Recombinant Gorilla gorilla gorilla Zinc finger and BTB domain-containing protein 25 (ZBTB25)

How does ZBTB25 interact with other proteins in transcriptional regulation complexes?

ZBTB25 functions as a transcriptional repressor by associating with histone deacetylase 1 (HDAC1) and the transcriptional corepressor Sin3a to form a silencing complex . In experimental systems, this interaction can be confirmed through reciprocal coimmunoprecipitation and immunofluorescence microscopy . The protein complex is recruited to specific gene promoters, where it hypoacetylates histone H3, leading to transcriptional downregulation of target genes such as IL-12B .

Molecular docking analyses have shown that ZBTB25 and HDAC1 interact strongly with a docking score of -914.5 Kcal/mol, indicating a stable and energetically favorable complex formation . The BTB/POZ domain of ZBTB25 is critical for this interaction, as it provides the interface for protein-protein binding.

What are the functional implications of zinc binding in ZBTB25?

The zinc finger domains in ZBTB25 are essential for its DNA-binding functionality and subsequent transcriptional regulation activities. These domains coordinate zinc ions through conserved cysteine and histidine residues, forming a structural motif that recognizes and binds to specific DNA sequences . Disruption of zinc coordination significantly impairs ZBTB25 function.

Research demonstrates that zinc ejectors like disulfiram (DP), disulfiram sodium salt (DS), and 2-nitrobenzaldehyde (2NB) can disrupt the zinc finger structure at different concentrations. For a 50% Zn²⁺ ejection from ZBTB25, the required concentrations are:

• DP: 4.61 ± 0.28 μM

• DS: 10.35 ± 0.30 μM

• 2NB: 25.54 ± 0.37 μM

This differential potency makes DP the strongest zinc ejector among these compounds, suggesting its potential utility in experimental inhibition of ZBTB25 function.

What are effective approaches for ZBTB25 knockdown in experimental systems?

Successful ZBTB25 knockdown has been achieved using both short hairpin RNA (shRNA) and small interfering RNA (siRNA) approaches. The efficiency of knockdown should be confirmed through multiple methods, including quantitative reverse transcription-PCR (qRT-PCR), Western blotting, and confocal microscopy .

Several validated sequences have been reported in the literature:

Control sequences such as shLuc (5′-GCGGTTGCCAAGAGGTTCCAT-3′) can be used as negative controls to validate knockdown specificity .

When performing knockdown experiments, researchers should consider:

• Cell-type specific optimization of transfection conditions

• Validation of knockdown at both mRNA and protein levels

• Assessment of potential off-target effects

• Inclusion of appropriate controls

How can researchers confirm ZBTB25-HDAC1 interactions in experimental settings?

To confirm interactions between ZBTB25 and HDAC1, several complementary approaches can be employed:

• Coimmunoprecipitation (Co-IP): Immunoprecipitate with antibodies against HDAC1 (particularly phosphorylated HDAC1 at serine 421 or 423) and detect ZBTB25 in the precipitate, or vice versa. Reciprocal Co-IPs strengthen interaction evidence .

• Immunofluorescence microscopy: Perform confocal imaging to visualize colocalization of ZBTB25 and HDAC1 within the nucleus. This is particularly effective in comparing infected versus uninfected cells in infection models .

• Chromatin Immunoprecipitation (ChIP): Determine whether ZBTB25 and HDAC1 co-occupy specific gene promoters, such as the IL-12B promoter .

• Mass spectrometry analysis: LC-MS/MS analysis of immunoprecipitated complexes can identify additional components of the ZBTB25-HDAC1 complex, as demonstrated in studies that identified Sin3a as part of this repressor complex .

• In silico molecular docking: Computational approaches can provide additional insights into the structural basis of ZBTB25-HDAC1 interactions .

What methods are available for ZBTB25 pharmacological inhibition?

Pharmacological inhibition of ZBTB25 can be achieved through zinc ejector compounds that disrupt the zinc finger domain's structure and function. The following methodological considerations are important:

• Zinc ejector selection: Disulfiram (DP) has been identified as the most potent zinc ejector for ZBTB25 inhibition, requiring only 4.61 ± 0.28 μM for 50% zinc ejection .

• Zinc ejection monitoring: The release of Zn²⁺ ions can be monitored using zinc-specific fluorophores such as FluoZin-3 .

• Cytotoxicity assessment: MTT assays should be performed to determine the cytotoxic threshold of the inhibitor. For instance, DP exhibits relatively low toxicity, with 85.33 ± 1.53% cell viability after exposure to 30 μM for 24 hours .

• Inhibition verification: The functional inhibition should be verified by assessing ZBTB25 recruitment to target promoters using ChIP assays. Complete abolishment of ZBTB25 recruitment to the IL-12B promoter has been observed at 20 μM DP concentration .

