Recombinant Mouse Erythroid differentiation-related factor 1 (Edrf1), partial

What is the basic structure and function of mouse Erdr1?

Erdr1 (Erythroid differentiation regulator 1) is a stress-induced secreted factor that is widely distributed and extremely conserved between humans and mice. It was initially identified as an inducer of hemoglobin synthesis but has emerged as a multifunctional protein with significant roles in immune cell function. Structurally, Erdr1 is classified as an intrinsically disordered protein (IDP), lacking a fixed structure, which allows it to interact with multiple binding partners dynamically and cooperatively .

To study this protein effectively, researchers should note that its flexible structure enables it to form different conformations depending on concentration and cellular context. When designing experiments, consider that:

• The protein's structural flexibility will affect purification methods

• Functional assays should account for concentration-dependent effects

• Binding studies must consider the dynamic nature of Erdr1-partner interactions

How should recombinant Erdr1 be stored and handled to maintain activity?

For optimal stability and activity of recombinant mouse Erdr1:

• Store lyophilized protein at -20°C

• After reconstitution, prepare single-use aliquots to avoid freeze-thaw cycles

• Reconstitute in sterile, buffered solutions (pH 7.2-7.4)

• For short-term storage (1 month), keep reconstituted protein at 4°C

• For long-term storage, store aliquots at -80°C

Activity testing should be performed routinely when using stored protein, as Erdr1's intrinsically disordered nature may lead to conformational changes affecting function. A hemoglobin induction assay using appropriate erythroid progenitor cells can serve as a reliable functional readout for activity confirmation.

What are the recommended cell types for studying Erdr1 function?

Based on current research, several cell types are appropriate for studying different aspects of Erdr1 function:

When designing experiments, consider that Erdr1 effects are both dose and context-dependent. Cell density has been shown to significantly influence experimental outcomes, with Erdr1 exhibiting dual roles in IL-1β production depending on concentration and cellular context .

How can I validate successful transfection or treatment with recombinant Erdr1?

To verify successful transfection or treatment with recombinant Erdr1:

• Western blot analysis using specific anti-Erdr1 antibodies

• qRT-PCR to measure Erdr1 mRNA expression

• ELISA to quantify Erdr1 protein in cell culture supernatants

• Functional readouts specific to the cell type:

  • In macrophages: measure IL-1β production

  • In erythroid progenitors: hemoglobin quantification

When validating experiments, include appropriate controls:

• Empty vector controls for transfection experiments

• Heat-inactivated protein controls for recombinant protein treatments

• Dose-response curves to account for concentration-dependent effects

How does Erdr1 concentration affect its dual role in regulating IL-1β production in macrophages?

Erdr1 exhibits a concentration-dependent dual role in regulating IL-1β production in macrophages, following a bell-shaped response curve . This dual functionality is linked to Erdr1's dynamic interactions with different adaptor proteins at varying concentrations.

Research findings indicate:

• At basal levels (M0 macrophages): Moderate IL-1β production

• At downregulated levels (M1 macrophages): Enhanced IL-1β production via:

  • Reduced interaction with YAP1

  • Increased interaction with Mid1

  • YAP1 nuclear translocation (YAP1 ON state)

• At upregulated levels (M2 macrophages): Inhibited IL-1β production via:

  • Direct sequestration of YAP1 in cytoplasm (YAP1 OFF state)

  • Reduced Mid1 interaction

Methodologically, to study this dual role:

• Use graded concentrations of recombinant Erdr1 (0.1-1000 ng/mL) in treatment protocols

• Monitor subcellular localization of YAP1 and Mid1 through immunofluorescence or subcellular fractionation

• Employ co-immunoprecipitation assays to detect protein-protein interactions at different concentrations

• Utilize CRISPR/Cas9 knockout models of YAP1 and Mid1 to validate specificity of pathways

The Erdr1-YAP1-Mid1 regulatory axis manifests differently depending on Erdr1 concentration, which researchers should carefully control in experimental design .

What experimental approaches can best elucidate the interaction between Erdr1 and the YAP1 pathway?

