Recombinant Rat THAP domain-containing protein 4 (Thap4)

What is the basic structure of Rat THAP4 protein?

Rat THAP4 belongs to the THAP (Thanatos-associated protein) domain family of proteins. The protein contains an N-terminal THAP domain, which is a sequence-specific DNA binding domain of approximately 90 residues. The C-terminal domain of THAP4 (cTHAP4) is evolutionarily conserved across all known THAP4 orthologs and forms a heme-binding nitrobindin domain . Structurally, cTHAP4 is composed almost exclusively of β-strands, with ten antiparallel strands forming a compact β-barrel capped on one end by a ten-residue long 3₁₀ α-helix . A large hydrophobic cavity is formed in the interior of the β-barrel, where a heme molecule binds and is coordinated by His567 .

What are the molecular characteristics of Rat THAP4?

Rat THAP4 (UniProt code: Q642B6) functions as a homodimer in solution . The dimerization interface is approximately 1312 Ų and is composed of conserved hydrophobic residues, suggesting this dimerization is biologically relevant . The protein demonstrates DNA binding and metal ion binding capabilities . The heme-binding property of THAP4 makes it unique among THAP proteins, as it is the only human THAP protein predicted to bind a cofactor . The similarity of its structure to nitrobindin suggests potential roles in nitric oxide (NO) transport, sensing, or metabolism .

What methods can be used to detect and quantify Rat THAP4?

ELISA (Enzyme-Linked Immunosorbent Assay) is a primary method for detecting and quantifying Rat THAP4 in various sample types. Commercial sandwich ELISA kits are available with the following specifications:

These kits offer high sensitivity and specificity for accurate quantification of THAP4 levels . Alternative methods include Western blotting using specific antibodies against THAP4, immunohistochemistry for tissue localization studies, and RT-PCR for gene expression analysis.

How can recombinant Rat THAP4 be produced and purified?

Recombinant Rat THAP4 can be produced using several expression systems:

Purification typically involves affinity chromatography based on the fusion tag used:

• His-tagged proteins can be purified using nickel or cobalt affinity columns

• GST-tagged proteins can be purified using glutathione-based affinity resins

• Fc-tagged proteins can be purified using Protein A/G columns

For crystallographic studies, as demonstrated with cTHAP4, additional purification steps such as size-exclusion chromatography may be necessary to achieve high purity and homogeneity required for crystal formation .

What are the optimal storage conditions for maintaining Rat THAP4 stability?

Recombinant Rat THAP4 should typically be stored at -80°C for long-term storage, with aliquoting recommended to avoid freeze-thaw cycles that can degrade protein activity. For short-term storage (1-2 weeks), 4°C is generally acceptable. Buffer conditions should maintain protein stability, typically including:

• A buffering agent (e.g., HEPES, Tris, or phosphate) at physiologically relevant pH (7.0-7.5)

• Salt (e.g., NaCl) at moderate concentration (50-150 mM) to maintain solubility

• Reducing agents (e.g., DTT or β-mercaptoethanol) if the protein contains free cysteines

• Protease inhibitors to prevent degradation

• Glycerol (10-20%) may be added as a cryoprotectant for frozen storage

For the specific case of cTHAP4, storage in 5 mM HEPES buffer at pH 5.0 containing 50 mM NaCl and 0.3 mM NaN₃ has been documented for experimental use .

What are the known protein interactions of THAP4?

THAP4 has been reported to interact with several proteins:

• HSP90AB1 (Heat Shock Protein 90 Alpha Family Class B Member 1) - a molecular chaperone involved in protein folding and stabilization

• MAPK6 (Mitogen-Activated Protein Kinase 6) - involved in signal transduction

• MOB4 (MOB Family Member 4) - involved in the regulation of the Hippo signaling pathway

These interactions suggest potential roles for THAP4 in cellular stress responses, signal transduction pathways, and transcriptional regulation. Further investigation of these protein-protein interactions would provide valuable insights into the functional networks in which THAP4 participates.

What role does THAP4 play in transcriptional regulation?

As a member of the THAP domain family, THAP4 is likely involved in transcriptional regulation through sequence-specific DNA binding . The THAP domain is characterized by a C2-CH (Cys-Xaa₂-Cys-Xaa₃₅-₅₀-Cys-Xaa₂-His) zinc-finger-like motif that mediates sequence-specific DNA binding .

