Recombinant Rat Putative small membrane protein NID67 (Nid67)

What is NID67 and what are its basic structural properties?

NID67 (NGF-induced differentiation clone 67 protein) is a 60-amino acid single-pass membrane protein originally identified as being induced by nerve growth factor (NGF). The rat NID67 protein (UniProt ID: Q99PE6) consists of a full-length sequence MDAISQSPVDVLLPKHILDIWAIVLIILATVVIMTSLFLCPATAVIIYRMRTHPVLNGAV with an expression region spanning amino acids 1-60 . The protein contains a transmembrane domain, suggesting its role in cellular membrane functions. NID67 is also known as C5orf62 in humans, where it maps to chromosome 5q33.1 . As a small membrane protein, its structure is characterized by a single membrane-spanning region with short cytoplasmic and extracellular domains.

How is the NID67 gene conserved across species and what are its homologs?

The NID67 gene demonstrates notable evolutionary conservation, particularly between rats (where it's located on chromosome 18) and humans (located on chromosome 5q33.1) . In mice, NID67 is part of the syntenic region that corresponds to the human chromosome 5q, specifically in the region associated with 5q- syndrome . Conservation analysis suggests functional importance across mammalian species. Comparative studies between rat, mouse, and human NID67 sequences show significant homology in the coding regions, indicating preserved functionality across species. Researchers should note that despite this conservation, species-specific differences in regulatory elements may affect expression patterns and responses to stimuli.

What are the most effective methods for studying NID67 expression in different tissue types?

For comprehensive NID67 expression analysis across tissues, multiple complementary approaches should be employed:

• qRT-PCR: Design primers specific to rat NID67 mRNA, using reference genes appropriate for the tissue being studied. Validate primers using standard curves to ensure specificity and efficiency.

• Western blotting: Use validated antibodies against NID67, with appropriate positive controls. Due to its small size (60 amino acids), use gradient gels (15-20%) to achieve proper separation, and consider using alternative visualization methods like chemiluminescence for low-abundance detection.

• Immunohistochemistry/Immunofluorescence: For tissue-specific localization, optimize fixation protocols (4% paraformaldehyde works well for membrane proteins), and use antigen retrieval methods as needed. Counter-stain with markers for specific cell types to determine exact cellular expression patterns.

• In situ hybridization: To detect mRNA expression in intact tissues, design RNA probes spanning unique regions of NID67 mRNA. Include positive and negative control probes to validate specificity.

• Single-cell RNA sequencing: For cell-type specific expression in heterogeneous tissues such as bone marrow, analyze NID67 expression patterns across different cell populations identified by canonical markers.

These methods should be used in combination, as each provides distinct and complementary information about NID67 expression patterns.

How should recombinant rat NID67 protein be handled and stored for optimal stability and activity?

Recombinant rat NID67 protein requires specific handling protocols to maintain stability and biological activity:

• Storage conditions: Store at -20°C for regular use, and at -80°C for long-term storage . Avoid repeated freeze-thaw cycles as this can lead to protein degradation and reduced activity.

• Working solutions: Prepare working aliquots and store at 4°C for up to one week to minimize freeze-thaw cycles . Use sterile, low-protein binding tubes for aliquoting.

• Buffer composition: The optimal storage buffer contains Tris buffer with 50% glycerol , which helps stabilize the protein structure. When diluting for experiments, maintain a minimum of 10% glycerol to prevent aggregation.

• Temperature sensitivity: Allow protein to thaw gradually on ice rather than at room temperature to prevent localized denaturation.

• Avoiding contamination: Use sterile techniques when handling the protein to prevent microbial contamination, which can lead to degradation.

• Activity testing: Before using in critical experiments, verify protein activity with appropriate functional assays to ensure the protein has maintained its biological properties.

Following these guidelines will help maintain the integrity and functionality of recombinant NID67 protein throughout your experimental workflows.

How does NID67 contribute to the pathophysiology of the 5q- syndrome mouse model?

The involvement of NID67 in 5q- syndrome pathophysiology is demonstrated through deletion models targeting the syntenic regions in mice. In the Cd74-Nid67 interval deletion mouse model, researchers observed:

• Hematopoietic defects: The deletion results in a 40-50% reduction in circulating red blood cells and decreased hemoglobin levels, recapitulating key features of human 5q- syndrome .

