Recombinant Chicken Nuclear factor erythroid 2-related factor 1 (NFE2L1), partial

What is chicken NFE2L1 and what are its primary functions?

NFE2L1 (also known as Nrf1) belongs to the cap'n'collar (CNC)-basic region leucine zipper (bZIP) subfamily of transcription factors. It plays crucial roles in:

• Regulating cellular responses to oxidative stress

• Maintaining redox homeostasis

• Controlling proteostasis through proteasome regulation

• Influencing metabolic processes and differentiation

The protein dimerizes with small Maf proteins (MAFF, MAFG, or MAFK) to bind antioxidant response elements (AREs) in target gene promoters . NFE2L1 is essential for development, as demonstrated by embryonic lethality in knockout models, and tissue-specific knockouts have shown it protects against conditions like neurodegeneration and steatohepatitis .

How does the structure of chicken NFE2L1 compare to its mammalian counterparts?

Chicken NFE2L1 shares the conserved functional domains found in mammalian NFE2L1, including:

• Acidic domains (AD1 and AD2) for transactivation

• Asparagine/serine/threonine-rich domain (NST) between AD1 and AD2

• CNC (cap'n'collar) motif for DNA binding specificity

• Basic-leucine zipper (bZIP) domain for dimerization and DNA binding

• Serine-rich domain near the CNC motif

• C-terminal domain (CTD) contributing to activation function

Interestingly, the chicken genome shows unique genomic arrangements, with evidence of fusion events involving the NFE2L1 gene on chromosome 27 and other genes, such as Ku70 on chromosome 1 .

What recombinant forms of chicken NFE2L1 are available for research?

Several recombinant forms are available:

• Full-length chicken NFE2L1 protein

• Partial constructs focusing on specific functional domains

• Tagged versions (commonly His-tagged) for purification and detection

For example, CUSABIO offers a partial recombinant chicken NFE2L1 (product code CSB-YP716980CH) with >85% purity expressed in yeast systems , while other manufacturers provide similar products with varying specifications.

How should recombinant chicken NFE2L1 be stored and handled for optimal stability?

For optimal stability and activity:

For reconstitution:

• Briefly centrifuge vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Avoid repeated freeze-thaw cycles

What methodologies can be used to study chicken NFE2L1 binding to antioxidant response elements (AREs)?

Several complementary approaches can be employed:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Prepare labeled oligonucleotides containing chicken ARE sequences

  • Incubate with recombinant chicken NFE2L1 (potentially with small Maf proteins)

  • Visualize binding through gel shift patterns

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link protein-DNA complexes in chicken cells

  • Immunoprecipitate using NFE2L1-specific antibodies

  • Analyze bound DNA sequences through PCR or sequencing

• Luciferase Reporter Assays:

  • Clone chicken ARE elements upstream of luciferase reporter genes

  • Co-transfect with NFE2L1 expression vectors into chicken cell lines (e.g., DF-1)

  • Measure reporter activity as shown in similar approaches with circGHR studies

How can the subcellular localization of NFE2L1 be investigated in chicken cells?

Based on studies in other species, NFE2L1 undergoes dynamic subcellular distribution:

• Immunofluorescence Microscopy:

  • Culture chicken cells (e.g., DF-1, LMH, primary hepatocytes)

  • Apply various stressors (oxidative agents, proteasome inhibitors)

  • Fix and stain using NFE2L1-specific antibodies

  • Co-stain with organelle markers (ER, nucleus)

• Subcellular Fractionation:

  • Separate nuclear, cytoplasmic, and ER membrane fractions

  • Detect NFE2L1 distribution by Western blotting

  • Compare fractions under normal vs. stress conditions

Under normal conditions, NFE2L1 is primarily localized to the ER membrane. Upon stress exposure, it undergoes cleavage and translocation to the nucleus to activate target genes .

What is the relationship between chicken NFE2L1 and the Keap1/Nrf2 signaling pathway?

While NFE2L2 (Nrf2) is the primary mediator of the Keap1/Nrf2 pathway, NFE2L1 plays important complementary roles:

• Both NFE2L1 and NFE2L2 regulate ARE-driven stress-responsive genes

• They often have overlapping yet distinct sets of target genes

• In chickens, the Keap1/Nrf2 pathway is crucial for intestinal oxidative stress responses

Studies with Bacillus amyloliquefaciens in broiler chickens have shown modulation of this pathway affects:

• Antioxidant enzyme levels (T-AOC, CAT, GSH-Px)

• Inflammatory markers (TNF-α, IL-1β)

• Intestinal barrier integrity genes (Claudin, Occludin1, ZO-1, MUC2)

This suggests that targeting NFE2L1 could provide complementary benefits to Nrf2 activation in poultry health management.

How does NFE2L1 regulate proteasome function in response to cellular stress?

