NEURL2 Human

What is NEURL2 and what is its primary function in human cells?

NEURL2 (Neuralized-like protein 2) is a 285 amino acid protein that functions as an E3 ubiquitin ligase, facilitating the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins . As a probable substrate-recognition component of a SCF-like ECS (Elongin BC-CUL2/5-SOCS-box protein) E3 ubiquitin-protein ligase complex, NEURL2 mediates the ubiquitination of specific target proteins . Its primary function involves contributing to catalysis through recognition and positioning of both the substrate and the ubiquitin-conjugating enzyme .

During myogenesis, NEURL2 plays a crucial role by controlling the ubiquitination and degradation of the specific pool of CTNNB1/beta-catenin located at the sarcolemma, making it an important regulator of myofiber differentiation and maturation .

What is the genomic location and structure of the human NEURL2 gene?

The human NEURL2 gene (also known as C20orf163) is located on chromosome 20 at cytogenetic position 20q13.12 . The gene encodes a protein with a molecular weight of approximately 34.1 kDa . The protein sequence contains specific domains that enable its function as an E3 ubiquitin ligase, including regions that mediate substrate recognition and interaction with the ubiquitination machinery.

How is NEURL2 expression regulated in different human tissues?

NEURL2 expression varies across different tissues and developmental stages. Notably, NEURL2 has been identified in thymic tissues, particularly in cortical thymic epithelial cells (cTECs) . Within the thymus, a specific subset of cells termed "NEURL2+" has been observed, defined by the expression of NEURL2 and DLL4 . Interestingly, these NEURL2+ cTECs show a higher resemblance to medullary thymic epithelial cells (mTECs) compared to other cTECs .

Research has indicated age-dependent expression patterns, with widespread expression observed in embryonic cTECs, suggesting developmental regulation of NEURL2 . In addition, studies of colorectal cancer have shown that related Neuralized (NEURL) expression can be epigenetically regulated and is often markedly suppressed in cancer contexts .

What is the role of NEURL2 in the Wnt/β-catenin signaling pathway?

While the search results focus more on NEURL rather than NEURL2 specifically in relation to Wnt/β-catenin signaling, they provide valuable insights into how this protein family functions. NEURL has been identified as a novel tumor suppressor that targets oncogenic Wnt/β-catenin signaling in human cancers, particularly colorectal cancer .

NEURL acts as an E3 ubiquitin ligase that interacts directly with oncogenic β-catenin and reduces its cytoplasmic levels. Importantly, this occurs in a GSK3β- and β-TrCP-independent manner, suggesting an alternative pathway for β-catenin degradation . Given the structural and functional similarities within the Neuralized protein family, NEURL2 may potentially share some of these regulatory capabilities, though specific research on NEURL2's role in Wnt signaling would be needed to confirm this hypothesis.

How does NEURL2 contribute to myofiber differentiation and maturation?

NEURL2 plays an important role in the process of myofiber differentiation and maturation through its function as part of the ubiquitin-proteasome system . Specifically, NEURL2 is involved in the regulation of myofibril organization and likely serves as the adaptor component of the E3 ubiquitin ligase complex in striated muscle .

A key mechanism through which NEURL2 influences myogenesis is by regulating the ubiquitin-mediated degradation of β-catenin . During myogenesis, NEURL2 controls the ubiquitination and degradation of a specific pool of CTNNB1/beta-catenin located at the sarcolemma . This targeted protein degradation is crucial for proper muscle development, as it helps maintain appropriate levels of signaling proteins during different stages of myofiber differentiation.

What are the implications of NEURL2 expression in thymic epithelial cells?

Recent multimodal human thymic profiling has revealed interesting patterns of NEURL2 expression in thymic epithelial cells (TECs). Specifically, a subset of cortical thymic epithelial cells (cTECs) expressing NEURL2 and DLL4 has been identified . These NEURL2+ cTECs show a higher resemblance to medullary thymic epithelial cells (mTECs) compared to other cTECs, suggesting they may represent a transitional cell state or have specialized functions .

The identification of NEURL2+ cTECs across multiple studies of young pediatric thymic samples suggests a potential developmental or age-dependent role for NEURL2 in thymic function . Given the thymus's critical role in T-cell development and central tolerance, NEURL2 expression patterns may have implications for understanding thymic development, T-cell selection processes, and potentially age-related thymic involution.

How does NEURL2 compare with other E3 ubiquitin ligases in substrate specificity?

NEURL2 functions as a substrate-recognition component of an E3 ubiquitin ligase complex, contributing to catalysis through the recognition and positioning of specific substrates and the ubiquitin-conjugating enzyme . While many E3 ubiquitin ligases recognize phosphorylated substrates through canonical degradation pathways (like the β-TrCP-dependent degradation of β-catenin), the Neuralized family appears to employ alternative recognition mechanisms.

For instance, NEURL has been shown to interact directly with β-catenin and reduce its cytoplasmic levels in a GSK3β- and β-TrCP-independent manner . This suggests that NEURL2 might also recognize structural features or post-translational modifications distinct from those targeted by other E3 ligases. Understanding these unique substrate recognition properties could provide insights into how NEURL2 achieves specificity for its targets during processes like myogenesis.