• Morphological abnormalities: The model displays macrocytic anemia with dysplastic features in the bone marrow, including the appearance of pencil-shaped cells in the peripheral blood .

• Progenitor cell deficiencies: A striking deficit in common myeloid progenitors (CMP), megakaryocytic-erythroid progenitors (MEP), and granulocyte-monocyte progenitors (GMP) lineages was observed in the bone marrow of deletion mice .

• p53-dependent mechanism: The hematopoietic defects were associated with increased p53 activity in bone marrow cells and elevated apoptosis. Remarkably, crossing these mice with p53-deficient mice (Trp53-/-) reversed the progenitor cell deficiencies, demonstrating that the pathophysiology operates through a p53-dependent mechanism .

What is the relationship between NID67 and neuronal differentiation in experimental models?

The relationship between NID67 and neuronal differentiation stems from its initial identification as an NGF-induced differentiation clone in PC12 pheochromocytoma cells . Key experimental findings include:

• NGF-specific induction: NID67 is preferentially induced by NGF in PC12 cells, a widely used model for neuronal differentiation . This specificity suggests a role in the NGF signaling pathway rather than in general differentiation processes.

• Temporal expression pattern: NID67 expression shows a distinct temporal pattern during NGF-induced differentiation, with expression levels changing at specific time points during the differentiation process.

• Membrane localization: As a single-pass membrane protein, NID67 may function in membrane remodeling or receptor complex formation during neurite extension and neuronal differentiation.

• Potential interaction with signaling pathways: Though not directly demonstrated in the available literature, the induction pattern suggests NID67 may interact with known NGF-responsive pathways such as the MAPK/ERK and PI3K/Akt pathways.

Experimental approaches to further study NID67 in neuronal differentiation would include knockdown or knockout studies in neuronal cell models, identification of interaction partners, and examination of downstream signaling effects when NID67 expression is modulated. These approaches would help elucidate whether NID67 is merely a marker of differentiation or plays a functional role in the differentiation process.

What are the methodological considerations for using shRNA to study NID67 function in different cell types?

When designing shRNA experiments to study NID67 function, researchers should consider:

• Target sequence selection: Design multiple shRNAs targeting different regions of NID67 mRNA to mitigate off-target effects. Focus on regions with unique sequences to avoid cross-targeting related genes. Commercial shRNA constructs like Santa Cruz Biotechnology's NID67 shRNA lentiviral particles (sc-91840-V) can be utilized as starting points .

• Delivery system optimization:

  • For neuronal cells (e.g., PC12): Lentiviral delivery typically achieves 60-80% transduction efficiency with minimal toxicity

  • For hematopoietic cells: Consider nucleofection for primary cells or lentiviral transduction with polybrene enhancement

  • For adherent cell lines: Lipid-based transfection may be sufficient for transient knockdown studies

• Knockdown validation:

  • Measure NID67 mRNA levels by qRT-PCR

  • Confirm protein reduction via Western blot (challenging due to small size; consider using epitope-tagged constructs)

  • Implement at least two independent validation methods

• Controls:

  • Include non-targeting shRNA controls with similar GC content

  • Consider rescue experiments with shRNA-resistant NID67 constructs to confirm specificity

  • Monitor cell viability to distinguish between specific phenotypes and general cytotoxicity

• Phenotypic analysis:

  • For neuronal cells: measure neurite outgrowth, NGF responsiveness, and expression of differentiation markers

  • For hematopoietic cells: analyze progenitor differentiation, cell cycle progression, and apoptosis markers

• Timing considerations: Establish optimal knockdown time course before phenotypic analyses, as membrane protein turnover rates may affect experimental outcomes.

How can researchers reconcile contradictory findings between NID67 knockout models and knockdown approaches?

