Recombinant Mouse NHL repeat-containing protein 2 (Nhlrc2)

What is NHL repeat-containing protein 2 (Nhlrc2) and what is its structural composition?

Nhlrc2 is an essential cytosolic protein containing three distinct domains: an N-terminal thioredoxin-like (Trx-like) domain, a six-bladed NHL repeat β-propeller domain, and a C-terminal β-stranded domain . The crystal structure of human NHLRC2 (which shares high homology with mouse Nhlrc2) reveals that the Trx-like and NHL repeat domains form a cleft containing a conserved CCINC motif . This motif appears at the position typically occupied by the CXXC motif in oxidoreductases, suggesting potential redox functionality .

The functional significance of this structure is suggested by the fact that conserved residues across species map primarily to the adjacent surfaces of the Trx-like and β-propeller domains that form this cleft, indicating this region likely represents the protein's functional site .

How is Nhlrc2 conserved across species and what does this suggest about its function?

Bioinformatics analyses have revealed that Nhlrc2 homologs exist across a wide range of eukaryotic and prokaryotic species, suggesting an evolutionarily conserved and fundamental biological role . The most conserved residues map to the potential active site of the Trx-like domain and the spatially adjacent binding surface of the β-propeller, indicating these regions are crucial for the protein's function .

The conservation pattern suggests that Nhlrc2 may function as a redox enzyme with a conserved mechanism across species . The presence of the CCINC motif in the position typically occupied by the CXXC motif in thioredoxin-like proteins further supports a potential role in thiol-disulfide exchange reactions .

What developmental processes require Nhlrc2 expression?

Nhlrc2 plays a critical role in early development, as evidenced by the embryonic lethality observed in mice with homozygous deletion of the Nhlrc2 gene . Specifically, loss of essential Nhlrc2 has been shown to lead to failed gastrulation, abnormal amniotic folding, and embryonic lethality in mice .

In cattle, a mutation in a conserved region of the β-propeller domain of Nhlrc2 has been associated with neural tube defects, further highlighting its importance in developmental processes . These findings collectively suggest that Nhlrc2 is essential for proper embryonic development, particularly in the formation of the neural tube and during gastrulation.

What are the optimal methods for detecting and quantifying Nhlrc2 expression in tissue samples?

Based on established protocols for NHLRC2 detection in human tissues, several complementary approaches can be employed to detect and quantify Nhlrc2 in mouse tissue samples:

• Immunohistochemistry (IHC): This technique allows visualization of Nhlrc2 protein expression patterns in tissue sections. Digital pathology image analysis software can be used to quantify the area of Nhlrc2-positive staining in relation to the total tissue area . The percentage of Nhlrc2-positive cells can be scored using a semiquantitative method (negative, <25%, 25-49%, 50-75%, >75% positive cells) .

• mRNA in situ hybridization: RNAscope assay can be used to detect Nhlrc2 mRNA in tissue sections, providing information about transcriptional activity . This method complements protein-level detection by IHC.

• Western blot analysis: This technique allows quantification of Nhlrc2 protein levels in tissue homogenates and cell lines . When analyzing experimental results, it's important to note that mRNA and protein levels may not always correlate, as demonstrated in studies comparing proteomic and gene expression data .

How can recombinant mouse Nhlrc2 be efficiently expressed and purified for structural studies?

For successful structural studies of recombinant mouse Nhlrc2, researchers should consider the following methodological approach:

• Construct design: Based on structural studies of human NHLRC2, separate constructs should be designed for different domains or combinations: the Trx-like domain alone (aa 1-220), the NHL repeat β-propeller domain (aa 221-572), the combined Trx-like and NHL repeat domains (aa 1-572), and the full-length protein .

• Expression system: E. coli expression systems have proven successful for human NHLRC2 fragments . For mouse Nhlrc2, optimize expression conditions including temperature, IPTG concentration, and induction time.

