Recombinant Mesocricetus auratus Myc-associated zinc finger protein (MAZ)

What is the structural composition of Mesocricetus auratus MAZ protein?

Mesocricetus auratus MAZ (Myc-associated zinc finger protein) is a transcription factor characterized by proline-rich regions, alanine repeats, and six C₂H₂-type zinc finger motifs. The protein contains five putative phosphorylation sites for casein kinase II (CKII). The full-length protein consists of 331 amino acids with a sequence that includes multiple alanine repeats and zinc finger domains essential for DNA binding. Structurally, MAZ forms a homotrimer, similar to its human counterpart, though with some minor structural variations . The sequence includes distinctive elements such as glycine-rich regions and multiple zinc-coordinating motifs that are critical for its function as a transcriptional regulator.

What are the primary functions of MAZ protein in normal cellular processes?

MAZ plays crucial roles in chromatin organization and gene transcription regulation. It functions primarily by binding to pyrimidine-rich DNA sequences, particularly in the nuclease-hypersensitive element (NHE) in the 5'-end promoter region of the c-myc gene. Phosphorylation of MAZ by CKII at serine residue 480 is required for maximum DNA-binding activity and subsequent enhancement of gene expression . Additionally, MAZ is involved in regulating immunity-related pathways, particularly through interaction with STAT1 signaling. Approximately 80% of occupied STAT1-binding sites colocalize with MAZ-binding sites in cells after IFN-γ stimulation, suggesting MAZ plays a significant role in modulating immune responses . Recent studies indicate that MAZ contributes to the epigenetic control of immunity-related gene expression by altering the chromatin landscape.

What expression systems are most effective for producing recombinant Mesocricetus auratus MAZ protein?

Multiple expression systems have been successfully employed for recombinant MAZ production, each with specific advantages for different research applications:

• Yeast expression system: This has been effectively used to produce recombinant Golden Syrian Hamster MAZ protein with high purity (>90%) . Yeast systems often provide proper protein folding and some post-translational modifications.

• Bacterial expression systems: While not explicitly mentioned for hamster MAZ in the search results, E. coli systems are commonly used for initial protein characterization studies due to their high yield and simplicity.

• Mammalian cell expression: For functional studies requiring proper post-translational modifications, particularly phosphorylation critical to MAZ function, mammalian cell systems such as HEK293 are preferable. These systems can achieve yields of over 5 g/L for recombinant proteins .

When selecting an expression system, researchers should consider the specific experimental requirements, particularly whether post-translational modifications like phosphorylation are essential for the intended functional studies.

What purification strategies yield the highest purity for recombinant MAZ protein?

For high-purity recombinant MAZ protein isolation, a multi-step purification strategy is recommended:

• Affinity chromatography: Utilizing His-tag affinity purification (as seen with commercially available recombinant hamster MAZ) . This approach allows for selective binding of the tagged protein to nickel or cobalt resins.

• Size exclusion chromatography: Essential for separating properly folded trimeric MAZ from monomers or aggregates.

• Ion exchange chromatography: As a polishing step to remove contaminants with similar molecular weights but different charge properties.

Quality assessment should include SDS-PAGE, Western blotting, and analytical SEC (HPLC) to confirm purity levels >90% . For functional studies, additional verification of proper folding through circular dichroism spectroscopy and DNA-binding assays is recommended.

How can the DNA-binding activity of recombinant MAZ be accurately assessed?

To evaluate the DNA-binding activity of recombinant MAZ protein, several complementary techniques provide robust analysis:

• Electrophoretic mobility shift assay (EMSA): This technique can determine binding affinity to pyrimidine-rich DNA sequences from the nuclease-hypersensitive element (NHE) in the c-myc promoter region. Adding increasing concentrations of phosphorylated versus non-phosphorylated MAZ protein can reveal the impact of phosphorylation on binding affinity .

• Chromatin immunoprecipitation (ChIP): For assessing genomic binding sites in cellular contexts, particularly important when examining colocalization with transcription factors like STAT1 .

• Luciferase reporter assays: These can be used to evaluate MAZ's ability to enhance expression from a c-myc promoter/luciferase reporter gene construct, providing functional validation of DNA-binding activity .