Resolving contradictory findings between NID67 knockout and knockdown approaches requires systematic analysis of several factors:

• Developmental compensation mechanisms:

  • Knockout models may activate compensatory pathways during development that mask phenotypes

  • Use inducible knockout systems (e.g., Cre-ERT2) to bypass developmental compensation

  • Compare acute (knockdown) versus chronic (knockout) loss of function

• Off-target effects:

  • Validate knockdown specificity using multiple siRNA/shRNA sequences

  • Perform rescue experiments with constructs resistant to the knockdown agent

  • Use CRISPR-mediated knockin of synonymous mutations to create knockdown-resistant alleles

• Genetic background influences:

  • Backcross knockout mice to multiple genetic backgrounds

  • Use isogenic cell lines for in vitro studies

  • Consider strain-specific modifier genes when interpreting phenotypes

• Dosage sensitivity:

  • Compare heterozygous and homozygous knockout phenotypes

  • Titrate knockdown efficiency to determine dosage thresholds for phenotypic manifestation

  • Generate hypomorphic alleles to assess intermediate expression levels

• Context-dependent functions:

  • Test phenotypes under different conditions (e.g., stress, differentiation cues)

  • Consider cell type-specific functions when interpreting tissue-specific phenotypes

  • Examine phenotypes at different developmental stages

• Methodological reconciliation approach:

  • Perform side-by-side comparisons using identical readouts

  • Apply both approaches in the same experimental system

  • Use complementary techniques like CRISPR interference for temporary repression

This systematic approach helps distinguish between technical artifacts and biologically meaningful differences in NID67 function under different experimental conditions.

What is the specific role of NID67 in hematopoietic progenitor cell development?

The specific contribution of NID67 to hematopoietic progenitor cell development can be inferred from studies of the Cd74-Nid67 interval deletion model, though its individual role remains to be fully elucidated:

• Progenitor cell population effects: The Cd74-Nid67 interval deletion results in significant deficits in multiple hematopoietic progenitor populations, including:

  • Common myeloid progenitors (CMP)

  • Megakaryocytic-erythroid progenitors (MEP)

  • Granulocyte-monocyte progenitors (GMP)

• Lineage-specific impacts: The most pronounced effects appear in the erythroid lineage, with a 40-50% reduction in circulating red blood cells and decreased hemoglobin levels . This suggests potential involvement in erythroid differentiation or survival.

• p53-dependent mechanism: The deficiencies observed in the deletion model are reversed by p53 deficiency, indicating that NID67 (or other genes in the interval) may normally function in regulating p53 activation or downstream effects . This suggests a potential role in cell cycle regulation or apoptosis in hematopoietic progenitors.

• Isolation of NID67-specific effects: While the deletion model provides valuable insights, it's important to note that the Cd74-Nid67 interval contains eight genes. To determine the specific contribution of NID67 alone, targeted gene knockout or knockdown approaches would be necessary.

• Potential molecular mechanisms: Given its membrane localization, NID67 might function in:

  • Cytokine or growth factor signaling relevant to hematopoiesis

  • Cell-cell interactions in the hematopoietic niche

  • Regulation of membrane dynamics during progenitor cell division or differentiation

Further research using conditional knockout models or CRISPR-based approaches targeting NID67 specifically would help clarify its individual contributions to hematopoietic development.

How does NID67 interact with p53 pathways in models of myelodysplastic syndrome?

The interaction between NID67 and p53 pathways in myelodysplastic syndrome models represents a significant mechanistic insight, though the precise molecular details remain to be fully characterized:

• Genetic evidence for p53 involvement: The reversal of hematopoietic progenitor cell deficiencies in Cd74-Nid67 deletion mice when crossed with Trp53-/- mice provides strong genetic evidence for p53 dependency . This indicates that the deletion leads to inappropriate p53 activation or prevents normal p53 inhibition.

• Cellular manifestations: The deletion model shows:

  • Increased p53-positive cells in the bone marrow

  • Elevated apoptosis in hematopoietic progenitors

  • Reduced progenitor cell development

• Potential molecular mechanisms:

  • NID67 might normally function to suppress p53 activation in hematopoietic progenitors

  • As a membrane protein, NID67 could mediate growth factor or cytokine signaling that regulates p53 activity

  • The deletion might activate cellular stress pathways that trigger p53-dependent cell cycle arrest or apoptosis

• Relevance to human 5q- syndrome: This p53-dependent mechanism identified in mouse models has important implications for human 5q- syndrome, suggesting that targeting p53 or its downstream effectors might have therapeutic potential .

• Broader implications: Understanding how NID67 interfaces with p53 pathways could provide insights into fundamental mechanisms of hematopoietic regulation and malignant transformation. This connection between a membrane protein and a key tumor suppressor suggests unexplored signaling connections worthy of further investigation.

To more precisely define the role of NID67 in p53 regulation, further studies could examine whether NID67 alone is sufficient to modify p53 activity, identify potential direct or indirect interactions, and characterize the signaling pathways that connect this membrane protein to nuclear p53 function.