To investigate the Erdr1-YAP1 interaction and its downstream effects:

• Protein-protein interaction studies:

  • Co-immunoprecipitation assays to confirm direct binding

  • Proximity ligation assays to visualize interactions in situ

  • FRET/BRET analyses for dynamic interaction studies

  • Domain mapping using truncated constructs to identify interaction regions

• Functional validation approaches:

  • YAP1 reporter assays (TEAD luciferase reporters) to measure transcriptional activity

  • ChIP-seq to identify YAP1 binding sites affected by Erdr1

  • RNA-seq to profile global transcriptional changes

  • YAP1 phosphorylation status assessment via Western blot

• Signaling pathway analysis:

  • Monitor expression of YAP1 target genes (CTGF, Cyr61) in response to Erdr1 modulation

  • Assess nuclear/cytoplasmic distribution of YAP1 using immunofluorescence

  • Evaluate activation status of upstream regulators (MST1/2, LATS1/2)

Research has demonstrated that Erdr1 regulates YAP1 through the non-classical Hippo pathway, and when Erdr1 is overexpressed at moderate levels, it promotes LPS-induced YAP1 expression and activation of downstream targets like CTGF and Cyr61 .

What are the methodological considerations for studying Erdr1-mediated macrophage polarization?

Studying Erdr1's role in macrophage polarization requires careful experimental design:

• Cell culture considerations:

  • Cell density is critical - maintain consistent plating density (suggested: 2-5×10^5 cells/mL)

  • Use primary bone marrow-derived macrophages for physiological relevance

  • Include RAW264.7 cells for high-throughput screening approaches

  • Control timing of LPS (M1) or IL-4 (M2) stimulation relative to Erdr1 treatment

• Polarization assessment:

  • Flow cytometry markers: CD80, CD86, MHC-II (M1); CD206, CD163 (M2)

  • Cytokine profiling: TNF-α, IL-6, IL-1β (M1); IL-10, TGF-β (M2)

  • Gene expression analysis: iNOS, CXCL10 (M1); Arg1, Ym1, Fizz1 (M2)

  • Metabolic assays: glycolysis (M1); oxidative phosphorylation (M2)

• Experimental validation:

  • Use Erdr1 knockout/knockdown models with rescue experiments

  • Compare dose-dependent effects of recombinant Erdr1 (10-1000 ng/mL range)

  • Implement YAP1 and Mid1 modulation to confirm pathway specificity

Research has shown that at decent cell density, Erdr1 is significantly downregulated in LPS-induced M1 macrophages but dramatically upregulated in IL-4-induced M2 macrophages compared to naive macrophages (M0) . This differential expression pattern is central to Erdr1's role in orchestrating macrophage polarization and should be carefully monitored in experimental protocols.

How can researchers accurately measure the dynamic interactions between Erdr1, YAP1, and Mid1?

The dynamic interactions between Erdr1, YAP1, and Mid1 require sophisticated methodological approaches:

• Real-time interaction analysis:

  • Live-cell imaging with fluorescently tagged proteins

  • FRET/BRET assays to detect proximity changes over time

  • Fluorescence correlation spectroscopy for quantitative binding kinetics

  • Single-molecule tracking to observe individual interaction events

• Domain-specific interaction mapping:

  • Generate domain deletion constructs of Erdr1, YAP1, and Mid1

  • Perform systematic co-immunoprecipitation with domain mutants

  • Use peptide arrays to identify specific binding motifs

  • Apply hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

• Contextual dependency evaluation:

  • Study interactions under varying conditions:

    • Different Erdr1 concentrations (10-1000 ng/mL)

    • Inflammatory stimuli (LPS, IL-4)

    • Cell density variations

    • Metabolic states

• Quantitative analysis methods:

  • Surface plasmon resonance for binding affinity measurements

  • Isothermal titration calorimetry for thermodynamic parameters

  • Analytical ultracentrifugation for complex formation analysis

Research has demonstrated that Erdr1 interacts with YAP1 and Mid1 through distinct domains, with the interactions changing dynamically based on Erdr1 concentration . These interactions govern the regulation of IL-1β production and determine macrophage polarization fate. The intrinsically disordered nature of Erdr1 enables these dynamic interactions, making careful methodological consideration essential.

What are the implications of Erdr1's role in high-altitude adaptation and hypoxia response?