When designing these experiments, it's crucial to account for the phosphorylation state of MAZ, as mutation of serine at position 480 to alanine has been shown to eliminate DNA-binding activity to the NHE element .

What methods effectively evaluate the phosphorylation status of MAZ protein?

Assessing MAZ phosphorylation status is critical given its impact on function. The following methodological approaches are recommended:

• In vitro kinase assays: Using purified casein kinase II (CKII) and recombinant MAZ protein with radioactive ³²P-ATP to measure phosphorylation levels. Site-directed mutagenesis of putative phosphorylation sites (particularly serine 480) can identify key regulatory residues .

• Phospho-specific antibodies: Development or use of antibodies that specifically recognize phosphorylated serine 480 for Western blot analysis.

• Mass spectrometry: For comprehensive mapping of all phosphorylation sites, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) provides the most detailed analysis.

• Phos-tag SDS-PAGE: This technique provides enhanced separation of phosphorylated protein species from non-phosphorylated forms, allowing visualization of multiple phosphorylation states.

For in vivo phosphorylation studies, combining immunoprecipitation with phospho-specific detection methods enables assessment of MAZ phosphorylation status under various cellular conditions and CKII inhibitor treatments .

How can recombinant MAZ protein be utilized in cancer research models using Mesocricetus auratus?

Recombinant MAZ protein is valuable for investigating cancer mechanisms in Syrian golden hamster models, particularly given MAZ's established links to multiple cancer types including glioblastoma, breast cancer, prostate cancer, and liposarcoma . Methodological approaches include:

• Xenograft and orthotopic tumor models: Syrian golden hamsters serve as clinically relevant animal models for human diseases . Recombinant MAZ can be used to study effects on tumor growth, as demonstrated in similar studies with other proteins like MIF that significantly enhanced pancreatic tumor growth and tumor-associated angiogenesis in hamsters .

• Molecular intervention studies: Examining how MAZ modulates the proinflammatory response in cancer models via STAT3 signaling pathways .

• Gene expression profiling: RNA-Seq analysis following MAZ modulation to identify downstream target genes relevant to cancer progression.

When designing these experiments, researchers should account for the species-specific interactions between hamster MAZ and other transcriptional regulators, as well as the high structural similarity between hamster and human MAZ that makes this model particularly relevant for translational research.

What role does MAZ play in inflammatory disease models, and how can recombinant protein be applied in these studies?

MAZ has been implicated in inflammatory disease mechanisms through its regulation of immune-related genes via the STAT1 pathway. Research applications include:

• Colitis and inflammatory bowel disease models: MAZ controls proinflammatory responses in colitis via STAT3 signaling . Recombinant MAZ can be used to investigate the molecular mechanisms underlying this regulation.

• Immune response modulation studies: Since MAZ depletion significantly suppresses immune response genes including IFN-stimulated genes like IRF8 and Absent in Melanoma 2 , recombinant protein can help clarify its role in immune regulation.

• Epigenetic landscape analysis: MAZ alters the epigenetic landscape in chromatin to control immunity-related gene expression . ChIP-seq studies using recombinant MAZ can map these changes across the genome.

Methodologically, researchers should:

• Conduct side-by-side comparisons with human MAZ to validate translational relevance

• Combine recombinant protein studies with genetic manipulation approaches (siRNA, CRISPR)

• Evaluate downstream effects using cytokine profiling and immune cell function assays

How does MAZ interact with the STAT1 pathway in regulating IFN-γ-stimulated genes?

Recent research has uncovered sophisticated interactions between MAZ and STAT1 in regulating interferon responses. Approximately 80% of occupied STAT1-binding sites colocalize with MAZ-binding sites in cells after IFN-γ stimulation, indicating coordinated genomic targeting . Methodologically, this interaction can be investigated through:

• Sequential ChIP (ChIP-reChIP): To confirm co-occupancy of MAZ and STAT1 at specific genomic loci.

• Proximity ligation assays: To detect physical interaction between MAZ and STAT1 proteins in situ.

• Protein interaction studies: Co-immunoprecipitation and FRET/BRET analyses can determine whether MAZ directly interacts with STAT1 or functions through indirect mechanisms.