What are the optimal methods for identifying protein interaction partners of NID67?

Identifying protein interaction partners of NID67 presents unique challenges due to its small size (60 amino acids) and membrane localization. A multi-faceted approach is recommended:

• Proximity-based labeling techniques:

  • BioID or TurboID: Fusion of biotin ligase to NID67 allows biotinylation of proximal proteins

  • APEX2 proximity labeling: Provides higher temporal resolution for capturing transient interactions

  • Optimization: For single-pass membrane proteins like NID67, both N- and C-terminal fusions should be tested to determine which preserves functionality

• Co-immunoprecipitation strategies:

  • Crosslinking: Use membrane-permeable crosslinkers (DSP, formaldehyde) to stabilize interactions

  • Detergent selection: Test multiple detergents (digitonin, CHAPS, DDM) optimized for membrane protein complexes

  • Epitope tagging: Use small tags (FLAG, HA) at either terminus with verification that tagging doesn't disrupt localization

• Membrane-specific yeast two-hybrid systems:

  • Split-ubiquitin membrane yeast two-hybrid for membrane protein interactions

  • MYTH (Membrane Yeast Two-Hybrid) system with NID67 as bait against cDNA libraries from relevant tissues

• Proteomic approaches:

  • SILAC or TMT labeling combined with immunoprecipitation to distinguish specific from non-specific interactions

  • Crosslinking mass spectrometry (XL-MS) to capture spatial relationships between interaction partners

• Validation methods:

  • Bimolecular fluorescence complementation (BiFC) to visualize interactions in living cells

  • FRET/FLIM to detect proximity between NID67 and candidate partners

  • Co-localization studies with super-resolution microscopy

  • Functional validation through mutagenesis of interaction interfaces

• Data analysis considerations:

  • Apply stringent statistical thresholds for proteomics data

  • Prioritize hits appearing in multiple complementary approaches

  • Consider topology when interpreting results (cytoplasmic vs. extracellular interactions)

This comprehensive approach accounts for the technical challenges presented by small membrane proteins like NID67 and provides multiple layers of validation.

How can researchers effectively study the role of NID67 in NGF signaling pathways?

To effectively study NID67's role in NGF signaling pathways, researchers should implement a systematic approach:

• Temporal expression profiling:

  • Perform time-course analysis of NID67 expression after NGF treatment in PC12 cells

  • Compare with other NGF-responsive genes to place NID67 in early, intermediate, or late response categories

  • Use transcription inhibitors to determine if NID67 is a direct or indirect NGF target

• Signaling pathway dissection:

  • Apply specific inhibitors of NGF signaling branches (MEK/ERK, PI3K/Akt, PLCγ) to identify which pathway(s) regulate NID67

  • Use constitutively active or dominant negative constructs of pathway components to confirm regulatory relationships

  • Examine post-translational modifications of NID67 in response to NGF using phospho-specific antibodies or mass spectrometry

• Loss-of-function approaches:

  • Implement NID67 knockdown or knockout in PC12 cells

  • Assess effects on:

    • NGF-induced neurite outgrowth (measure length, branching, stability)

    • Expression of neuronal differentiation markers (GAP43, β-III tubulin)

    • Activation of downstream signaling pathways (phosphorylation of ERK, Akt, CREB)

    • Cell survival under stress conditions

• Gain-of-function studies:

  • Overexpress NID67 in PC12 cells and assess whether it enhances or inhibits NGF responses

  • Test whether NID67 overexpression can sensitize cells to sub-threshold NGF concentrations

  • Examine effects on NGF receptor (TrkA) trafficking, localization, and degradation

• Membrane dynamics and localization:

  • Use live cell imaging with fluorescently tagged NID67 to track its localization during NGF stimulation

  • Determine if NID67 co-localizes with NGF receptors or signaling endosomes

  • Apply super-resolution microscopy to analyze nanoscale distribution in membrane microdomains

• Comparative analysis across cell types:

  • Compare NID67 function in NGF-responsive versus non-responsive cell types

  • Assess conservation of NID67 function in primary neurons versus PC12 cells

  • Examine potential redundancy with related proteins in different neural cell types

This multi-dimensional approach will help establish whether NID67 is merely a marker of NGF response or plays a functional role in NGF signaling and neuronal differentiation.