• Purification strategy: A multi-step purification protocol should include:

  • Initial capture using affinity chromatography (His-tag or GST-tag)

  • Intermediate purification using ion exchange chromatography

  • Final polishing using size exclusion chromatography

• Protein quality assessment: Evaluate protein homogeneity using SDS-PAGE, dynamic light scattering, and thermal shift assays. For structural studies, protein stability is critical; therefore, optimize buffer conditions to enhance stability .

What functional insights can be derived from the crystal structure of Nhlrc2?

The crystal structure of human NHLRC2 provides valuable insights that can be extrapolated to mouse Nhlrc2 due to high sequence conservation:

• Domain arrangement: The Trx-like domain is connected to the β-propeller via a flexible loop, suggesting potential conformational flexibility during function .

• Active site identification: The CCINC motif of the Trx-like domain is located in close proximity to the binding interface of the β-propeller domain, forming a cleft that likely represents the functional site of the protein .

• Surface properties: Analysis reveals an extended negative electrostatic potential in the surface of the cleft formed by the two domains, suggesting this region likely forms a binding site for a ligand or interaction partner(s) .

• C-terminal domain positioning: SAXS analysis shows that the non-conserved C-terminal domain does not pack against the N-terminal domains, indicating potential independent functionality .

These structural features support the hypothesis that Nhlrc2 may function as a redox-active protein with the conserved CCINC motif participating in thiol-disulfide exchange reactions, possibly involving interaction partners that bind to the negatively charged cleft.

How do disease-associated mutations affect Nhlrc2 structure and function?

While specific disease-associated mutations in mouse Nhlrc2 have not been extensively characterized, insights from human NHLRC2 mutations provide valuable information:

• Asp148Tyr mutation: This mutation destabilizes the protein structure by 2°C, potentially affecting protein stability and function . Located in the Trx-like domain, this residue is not conserved across species, suggesting species-specific functional implications .

• R201GfsTer6 mutation: This frameshift mutation results in a truncated protein, likely causing loss of function .

• β-propeller domain mutations: Mutations in the conserved regions of the β-propeller domain have been associated with neural tube defects in cattle, highlighting the importance of this domain for proper development .

When studying mouse models with equivalent mutations, researchers should consider:

• Thermal stability assessments to detect structural perturbations

• Activity assays to measure functional consequences

• Interaction studies to identify disrupted protein-protein interactions

• In vivo phenotypic analyses to correlate structural changes with developmental or disease outcomes

What is the role of Nhlrc2 in cancer biology, particularly in lung cancer models?

Based on human studies, Nhlrc2 may play significant roles in cancer biology that warrant investigation in mouse models:

For mouse lung cancer models, researchers should consider:

• Comparing Nhlrc2 expression between different histological subtypes

• Correlating Nhlrc2 expression with markers of proliferation

• Investigating the effects of Nhlrc2 knockdown or overexpression on cancer cell growth, invasion, and metastasis

• Examining the role of Nhlrc2 in tumor-associated immune cells

What is known about Nhlrc2's role in fibrotic diseases and how can it be studied in mouse models?

Fibrosis represents a potential area of research for Nhlrc2 function:

• Association with pulmonary fibrosis: Human NHLRC2 expression was increased in lung tissues of patients with idiopathic pulmonary fibrosis (IPF) . Certain variants of human NHLRC2 have been linked to severe fibrotic interstitial lung disease in children (FINCA disease) .

• Potential mechanisms: The thioredoxin-like domain of Nhlrc2 suggests potential involvement in redox processes, which are known to play roles in fibrotic diseases through oxidative stress pathways .

To study Nhlrc2 in mouse models of fibrosis, researchers could:

• Develop conditional knockout models to circumvent embryonic lethality

• Utilize bleomycin-induced pulmonary fibrosis models to assess changes in Nhlrc2 expression

• Investigate the effects of Nhlrc2 modulation on fibroblast activation and extracellular matrix production

• Explore potential interactions between Nhlrc2 and known pro-fibrotic signaling pathways (TGF-β, Wnt, etc.)