• Genome-wide binding analysis: ChIP-seq for both MAZ and STAT1 under various stimulation conditions reveals the temporal dynamics of their co-regulation .

The functional consequence of MAZ depletion is significant reduction in STAT1 binding across the genome, suggesting MAZ may function as a pioneer factor or stabilize STAT1 binding to chromatin. This relationship appears particularly important for regulation of immune response genes, including IRF8 and Absent in Melanoma 2 .

What epigenetic mechanisms does MAZ employ to regulate gene expression in Mesocricetus auratus?

MAZ contributes to gene regulation through sophisticated epigenetic mechanisms that alter chromatin structure. Research strategies to investigate these mechanisms include:

• Integrated ChIP-seq and ATAC-seq analysis: To correlate MAZ binding with changes in chromatin accessibility.

• CUT&RUN or CUT&Tag: For high-resolution mapping of MAZ binding sites with lower background than traditional ChIP.

• ChIP-seq for histone modifications: To assess how MAZ binding correlates with activating (H3K4me3, H3K27ac) or repressive (H3K27me3, H3K9me3) histone marks.

• 3D chromatin organization studies: Using Hi-C or related techniques to determine if MAZ influences long-range chromatin interactions.

Studies have shown that MAZ controls expression of immunity-related genes by changing the epigenetic landscape in chromatin . This suggests MAZ may recruit chromatin modifiers or pioneer the opening of condensed chromatin regions. Since MAZ depletion significantly alters gene expression profiles, particularly of immune response genes, it likely plays a critical role in maintaining proper epigenetic states at its target loci.

What are the main challenges in producing functional recombinant MAZ protein, and how can they be overcome?

Producing fully functional recombinant MAZ protein presents several technical challenges:

• Post-translational modification requirements: MAZ function depends on proper phosphorylation, particularly at serine 480 . Solutions include:

  • Using mammalian expression systems that provide appropriate kinase activity

  • Co-expression with CKII or in vitro phosphorylation following purification

  • Implementing phosphomimetic mutations (S→D or S→E) for functional studies

• Solubility and proper folding: Zinc finger proteins often face solubility issues. Strategies include:

  • Optimization of expression conditions (temperature, induction parameters)

  • Use of solubility tags (MBP, SUMO, etc.) with TEV cleavage sites

  • Refolding protocols in the presence of zinc ions

• Protein stability during purification: Maintaining zinc finger integrity requires:

  • Including zinc in all buffers (typically 10-50 μM ZnCl₂)

  • Adding reducing agents to prevent cysteine oxidation

  • Implementing rapid purification protocols at 4°C

• Functional verification: Confirming proper activity through:

  • DNA-binding assays using known MAZ targets

  • Reporter gene assays in cellular systems

  • Structure validation through circular dichroism or limited proteolysis

How can researchers address antibody specificity issues when studying MAZ in Syrian golden hamster models?

Antibody specificity is a significant challenge when working with Syrian golden hamster proteins due to limited commercial reagents . Methodological solutions include:

• Validation of cross-reactive antibodies:

  • Test human/mouse anti-MAZ antibodies for cross-reactivity with hamster MAZ

  • Validate using Western blot against recombinant hamster MAZ protein

  • Perform peptide competition assays to confirm specificity

• Development of hamster-specific antibodies:

  • Generate custom antibodies using recombinant hamster MAZ as immunogen

  • Validate using tissues from multiple hamster specimens with appropriate controls

  • Perform immunohistochemistry with peptide blocking to confirm specificity

• Alternative detection methods:

  • Epitope tagging (HA, FLAG, etc.) of MAZ in expression constructs

  • RNA-based detection methods (RNA-FISH, qRT-PCR) to bypass antibody requirements

  • CRISPR knock-in of tags into endogenous loci

• Protein engineering approaches:

  • Production of recombinant MAZ protein with various epitope tags that can be detected with well-characterized tag antibodies

  • Development of activity-based probes for detecting functional MAZ

When designing experiments, researchers should implement rigorous controls and validation steps as demonstrated in other hamster protein studies, which used multiple antibodies and complementary techniques like qRT-PCR to confirm expression patterns .