While the exact transcriptional targets of THAP4 have not been fully characterized, other THAP proteins have been shown to regulate genes involved in cell proliferation, apoptosis, and cell cycle control . Given THAP4's upregulation in response to heat shock and its high expression in heart cells and lymphoma cells, it may regulate genes involved in stress responses and tissue-specific functions .

To identify THAP4 transcriptional targets, researchers could employ techniques such as:

• Chromatin Immunoprecipitation followed by sequencing (ChIP-seq)

• Gene expression profiling after THAP4 knockdown or overexpression

• Reporter assays using predicted THAP4 binding sites

What is the significance of heme binding in THAP4 function?

THAP4 is unique among THAP proteins in containing a heme-binding nitrobindin domain . The crystal structure reveals that the heme molecule is bound in a hydrophobic cavity within the β-barrel structure of cTHAP4 and is coordinated by His567 . This structural feature suggests potential roles in:

• Nitric oxide (NO) signaling: Similar to nitrophorins, THAP4 might be involved in NO storage, transport, or release in a pH-dependent manner .

• Redox sensing: The heme group could function as a redox sensor, with changes in the oxidation state potentially affecting THAP4's DNA binding or protein interaction capabilities.

• Gas sensing: Beyond NO, heme proteins can sense other diatomic gases like CO and O₂, suggesting potential roles in cellular responses to these molecules.

• Structural stabilization: The heme group may serve to stabilize the protein structure, with implications for THAP4's functional regulation.

Experimental approaches to investigate the role of heme binding could include site-directed mutagenesis of the heme-coordinating His567, spectroscopic analyses of heme-binding properties, and functional assays comparing wild-type THAP4 with heme-binding deficient mutants.

How do post-translational modifications affect THAP4 function?

While specific post-translational modifications (PTMs) of Rat THAP4 have not been extensively characterized in the provided search results, PTMs are likely to play crucial roles in regulating THAP4's function, similar to other transcription factors. Based on sequence analysis and comparison with related proteins, potential PTMs might include:

• Phosphorylation: Protein kinases may phosphorylate specific serine, threonine, or tyrosine residues, potentially affecting THAP4's DNA binding affinity, protein-protein interactions, or subcellular localization.

• Ubiquitination: This modification could regulate THAP4 protein levels through proteasomal degradation.

• SUMOylation: This modification often affects transcription factor activity and nuclear localization.

• Acetylation: This could influence DNA binding properties or protein stability.

To investigate PTMs of THAP4, researchers could employ mass spectrometry-based proteomics approaches, including:

• Enrichment strategies specific for phosphopeptides, ubiquitinated peptides, etc.

• Comparison of PTM profiles under different cellular conditions (e.g., normal vs. stress conditions)

• Functional analysis of site-specific mutants where potential PTM sites are replaced with non-modifiable residues

What are the challenges in crystallizing full-length THAP4?

The crystal structure of the C-terminal domain of THAP4 (cTHAP4) has been determined to 1.79 Å resolution , but crystallizing the full-length protein presents several challenges:

• Domain flexibility: The presence of potentially flexible regions between the N-terminal THAP domain and the C-terminal nitrobindin domain might hinder crystal formation by introducing conformational heterogeneity.

• Solubility issues: Full-length transcription factors often contain regions with lower solubility than individual domains, which can lead to aggregation during concentration steps required for crystallization.

• Multiple functional states: THAP4 may adopt different conformations when bound to DNA versus its unbound state, adding another layer of complexity.

• Post-translational modifications: If THAP4 undergoes various PTMs, this heterogeneity can impede crystal formation.

Strategies to overcome these challenges might include:

• Limited proteolysis followed by crystallization of stable fragments

• Surface entropy reduction through mutation of surface residues with high conformational entropy

• Co-crystallization with binding partners (DNA, protein interactors) to stabilize specific conformations

• Using fusion proteins or crystallization chaperones to promote crystal contacts

How can CRISPR-Cas9 be optimized for studying THAP4 function?