• Assess the redox status and oxidative stress markers in relation to Nhlrc2 expression in fibrotic tissues

What are recommended methods for studying protein-protein interactions involving Nhlrc2?

To elucidate Nhlrc2's functional network, several complementary approaches are recommended:

• Co-immunoprecipitation (Co-IP): This can identify endogenous interaction partners of Nhlrc2 in mouse tissue lysates or cell lines. Use specific antibodies against Nhlrc2 for pull-down experiments, followed by mass spectrometry analysis.

• Proximity-dependent biotin identification (BioID): This technique allows identification of proximal and transient interactors by fusing Nhlrc2 to a biotin ligase, which biotinylates nearby proteins that can then be purified and identified.

• Yeast two-hybrid screening: This approach can identify direct protein-protein interactions using the different domains of Nhlrc2 as bait.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): These biophysical methods can quantitatively characterize the binding parameters of Nhlrc2 with identified interaction partners.

• Structural analysis of complexes: X-ray crystallography or cryo-electron microscopy of Nhlrc2 in complex with interaction partners can provide atomic-level insights into binding interfaces and mechanisms.

The cleft formed by the Trx-like and NHL repeat domains, which shows an extended negative electrostatic potential, should be a focal point for these interaction studies as it likely represents a binding site for interaction partners .

How can the redox properties of the CCINC motif in recombinant mouse Nhlrc2 be investigated?

The CCINC motif in the Trx-like domain of Nhlrc2 represents a potential functional site for redox activity . To investigate its properties:

• Thiol reactivity assays: Measure the reactivity of the cysteine residues using thiol-reactive probes such as DTNB (Ellman's reagent) or maleimide-based fluorescent dyes.

• Redox potential determination: Use redox buffers with different ratios of reduced and oxidized glutathione to determine the redox potential of the CCINC motif.

• Site-directed mutagenesis: Create mutants where one or both cysteines in the CCINC motif are replaced with serines to assess their contribution to any observed redox activity.

• Mass spectrometry analysis: Use this technique to identify the oxidation states of the cysteines and potential post-translational modifications under different redox conditions.

• Functional rescue experiments: In cell models with reduced endogenous Nhlrc2, compare the ability of wild-type versus CCINC motif mutants to rescue phenotypes, connecting redox activity to biological function.

• Hydrogen peroxide sensitivity assays: Assess whether Nhlrc2 can protect cells from oxidative stress and whether this protection depends on the CCINC motif.

How do mouse and human Nhlrc2/NHLRC2 compare in terms of structure, expression, and function?

Understanding the similarities and differences between mouse and human orthologs is crucial for translational research:

What genetic models are available to study Nhlrc2 function in vivo?

Researchers investigating Nhlrc2 function have several genetic approaches available:

• Conventional knockout models: Complete Nhlrc2 knockout leads to embryonic lethality, limiting its utility for studying later developmental stages or adult functions .

• Conditional knockout systems: Cre-loxP systems allow tissue-specific or temporal control of Nhlrc2 deletion, circumventing embryonic lethality. Consider targeting:

  • Specific lung cell populations (type II pneumocytes, bronchial epithelium)

  • Immune cells (macrophages)

  • Central nervous system components

• Knock-in models of disease-associated mutations: Creating mouse models carrying equivalents of human disease mutations (e.g., Asp148Tyr) can provide insights into pathological mechanisms .

• Transgenic overexpression models: Tissue-specific overexpression can assess gain-of-function effects and potential therapeutic applications.

• Reporter models: Knock-in of reporter genes (GFP, LacZ) at the Nhlrc2 locus allows visualization of expression patterns during development and in adult tissues.

When designing genetic models, researchers should consider the three-domain structure of Nhlrc2 and the potential for domain-specific functions. Targeting specific domains through precise genetic engineering may reveal more nuanced insights than complete gene knockout.