While direct evidence specifically linking Erdr1 to high-altitude adaptation is limited, several research findings suggest potential involvement:

• Erythropoietic regulation:

  • Erdr1 was initially identified as an inducer of hemoglobin synthesis

  • High-altitude adaptation in Tibetans involves genes regulating hemoglobin levels (EGLN1, EPAS1)

  • Research methodology should include:

    • Erythroid colony formation assays under hypoxic conditions

    • Hemoglobin quantification in Erdr1-treated cells

    • Gene expression analysis of globin genes

• Hypoxia signaling interface:

  • Potential interactions between Erdr1 and hypoxia pathways:

    • Examine effects on HIF-1α and HIF-2α stability and activity

    • Assess oxygen-dependent protein hydroxylation

    • Evaluate transcriptional effects on hypoxia-responsive genes

• Population genetics approach:

  • Analyze Erdr1 genetic variants in high-altitude populations

  • Perform association studies between Erdr1 variants and physiological traits

  • Conduct functional validation of altitude-associated variants

• Experimental models:

  • Hypobaric hypoxia chambers to simulate high altitude

  • Genetically modified mouse models with Erdr1 modulation

  • Cell culture systems with controlled oxygen tension

To properly investigate this relationship, researchers should design experiments that specifically assess Erdr1's function under hypoxic conditions and compare its activity between lowland and highland adapted cell models.

What are the optimal expression systems for producing recombinant mouse Erdr1?

Different expression systems offer various advantages for recombinant mouse Erdr1 production:

For functional studies, mammalian expression systems are recommended despite lower yields, as Erdr1's intrinsically disordered nature may be sensitive to proper folding and post-translational modifications. When using E. coli systems, extensive validation of protein functionality is essential.

Purification protocols should include:

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

• Intermediate purification by ion exchange chromatography

• Polishing step using size exclusion chromatography

• Endotoxin removal for immune cell applications

• Sterile filtration and quality control testing

How can researchers accurately measure Erdr1 concentration-dependent effects in experimental systems?

Due to Erdr1's bell-shaped response curve in many biological systems , accurately measuring concentration-dependent effects requires specific methodological approaches:

• Concentration range determination:

  • Perform initial broad-range dose-response experiments (0.1-1000 ng/mL)

  • Narrow down to critical concentration ranges based on observed inflection points

  • Use at least 5-7 concentration points across the functional range

• Quantification methods:

  • Develop and validate a sensitive ELISA for Erdr1 quantification

  • Use quantitative Western blotting with recombinant protein standards

  • Consider MS-based absolute quantification methods

• Experimental design considerations:

  • Include concentration controls in all experiments

  • Account for cell density effects on Erdr1 function

  • Monitor protein stability throughout experimental timeframes

  • Control for potential carrier protein effects

• Cell-based reporter systems:

  • Develop cell lines with fluorescent/luminescent reporters for Erdr1-responsive elements

  • Monitor YAP1 translocation as a readout for Erdr1 activity

  • Measure IL-1β production in macrophage systems

When interpreting results, graph all data points rather than selecting specific concentrations to fully visualize the bell-shaped response curve. Statistical analysis should include non-linear regression models appropriate for non-monotonic dose-response relationships.

What controls should be included when studying Erdr1's interaction with YAP1 and Mid1 pathways?

Robust controls are essential when investigating the complex interactions between Erdr1, YAP1, and Mid1:

• Genetic controls:

  • YAP1 knockout/knockdown cells (negative control for YAP1-dependent effects)

  • Mid1 knockout/knockdown cells (negative control for Mid1-dependent effects)

  • YAP1/Mid1 double knockout cells (to assess pathway independence)

  • Rescue experiments with wild-type and mutant constructs

• Protein interaction controls:

  • Irrelevant protein of similar size/structure (negative control)

  • Known YAP1/Mid1 interacting proteins (positive control)

  • Heat-denatured Erdr1 (to control for non-specific binding)

  • Competition assays with unlabeled proteins

• Pathway activation controls:

  • Hippo pathway activators (e.g., high cell density, contact inhibition)

  • Hippo pathway inhibitors (e.g., mechanical stress, serum stimulation)

  • YAP1 inhibitors (e.g., verteporfin)

  • Mid1 pathway modulators

• Experimental technique controls:

  • Input controls for co-immunoprecipitation

  • Subcellular fractionation purity controls

  • Antibody specificity validation

  • Isotype controls for immunofluorescence

Research has shown that Erdr1's moderate overexpression promotes LPS-induced IL-1β production, but this promotion is effectively blocked in Mid1 knockout, YAP1 knockout, or Mid1/YAP1 double knockout macrophages . These genetic models serve as critical controls for validating pathway specificity.

How might Erdr1 be utilized in research on inflammatory diseases?