NFE2L1 plays a critical role in proteasome homeostasis through a feedback mechanism:

• Under normal conditions, NFE2L1 is localized to the ER membrane and rapidly degraded via ERAD (ER-associated degradation)

• During proteasome impairment:

  • NFE2L1 degradation decreases

  • It undergoes proteolytic processing

  • The processed form translocates to the nucleus

  • It binds ARE elements in promoters of proteasome subunit genes

  • This induces expression of proteasome components

This "bounce-back effect" helps restore proteasomal function during stress conditions. This mechanism may be especially relevant in aging and neurodegenerative contexts, where enhancing NFE2L1 function could potentially upregulate proteasome activity and reduce pathology .

What approaches can be used to study NFE2L1 isoforms in chicken tissues?

Multiple NFE2L1 isoforms have been identified in mammals, and similar diversity likely exists in chickens:

• RT-qPCR with Isoform-Specific Primers:

  • Design primers targeting unique exons or exon junctions

  • Perform quantitative PCR across different chicken tissues

  • Compare expression patterns as done for NFE2L1-616 in human tissues

• Western Blotting:

  • Use antibodies targeting common regions or isoform-specific epitopes

  • Compare migration patterns in different chicken tissues

  • Validate with recombinant controls of known isoforms

• RNA-Seq Analysis:

  • Perform deep sequencing of chicken tissue transcriptomes

  • Analyze for alternative transcription start sites and splicing events

  • Validate findings with targeted RT-PCR

Different NFE2L1 isoforms may have tissue-specific expression patterns and distinct functions in stress response regulation .

How can chicken NFE2L1 be used to study the molecular basis of oxidative stress responses in avian systems?

Chicken NFE2L1 provides a valuable model for comparative studies of oxidative stress response:

• Tissue-Specific Knockout/Knockdown Studies:

  • Use CRISPR/Cas9 to generate NFE2L1-deficient chicken cell lines

  • Create conditional knockout models in specific tissues

  • Challenge with oxidative stressors and assess phenotypes

• Comparative Transcriptomics:

  • Compare gene expression profiles between wild-type and NFE2L1-modified cells

  • Identify chicken-specific vs. conserved NFE2L1 target genes

  • Map the avian antioxidant response network

• Metabolic Flux Analysis:

  • Measure changes in redox-related metabolites after NFE2L1 modulation

  • Trace isotope-labeled precursors through antioxidant pathways

  • Compare with mammalian systems to identify avian-specific features

These approaches can reveal evolutionary adaptations in avian stress responses with potential applications in poultry health and comparative physiology.

How might the fusion events between chicken NFE2L1 and Ku70 impact cellular function?

The discovered fusion between NFE2L1 and Ku70 genes in chickens represents an intriguing area for investigation:

• Structural Implications:

  • The fusion results in Ku70 with 18 additional N-terminal amino acids

  • This modified protein still localizes to the nucleus and forms heterodimers with Ku80

  • The protein maintains its ability to rapidly accumulate at DNA damage sites

• Functional Consequences:

  • Potential cross-regulation between DNA repair and oxidative stress pathways

  • May influence how chickens respond to genotoxic and oxidative stressors

  • Could provide evolutionary advantages specific to avian physiology

• Research Applications:

  • Previous studies using GdKu70 cDNA may need reinterpretation

  • The fusion provides a natural model to study domain interactions

  • May reveal novel regulatory mechanisms between stress response pathways

This fusion phenomenon highlights the importance of species-specific considerations when working with recombinant proteins and interpreting cross-species functional studies .

What are the most promising applications of recombinant chicken NFE2L1 in agricultural and biomedical research?

Recombinant chicken NFE2L1 offers valuable opportunities in several research areas:

• Poultry Health and Productivity:

  • Development of nutritional supplements targeting NFE2L1 pathways

  • Creation of biomarkers for stress resilience in breeding programs

  • Design of management strategies to enhance natural antioxidant defenses

• Comparative Biology:

  • Understanding evolutionary adaptations in stress response mechanisms

  • Identifying avian-specific regulatory pathways

  • Elucidating the functional significance of gene fusion events

• Biomedical Applications:

  • Using chicken systems as alternative models for studying proteostasis

  • Investigating species-specific differences in age-related protein degradation

  • Developing therapeutic approaches targeting conserved NFE2L1 functions

Future research will likely focus on integrating multi-omics approaches to fully map the NFE2L1 regulatory network in chickens and compare it with other species to identify both conserved and divergent features.

What technical challenges remain in working with recombinant chicken NFE2L1?

Several technical challenges should be addressed in future research:

• Post-translational Modifications:

  • Current recombinant systems may not reproduce all native modifications

  • Understanding glycosylation patterns at the NST domain

  • Characterizing phosphorylation and ubiquitination dynamics

• Structural Analysis:

  • Obtaining crystal structures of chicken-specific domains

  • Resolving membrane topology of the full-length protein

  • Characterizing conformational changes during activation

• Isoform-Specific Functions:

  • Developing tools to study individual isoforms separately

  • Creating isoform-specific antibodies

  • Establishing chicken cell models expressing specific variants

Addressing these challenges will enhance our understanding of NFE2L1 biology and improve the utility of recombinant proteins for research applications.