What are the optimal methods for studying NEURL2 protein-protein interactions?

When studying NEURL2 protein-protein interactions, researchers should consider a multi-faceted approach combining the following methodologies:

• Co-immunoprecipitation (Co-IP): This technique can be used to isolate NEURL2 along with its interacting partners from cell lysates. When designing Co-IP experiments, researchers should:

  • Use antibodies specific to NEURL2 or tag NEURL2 with epitopes like His-tag for pull-down assays

  • Include appropriate controls (IgG controls, lysates from cells not expressing NEURL2)

  • Consider crosslinking approaches for transient interactions

• Proximity Ligation Assays: These can detect protein interactions in situ with high sensitivity and specificity, providing spatial information about where NEURL2 interactions occur within cells.

• Yeast Two-Hybrid Screening: For identifying novel interaction partners of NEURL2, particularly those involved in ubiquitination pathways or myofiber differentiation.

• Proteomic Analysis: Mass spectrometry-based approaches following immunoprecipitation can identify both known and novel NEURL2 interaction partners.

When specifically studying NEURL2 interactions with β-catenin or components of the ubiquitination machinery, researchers should design experiments that can distinguish direct from indirect interactions and consider the subcellular localization of these interactions, particularly at the sarcolemma in muscle cells .

What experimental design would best evaluate NEURL2's role in protein ubiquitination?

An effective experimental design to evaluate NEURL2's role in protein ubiquitination would involve:

Table 1: Experimental Design for Studying NEURL2-Mediated Ubiquitination

For optimal results, experiments should include appropriate positive controls (known E3 ligases and their substrates) and negative controls (catalytically inactive NEURL2 mutants). When investigating NEURL2's role in β-catenin ubiquitination specifically, researchers should design experiments that can distinguish NEURL2-mediated ubiquitination from the canonical GSK3β/β-TrCP pathway .

What are the best approaches for manipulating NEURL2 expression in experimental models?

When manipulating NEURL2 expression in experimental models, researchers should consider the following approaches based on their specific research questions:

• Overexpression Systems:

  • Transient transfection with expression vectors containing full-length NEURL2 cDNA

  • Stable cell lines with inducible NEURL2 expression (e.g., Tet-On/Off systems)

  • Viral delivery systems (lentivirus, adenovirus) for difficult-to-transfect cells

  • Expression of tagged versions (His-tag, FLAG, GFP) for detection and purification purposes

• Knockdown/Knockout Approaches:

  • siRNA or shRNA targeting NEURL2 for temporary knockdown

  • CRISPR-Cas9 genome editing for complete knockout

  • Conditional knockout models for tissue-specific or temporal control

• Model Systems Selection:

  • For muscle-related studies: C2C12 myoblasts, primary myoblasts, or muscle-derived stem cells

  • For thymic studies: thymic epithelial cell cultures or thymic organoids

  • For in vivo studies: transgenic mouse models with NEURL2 manipulation

When designing these experiments, researchers should include appropriate validation steps, such as confirming NEURL2 expression levels by Western blot, qPCR, or immunofluorescence. Given NEURL2's role in specific cellular contexts like myogenesis, experiments should be designed to capture relevant developmental stages or physiological conditions.

How can researchers distinguish between direct and indirect effects of NEURL2 on target proteins?

Distinguishing between direct and indirect effects of NEURL2 on target proteins requires a systematic approach combining multiple experimental strategies:

• In vitro binding assays using purified recombinant NEURL2 and putative target proteins to establish direct physical interactions.

• Domain mapping and mutagenesis studies to identify specific interaction interfaces between NEURL2 and targets. If mutations that disrupt binding also eliminate functional effects, this supports a direct mechanism.

• Temporal analysis of protein-protein interactions and subsequent effects. Direct effects typically occur more rapidly than indirect ones, which require intermediate steps.

• Proximity labeling techniques such as BioID or APEX to identify proteins in close physical proximity to NEURL2 in living cells.

• Reconstitution experiments in simplified systems. For example, if NEURL2-mediated ubiquitination of a target can be reconstituted with purified components in vitro, this strongly suggests a direct effect.

For studies focusing on NEURL2's role in β-catenin regulation, researchers should specifically investigate whether NEURL2 directly binds to β-catenin and catalyzes its ubiquitination, or whether it acts through intermediate proteins or signaling pathways. Comparing the effects of NEURL2 to those of related proteins like NEURL, which has been shown to directly interact with β-catenin , can provide valuable insights.

What are the best statistical approaches for analyzing NEURL2 expression data across different tissues or experimental conditions?

