • Combinatorial inhibition of multiple membrane insertion pathways simultaneously

  • Temperature-sensitive mutants of insertion machinery components

How might alterations in LRRC59 contribute to therapeutic resistance mechanisms?

Given LRRC59's roles in cancer progression and correlation with immune checkpoint genes, its potential contribution to therapeutic resistance warrants investigation:

• Experimental approaches to study LRRC59 in therapy resistance:

  • Generating therapy-resistant cell lines and analyzing LRRC59 expression changes

  • CRISPR-mediated LRRC59 knockout or overexpression in cells undergoing treatment

  • Patient-derived xenograft models comparing treatment responses with LRRC59 status

• Potential resistance mechanisms related to LRRC59:

  • EMT-mediated resistance: LRRC59's connection to Snail, vimentin, and E-cadherin suggests it may promote EMT-driven therapy resistance

  • Immune evasion: Correlations with immune checkpoint genes indicate potential roles in immunotherapy resistance

  • Nuclear transport alterations: LRRC59's ability to reach the inner nuclear membrane suggests possible influences on nuclear drug transport or nuclear receptor signaling

• Methodological considerations:

  • Temporal analysis: Monitoring LRRC59 expression changes during treatment and resistance development

  • Combinatorial targeting: Testing LRRC59 inhibition alongside standard therapies

  • Biomarker validation: Assessing whether LRRC59 status predicts response to specific therapeutic approaches

What methodological approaches should be employed to study LRRC59's protein interaction network comprehensively?

Understanding LRRC59's complete protein interaction network requires multi-faceted approaches:

• Domain-specific interaction mapping:

  • Leucine-rich repeat domain interactions

  • Transmembrane domain associations

  • C-terminal luminal domain binding partners

• Compartment-specific interactome analysis:

  • ER membrane interactions versus inner nuclear membrane interactions

  • Proximity labeling approaches (BioID, APEX) to capture transient interactions in specific cellular compartments

  • Fractionation-based comparative proteomics

• Dynamic interaction profiling:

  • Stimulus-dependent interaction changes (growth factors, stress conditions)

  • Cell cycle-dependent interaction variations

  • Differentiation or disease progression-associated alterations

• Technical considerations:

  • Membrane protein-specific pull-down conditions

  • Crosslinking strategies to capture transient interactions

  • Label-free quantification versus isotope labeling approaches

  • Validation through orthogonal methods (co-immunoprecipitation, FRET, PLA)

• Network integration:

  • Combining experimental interaction data with predictive algorithms

  • Integration with transcriptional networks and signaling pathways

  • Systems biology modeling of LRRC59-centered networks

This comprehensive approach would help resolve existing contradictions and provide a more complete understanding of LRRC59's functional roles across cellular compartments and disease states.