Given Erdr1's role in macrophage polarization and IL-1β regulation, it has significant potential applications in inflammatory disease research:

• Mechanistic studies:

  • Investigate Erdr1 expression in inflammatory disease tissues

  • Analyze correlation between Erdr1 levels and disease severity

  • Examine genetic variants in Erdr1 associated with inflammatory conditions

  • Study the YAP1-Erdr1-Mid1 axis in disease models

• Therapeutic exploration:

  • Use recombinant Erdr1 to modulate macrophage polarization

  • Develop Erdr1 mimetics targeting specific interaction domains

  • Create blocking antibodies against Erdr1 for pathway inhibition

  • Explore gene therapy approaches to modulate Erdr1 expression

• Disease models for investigation:

  Disease	Relevant Erdr1 Function	Experimental Model
  Rheumatoid arthritis	Macrophage M1/M2 balance	Collagen-induced arthritis
  Inflammatory bowel disease	IL-1β regulation	DSS-induced colitis
  Atherosclerosis	Foam cell formation	ApoE-/- mice on high-fat diet
  Asthma/allergic inflammation	M2 polarization	OVA sensitization model
  Sepsis	Cytokine storm regulation	LPS challenge model

• Biomarker development:

  • Assess Erdr1 as a prognostic or diagnostic marker

  • Monitor Erdr1 levels during therapy response

  • Develop assays for Erdr1-YAP1-Mid1 pathway activation

Research has demonstrated that Erdr1 orchestrates macrophage polarization and determines cell fate via dynamic interaction with YAP1 and Mid1 , making it a promising target for inflammatory disease research.

What are the emerging applications of Erdr1 in olfactory and gustatory research?

Recent evidence suggests that Erdr1 may play a role in olfactory and gustatory function, particularly in pathological conditions:

• Olfactory/gustatory dysfunction research:

  • Erdr1 regulation has been implicated in COVID-19-related anosmia through GATA-1-mediated pathways

  • Erythroid membrane antigens associated with Erdr1 function are linked to Type II congenital smell loss

  • Research approaches should include:

    • Erdr1 expression analysis in olfactory epithelium

    • Functional studies in olfactory sensory neurons

    • Investigation of Erdr1 in taste bud regeneration

• Methodology for olfactory/gustatory research:

  • Olfactory epithelium organoid systems with Erdr1 modulation

  • Calcium imaging in olfactory neurons treated with recombinant Erdr1

  • Taste bud organoid development studies

  • Behavioral testing in Erdr1 conditional knockout mice

• Potential mechanisms:

  • Erdr1 may influence olfactory neuron development or regeneration

  • Potential modulation of inflammatory responses in olfactory epithelium

  • Possible regulation of taste bud cell turnover

  • Indirect effects through GATA-1 transcriptional regulation

When designing experiments to study Erdr1's role in sensory systems, researchers should consider both developmental contexts and regenerative processes, as well as the impact of inflammatory conditions on sensory function.

How can researchers investigate the evolutionary significance of Erdr1 in high-altitude adaptation?

To investigate the evolutionary significance of Erdr1 in high-altitude adaptation:

• Population genetics approaches:

  • Sequence the Erdr1 gene locus in high-altitude and lowland populations

  • Perform haplotype analysis to identify selection signatures

  • Calculate FST and other measures of genetic differentiation

  • Compare with known high-altitude adaptation genes like EGLN1 and EPAS1

• Functional genomics methods:

  • Generate cell lines carrying altitude-associated Erdr1 variants

  • Test variant function under normoxic and hypoxic conditions

  • Assess hemoglobin regulation in erythroid progenitors

  • Investigate oxygen sensing pathway interactions

• Physiological studies:

  • Measure Erdr1 expression in response to hypoxia

  • Assess the impact of Erdr1 variants on erythropoiesis

  • Examine effects on oxygen transport and utilization

  • Study interaction with hypoxia-inducible factors

• Comparative biology:

  • Compare Erdr1 sequences and function across species adapted to different altitudes

  • Analyze convergent evolution in multiple high-altitude populations

  • Examine related genes in the same pathway for parallel selection

While Erdr1 has not been directly identified in genome-wide selection scans of high-altitude populations, its role in erythroid differentiation and hemoglobin regulation suggests potential involvement in adaptation mechanisms. Future research should explore potential epistatic interactions between Erdr1 and known altitude adaptation genes like EGLN1 and EPAS1 .

What approaches should be used to investigate the translational potential of Erdr1 in therapeutic applications?