When analyzing NEURL2 expression data across different tissues or experimental conditions, researchers should employ the following statistical approaches:

• For bulk RNA-seq or microarray data:

  • Normalization methods appropriate for the platform (e.g., RPKM/FPKM/TPM for RNA-seq)

  • Differential expression analysis using DESeq2, edgeR, or limma

  • Multiple testing correction (Benjamini-Hochberg procedure) to control for false discovery rate

  • Power analysis to ensure adequate sample size for detecting biologically meaningful differences

• For single-cell RNA-seq data (particularly relevant for thymic studies ):

  • Specialized normalization methods for single-cell data (e.g., SCTransform)

  • Dimensionality reduction techniques (PCA, t-SNE, UMAP) for visualization

  • Clustering algorithms to identify cell populations with distinct NEURL2 expression patterns

  • Trajectory analysis to understand developmental relationships between NEURL2+ and NEURL2- cells

• For spatial transcriptomics data:

  • Deconvolution methods like SPOTlight to estimate cell type compositions in tissue sections

  • Spatial statistics to identify significant co-localization patterns

  • Integration with single-cell data to map NEURL2 expression to specific tissue regions

• For protein expression data:

  • Appropriate normalization to loading controls or total protein content

  • Non-parametric tests when data does not meet normality assumptions

  • Correlation analyses to identify relationships between NEURL2 expression and biological variables

When analyzing NEURL2 expression specifically in thymic tissues, researchers should consider the heterogeneity of TEC populations and the potential for age-dependent expression patterns, as suggested by the observed differences between embryonic and pediatric samples .

How should researchers interpret contradictory findings regarding NEURL2 function in different experimental systems?

When faced with contradictory findings regarding NEURL2 function, researchers should implement the following analytical framework:

• Systematic comparison of experimental conditions:

  • Cell types/tissues used (NEURL2 may have context-specific functions)

  • Developmental stages or physiological states (expression patterns may be age-dependent )

  • Methodology differences (antibody specificity, knockout vs. knockdown approaches)

  • NEURL2 expression levels (overexpression may lead to non-physiological effects)

• Integration of multiple data types:

  • Combine insights from transcriptomic, proteomic, and functional studies

  • Consider protein-protein interaction networks to identify context-specific partners

  • Evaluate results in light of known biological pathways and processes

• Consideration of redundancy with related proteins:

  • Assess potential compensatory mechanisms by other Neuralized family members

  • Evaluate whether contradictory results might reflect differences in redundancy across systems

• Meta-analysis approaches:

  • Systematically review existing literature using rigorous inclusion criteria

  • Apply statistical methods to synthesize findings across multiple studies

  • Identify moderating variables that might explain discrepant results

When specifically examining contradictory findings related to NEURL2's role in ubiquitination pathways or β-catenin regulation, researchers should consider the possibility that NEURL2 targets specific subcellular pools of proteins (e.g., sarcolemma-associated β-catenin during myogenesis ) rather than affecting total cellular levels, which could explain apparent discrepancies in results.

What are the potential therapeutic implications of NEURL2 research in human diseases?

The emerging understanding of NEURL2 and related proteins suggests several potential therapeutic implications:

• Cancer therapeutics: Given that related protein NEURL functions as a tumor suppressor by targeting oncogenic Wnt/β-catenin signaling in colorectal cancer , NEURL2 might have similar properties or applications. If NEURL2 shares the ability to promote β-catenin degradation through a GSK3β- and β-TrCP-independent mechanism, it could potentially serve as a novel therapeutic target for cancers with dysregulated Wnt signaling that are resistant to existing approaches.

• Muscular disorders: NEURL2's important role in myofiber differentiation and maturation suggests potential applications in muscular dystrophies or other muscle-related disorders. Therapeutic strategies might involve modulating NEURL2 activity to promote proper muscle development or repair.

• Immunological applications: The identification of NEURL2+ cTECs in thymic tissue points to potential roles in T-cell development and selection. Understanding these functions could inform therapies targeting autoimmune diseases, immunodeficiencies, or thymic involution.

Further research is needed to fully characterize NEURL2's roles in these contexts and to develop specific modulators of its activity for therapeutic applications.

What are the most promising future research directions for understanding NEURL2 biology?

Based on current knowledge, the most promising future research directions for NEURL2 include:

• Comprehensive substrate identification: Employing proteomics approaches to identify the complete set of proteins targeted by NEURL2 for ubiquitination, particularly in muscle and thymic tissues.

• Structural studies: Determining the three-dimensional structure of NEURL2, especially in complex with substrates or other components of the ubiquitination machinery, to understand the molecular basis of its specificity.

• Developmental biology: Investigating the role of NEURL2 in thymic development, particularly the significance of NEURL2+ cTECs and their relationship to mTECs .

• Signaling pathway integration: Elucidating how NEURL2-mediated ubiquitination interfaces with other signaling pathways, particularly Wnt/β-catenin signaling, given the established role of related protein NEURL in this pathway .

• Therapeutic targeting: Developing specific modulators (activators or inhibitors) of NEURL2 activity for potential therapeutic applications in cancer, muscular disorders, or immune-related conditions.

• Comparative studies with related proteins: Systematically comparing NEURL2 with other Neuralized family members to understand functional similarities, differences, and potential redundancy.

Advancing these research directions will require interdisciplinary approaches combining biochemistry, structural biology, cell biology, developmental biology, and translational research.