CRISPR-Cas9 technology offers powerful approaches for investigating THAP4 function through gene editing. Optimization strategies specific for THAP4 studies might include:

• Guide RNA design:

  • Target exons encoding functional domains (THAP domain, heme-binding site)

  • Avoid regions with high homology to other THAP family members

  • Use algorithms that predict off-target effects and select guides with high specificity

  • Consider the chromatin accessibility of target regions

• Knockin strategies:

  • Design fluorescent fusion proteins that maintain THAP4 function

  • Create tagged versions for ChIP-seq or immunoprecipitation studies

  • Introduce specific mutations to disrupt heme binding (e.g., His567 mutations)

  • Generate conditional alleles using loxP sites for tissue-specific deletion

• Validation methods:

  • Confirm editing efficiency using T7 endonuclease assays or deep sequencing

  • Verify protein expression changes by Western blot

  • Assess functional consequences through DNA binding assays, transcriptional reporter assays

  • Evaluate phenotypic changes in relevant cell types (e.g., cardiomyocytes given THAP4's high expression in heart)

• Cell type considerations:

  • Optimize transfection protocols for cell types where THAP4 has high functional relevance

  • Consider using primary cells versus cell lines depending on the research question

  • For in vivo studies, evaluate tissue-specific delivery methods for CRISPR components

What is the link between THAP4 and disease pathogenesis?

THAP4 has been implicated in several disease states, although the specific mechanisms are still being elucidated. Based on the available information and knowledge about related THAP proteins:

• Cancer: THAP4 is upregulated in lymphoma cells , suggesting a potential role in oncogenesis. Other THAP family members have been implicated in various cancers , indicating a broader role for THAP proteins in malignancy.

• Neurodegenerative disorders: THAP4 has been linked to neurodegenerative conditions , possibly through its roles in transcriptional regulation, stress responses, or potential NO signaling functions.

• Cardiovascular disease: Given THAP4's high expression in heart cells and the involvement of other THAP proteins in heart disease , THAP4 might play a role in cardiac pathophysiology.

Research approaches to investigate these connections might include:

• Analysis of THAP4 expression and mutation in patient samples

• Generation of disease models with altered THAP4 function

• Identification of disease-relevant transcriptional targets

How can variations in THAP4 be analyzed in different disease models?

Analyzing THAP4 variations across disease models requires a multi-faceted approach:

• Expression analysis:

  • RT-qPCR to quantify THAP4 mRNA levels

  • Western blotting or ELISA to measure protein levels

  • Immunohistochemistry to assess tissue localization and expression patterns

  • Single-cell RNA sequencing to identify cell type-specific expression changes

• Functional variations:

  • DNA binding assays to assess changes in transcriptional activity

  • Protein-protein interaction studies to identify altered molecular associations

  • Subcellular localization studies using fluorescence microscopy

  • Heme-binding studies to evaluate cofactor interactions

• Genetic variations:

  • Sequencing to identify mutations or polymorphisms

  • CRISPR-based modeling of patient-specific mutations

  • Analysis of epigenetic modifications affecting THAP4 expression

• Cross-species analysis:

  • Comparison of THAP4 function in different model organisms

  • Evaluation of conservation of disease-associated features

Data integration across these approaches can provide comprehensive insights into how THAP4 variations contribute to disease pathogenesis and potentially identify therapeutic targets.

How might the heme-binding property of THAP4 be exploited for therapeutic development?

The unique heme-binding capability of THAP4 presents intriguing possibilities for therapeutic development:

• Targeted small molecule modulators:

  • Design of compounds that specifically bind to the heme pocket of THAP4

  • Development of molecules that compete with heme binding to alter THAP4 function

  • Creation of allosteric modulators that affect heme binding indirectly

• NO-related therapeutics:

  • If THAP4 is involved in NO signaling, development of drugs that modulate this function

  • Design of THAP4-targeted NO donors or scavengers for tissue-specific effects

  • Engineering of modified THAP4 proteins as potential NO delivery systems

• Diagnostic applications:

  • Development of assays that detect THAP4-heme interactions as disease biomarkers

  • Creation of imaging probes that bind to THAP4 for visualization in tissues

• Protein-protein interaction targeting:

  • Design of molecules that disrupt or enhance interactions between THAP4 and binding partners

  • Development of peptide mimetics based on interaction interfaces

These approaches would require detailed structural knowledge of THAP4's heme-binding domain, which has been characterized at 1.79 Å resolution , providing a solid foundation for structure-based drug design.