To evaluate Erdr1's therapeutic potential:

• Pre-clinical model development:

  • Generate conditional Erdr1 knockout mouse models

  • Develop targeted delivery systems for recombinant Erdr1

  • Establish disease models relevant to Erdr1 function

  • Create humanized mouse models to bridge species differences

• Therapeutic strategy evaluation:

  Approach	Mechanism	Target Conditions	Assessment Methods
  Recombinant Erdr1	Supplement/replace function	Inflammatory disorders	Efficacy in disease models, PK/PD profiling
  Erdr1 mimetic peptides	Target specific interactions	Autoimmune diseases	Binding assays, functional validation
  Gene therapy	Regulate endogenous expression	Genetic disorders	Vector delivery efficiency, expression stability
  Pathway modulators	Target YAP1-Mid1 interaction	Cancer, inflammation	Target engagement, pathway activation

• Safety and efficacy assessment:

  • Determine optimal dosing based on bell-shaped response curve

  • Evaluate potential off-target effects across multiple tissues

  • Assess immunogenicity of recombinant protein

  • Conduct toxicology studies in multiple species

• Biomarker development:

  • Identify patient populations likely to respond

  • Develop companion diagnostics for Erdr1 pathway activation

  • Establish pharmacodynamic markers for clinical trials

  • Create assays for monitoring treatment response

When designing translational studies, researchers must account for Erdr1's concentration-dependent dual functions and ensure that therapeutic approaches achieve the intended biological effect rather than the opposite response due to concentration variations.

How can researchers address the challenges of Erdr1's intrinsically disordered structure in experimental studies?

The intrinsically disordered nature of Erdr1 presents unique challenges that require specific strategies:

When interpreting experimental results, researchers should explicitly consider that observed effects might result from an ensemble of Erdr1 conformations rather than a single structure. This is particularly important when designing interaction studies with YAP1 and Mid1.

What are common pitfalls in studying Erdr1's dual role in macrophage polarization, and how can they be avoided?

Several challenges can complicate the study of Erdr1's role in macrophage polarization:

Research has shown that Erdr1's effects on IL-1β production follow a bell-shaped curve, with promotion at moderate levels and inhibition at higher levels . This non-monotonic response emphasizes the importance of comprehensive concentration testing in experimental designs.

What strategies help ensure reproducible results when working with recombinant Erdr1?

To enhance reproducibility in Erdr1 research:

• Protein quality control:

  • Implement batch-to-batch consistency testing

  • Verify protein integrity by SDS-PAGE before each experiment

  • Conduct activity assays on each preparation

  • Store reference aliquots for comparative testing

• Experimental standardization:

  • Develop and follow detailed standard operating procedures

  • Control environmental variables (temperature, CO2, humidity)

  • Use consistent cell passage numbers (preferably p3-p10)

  • Standardize reagent sources and preparation methods

• Quantification and validation:

  • Employ multiple methodologies for key measurements

  • Include internal controls in each experiment

  • Blind analysis where possible

  • Perform biological replicates across different days/preparations

• Reporting and documentation:

  • Document all experimental conditions in detail

  • Report protein concentration determination method

  • Include information on storage conditions and time

  • Specify exact buffer compositions and cell culture conditions

A practical approach is to prepare a single large batch of recombinant Erdr1, divide into single-use aliquots, and use the same batch for an entire study. This eliminates variation introduced by different protein preparations.

How can conflicting results in Erdr1 research be reconciled and interpreted?

When faced with conflicting results in Erdr1 research:

• Systematic reconciliation approach:

  • Create a comparison table of experimental conditions across studies

  • Identify key methodological differences that may explain discrepancies

  • Perform bridging studies that systematically vary critical parameters

  • Develop standardized protocols based on reconciliation findings

• Critical variables to examine:

  • Erdr1 concentration ranges used

  • Cell types and their activation states

  • Cell density and culture conditions

  • Timing of measurements

  • Presence of additional stimuli or inhibitors

• Mechanistic investigation:

  • Determine if contextual factors explain different outcomes

  • Assess whether concentration-dependent dual effects are responsible

  • Examine potential involvement of different signaling partners

  • Consider post-translational modifications or isoform differences

• Collaborative approach:

  • Establish multi-laboratory validation studies

  • Share reagents and protocols between research groups

  • Create community standards for Erdr1 research

  • Develop reference datasets for benchmarking