How does Mesocricetus auratus MAZ compare structurally and functionally to human and mouse homologs?

Comparative analysis of MAZ across species reveals important insights for translational research:

Functionally, hamster MAZ shows significant conservation in its regulatory roles, particularly in immune pathway regulation through STAT1 and STAT3 signaling . The high structural and functional similarity between hamster and human MAZ makes the Syrian golden hamster a valuable model for studying MAZ-related human diseases. Researchers should leverage this cross-species conservation while remaining attentive to subtle species-specific differences that may affect experimental interpretation.

What are the optimal experimental designs for translational studies using hamster MAZ findings for human applications?

For maximizing translational relevance when studying hamster MAZ:

• Parallel validation approach:

  • Design experiments that simultaneously test hamster and human MAZ

  • Compare DNA-binding profiles using ChIP-seq or similar techniques

  • Validate key findings in both species' cell lines

• Domain-specific functional analysis:

  • Perform domain-swapping experiments to identify regions responsible for species-specific functions

  • Create chimeric proteins to isolate functionally distinct regions

  • Use site-directed mutagenesis to examine conservation of key residues

• Disease-relevant model systems:

  • Employ hamster models for diseases where MAZ plays a documented role (cancer, inflammatory conditions)

  • Validate findings using patient-derived samples or human cell lines

  • Use RNA-seq to compare transcriptional responses to MAZ modulation across species

• Controlled environmental factors:

  • Standardize experimental conditions when comparing across species

  • Account for species-specific cellular environments that may influence MAZ function

  • Consider differences in post-translational modification machinery

What emerging technologies will advance our understanding of MAZ function in epigenetic regulation?

Several cutting-edge technologies show promise for elucidating MAZ's role in epigenetic regulation:

• Single-cell multi-omics: Integrating single-cell RNA-seq, ATAC-seq, and protein analysis to understand cell-specific roles of MAZ in heterogeneous populations.

• CUT&Tag and CUT&RUN: These techniques offer higher resolution and lower background than traditional ChIP-seq for mapping MAZ binding sites and associated histone modifications .

• HiChIP and Micro-C: Advanced chromatin conformation capture techniques to understand how MAZ influences 3D genome organization and enhancer-promoter interactions.

• CRISPR epigenome editing: Using catalytically dead Cas9 fused to epigenetic modifiers to alter specific MAZ binding sites and observe functional consequences.

• Live-cell imaging of chromatin dynamics: Techniques like FRAP (Fluorescence Recovery After Photobleaching) with fluorescently tagged MAZ to study its dynamics at chromatin in real-time.

These technologies will help resolve how MAZ controls expression of immunity-related genes by changing the epigenetic landscape in chromatin , potentially revealing therapeutic targets for diseases associated with dysregulated MAZ expression.

How might research on MAZ in Syrian golden hamsters contribute to therapeutic development for human diseases?

Research on MAZ in Syrian golden hamster models has significant potential for therapeutic applications:

• Cancer therapeutics:

  • MAZ is implicated in multiple cancer types including glioblastoma, breast cancer, prostate cancer, and liposarcoma

  • Hamster models can validate therapeutic approaches targeting MAZ-regulated pathways

  • Small molecule inhibitors of MAZ-DNA interaction could be screened in hamster cancer models

• Anti-inflammatory approaches:

  • Since MAZ controls proinflammatory responses via STAT3 signaling , modulation of this pathway could yield new anti-inflammatory treatments

  • The role of MAZ in regulating IFN-γ responses suggests potential applications for autoimmune diseases

• Precision medicine strategies:

  • Understanding MAZ's role in STAT1 signaling and immune gene regulation could lead to targeted therapies for patients with specific immune dysfunction profiles

  • Biomarker development based on MAZ activation status or target gene expression

• Drug delivery and target validation:

  • Recombinant MAZ protein could be used to validate potential binding sites for therapeutic compounds

  • Syrian golden hamster models provide a clinically relevant system to test MAZ-targeted therapies before human trials

The Syrian golden hamster's value as a translational model is enhanced by the structural similarity between hamster and human MAZ, making it particularly useful for therapeutic development pipelines .