• Functional readouts: The biological effects of ZBTB25 inhibition can be assessed using relevant functional assays, such as measuring intracellular pathogen survival. Treatment with 20 μM DP reduced intracellular M. tuberculosis survival to 35.11 ± 4.32% .

How does ZBTB25 modulate host-pathogen interactions during Mycobacterium tuberculosis infection?

ZBTB25 plays a critical role in tuberculosis pathogenesis through its involvement in multiple host defense pathways:

• IL-12B gene repression: Following M. tuberculosis infection, ZBTB25 associates with phosphorylated HDAC1 and Sin3a, forming a repressor complex that is recruited to the IL-12B gene promoter. This complex hypoacetylates histone H3, leading to downregulation of IL-12B expression .

• Autophagy regulation: ZBTB25 inhibition promotes colocalization of M. tuberculosis and LC3 (microtubule-associated protein 1A/1B-light chain 3) in autophagosomes, thereby enhancing autophagy-mediated killing of intracellular pathogens .

• JAK2/STAT4 pathway modulation: Enhanced phosphorylation of JAK2 and STAT4 is observed in macrophages upon treatment with HDAC1 and ZBTB inhibitors. Inhibition of this pathway negates the killing of intracellular M. tuberculosis, suggesting its role in autophagy-mediated pathogen clearance .

• Potential therapeutic target: Given its role in suppressing host defense mechanisms, ZBTB25 represents a promising target for host-directed anti-TB therapy, particularly valuable in the context of drug-resistant tuberculosis .

Experimental evidence shows that knockdown of ZBTB25 prevents recruitment of the repressor complex to the IL-12B promoter, enhancing gene expression and increasing IL-12p40 release from infected macrophages .

What is the role of ZBTB25 in viral replication processes?

ZBTB25 has been identified as a positive regulator of Influenza A Virus (IAV) replication, functioning through several mechanisms:

• Enhancement of viral RdRp activity: ZBTB25 enhances viral RNA-dependent RNA polymerase (RdRp) activity by binding to both viral RdRp and viral RNA, thereby stimulating viral RNA synthesis .

• Dual transcription functions: ZBTB25 exhibits a unique dual functionality, promoting viral RNA transcription through binding to the U-rich region of viral RNA while simultaneously suppressing cellular interferon production .

• Zinc finger domain importance: The zinc finger domain of ZBTB25 is required for its RNA-inhibitory activity through zinc ion chelation. Compounds like disulfiram that disrupt zinc finger function effectively repress IAV replication .

This multifaceted role makes ZBTB25 an attractive target for antiviral therapeutic development, particularly for addressing viral resistance issues due to high mutation rates in IAV .

How does Gorilla gorilla gorilla ZBTB25 compare with human ZBTB25?

While direct comparative data between Gorilla gorilla gorilla ZBTB25 and human ZBTB25 is limited in the provided search results, general principles of molecular evolution suggest:

• Sequence conservation: As closely related primates, gorillas and humans likely share high sequence homology in ZBTB25. Comparative analysis would focus on amino acid variations in functional domains, particularly the zinc finger and BTB/POZ domains.

• Functional conservation: The fundamental transcriptional repressor function of ZBTB25 is likely conserved between species, although subtle differences in binding affinities or regulatory networks may exist.

• Transcriptional targets: While core functions may be preserved, the specific gene targets regulated by ZBTB25 could vary between species due to genome divergence and species-specific adaptations.

Methodologically, researchers investigating cross-species differences should consider:

• Sequence alignment and phylogenetic analysis

• Comparative structural modeling

• Cross-species functional assays

• Assessment of binding site conservation in target genes

What approaches can be used to study ZBTB25 binding specificity?

To characterize ZBTB25 binding specificity, researchers can employ several complementary methodologies:

• Chromatin Immunoprecipitation (ChIP): ChIP assays can identify genomic regions bound by ZBTB25 in vivo. In studies of M. tuberculosis infection, ChIP has successfully demonstrated ZBTB25 binding to the IL-12B promoter .

• ChIP-seq: This genome-wide approach can identify all binding sites of ZBTB25 across the genome, providing insights into global regulatory networks.

• Electrophoretic Mobility Shift Assay (EMSA): EMSA can determine direct binding of recombinant ZBTB25 to specific DNA sequences in vitro.

• Protein Binding Microarrays: This high-throughput approach can identify consensus binding sequences for ZBTB25.

• Reporter gene assays: Functional validation of ZBTB25 binding sites can be achieved using reporter constructs containing putative binding sequences.

• Mutagenesis studies: Systematic mutation of ZBTB25 zinc finger domains can identify specific residues critical for DNA recognition.

When studying binding specificity across species, researchers should consider evolutionary conservation of binding motifs and potential species-specific adaptations in the DNA recognition domains.

What are the considerations for developing ZBTB25-targeted therapeutics?

Development of ZBTB25-targeted therapeutics presents both opportunities and challenges that researchers should consider:

• Target validation:

  • Confirm ZBTB25's role in disease pathogenesis through multiple approaches including genetic knockdown and pharmacological inhibition

  • Validate across different experimental systems and disease models

• Compound development:

  • Zinc ejector compounds like disulfiram represent promising starting points

  • Structure-based drug design focusing on the zinc finger domain may yield selective inhibitors

  • Consider the differential potency of zinc ejectors: DP (4.61 ± 0.28 μM) > DS (10.35 ± 0.30 μM) > 2NB (25.54 ± 0.37 μM)

• Therapeutic window:

  • Assess cytotoxicity thoroughly (e.g., DP maintains 85.33 ± 1.53% cell viability at 30 μM for 24h)

  • Determine minimum effective concentration (e.g., 20 μM DP completely abolishes ZBTB25 recruitment to target promoters)

• Disease applications:

  • Infectious diseases: ZBTB25 inhibition shows promise for host-directed therapy in tuberculosis and potentially for IAV infections

  • Consider potential applications in other diseases where ZBTB25-mediated transcriptional repression contributes to pathology

• Delivery challenges:

  • Design delivery systems capable of targeting specific cell types (e.g., macrophages for TB therapy)

  • Consider bioavailability and tissue distribution requirements

• Biological complexity:

  • Account for potential compensatory mechanisms through other ZBTB family members

  • Consider context-dependent functions of ZBTB25 in different tissues and disease states

How can systems biology approaches enhance our understanding of ZBTB25 regulatory networks?

Systems biology offers powerful frameworks to understand the complex regulatory networks involving ZBTB25:

• Multi-omics integration:

  • Combine ChIP-seq (ZBTB25 binding sites), RNA-seq (transcriptional effects), and proteomics (protein interactions) to build comprehensive regulatory models

  • Integrate epigenomic data (histone modifications, chromatin accessibility) to understand the chromatin context of ZBTB25 binding

• Network modeling:

  • Construct gene regulatory networks incorporating ZBTB25, HDAC1, Sin3a, and their target genes

  • Use mathematical modeling to predict network responses to perturbations

  • Apply Bayesian networks to infer causal relationships within the regulatory system

• Single-cell approaches:

  • Apply single-cell transcriptomics to capture heterogeneity in ZBTB25-mediated responses

  • Combine with spatial transcriptomics to understand tissue-specific regulatory patterns

• Comparative systems biology:

  • Compare ZBTB25 regulatory networks across species to identify conserved core functions versus species-specific adaptations

  • Evaluate network conservation between gorilla and human systems

• Perturbation biology:

  • Systematically perturb the system through ZBTB25 knockdown, overexpression, and pharmacological inhibition

  • Measure global network responses to understand system robustness and identify potential compensatory mechanisms

By applying these approaches, researchers can move beyond individual gene effects to understand how ZBTB25 functions within larger regulatory systems controlling cellular processes in both normal physiology and disease states.

What are the future directions for ZBTB25 research in immunology and infectious diseases?

Future research on ZBTB25 in immunology and infectious diseases should address several key knowledge gaps:

• Expanded pathogen repertoire:

  • Investigate ZBTB25's role beyond M. tuberculosis and IAV to other bacterial, viral, and parasitic infections

  • Determine if ZBTB25 represents a common immune evasion target across diverse pathogens

• Mechanistic dissection:

  • Elucidate the full spectrum of genes regulated by ZBTB25 during infection using genome-wide approaches

  • Define the structural basis of ZBTB25 interactions with HDAC1/Sin3a complex and target DNA sequences

  • Investigate post-translational modifications of ZBTB25 that regulate its activity during infection

• Therapeutic development:

  • Screen compound libraries for novel ZBTB25 inhibitors with improved potency and specificity

  • Develop combination therapies targeting ZBTB25 alongside conventional antimicrobials

  • Explore the therapeutic potential in drug-resistant infections where conventional treatments fail

• Translational research:

  • Validate ZBTB25 as a biomarker for disease progression or treatment response

  • Develop animal models to test ZBTB25-targeted therapies in vivo

  • Investigate population genetic variations in ZBTB25 that might influence infection susceptibility

• Comparative immunology:

  • Compare ZBTB25 function across different species to understand evolutionary adaptation in host-pathogen interactions

  • Investigate potential species-specific differences in ZBTB25 regulation between gorillas and humans

These research directions will not only expand our fundamental understanding of ZBTB25 biology but also accelerate the development of novel therapeutic strategies for infectious diseases, particularly those with limited treatment options due to antimicrobial resistance.