TARDBP (1-414) Human, His

What is the functional significance of the glycine-rich domain in TARDBP (1-414) Human, His protein?

The glycine-rich domain of TARDBP (1-414) Human, His, located in the C-terminal region of the protein, plays crucial roles in regulating gene expression and mediating protein-protein interactions. Specifically, this domain facilitates TDP-43 binding to heterogeneous ribonucleoproteins (hnRNPs) . Pathogenic mutations, such as p.Gly290Ala and p.Gly298Ser, have been identified within this domain in familial ALS cases, suggesting its importance in disease pathogenesis .

When designing experiments with TARDBP (1-414) Human, His, researchers should consider that alterations in this domain may significantly impact protein function. Methodologically, site-directed mutagenesis targeting this region can help elucidate structure-function relationships and potentially recapitulate disease-associated phenotypes in experimental models.

How does nuclear-cytoplasmic shuttling affect TARDBP (1-414) Human, His function in experimental systems?

TDP-43 is predominantly a nuclear protein, but pathological conditions can trigger its mislocalization to the cytoplasm. In experimental models overexpressing TARDBP, approximately 1% of cells show cytoplasmic translocation of the protein, mirroring disease pathology . This subcellular redistribution appears to be an early event in TDP-43 proteinopathies.

When working with TARDBP (1-414) Human, His in cell culture systems, researchers should employ nuclear and cytoplasmic fractionation techniques followed by immunoblotting to quantify distribution patterns. Immunofluorescence microscopy with antibodies against the His-tag or TDP-43 epitopes provides complementary spatial information. Treatments affecting nuclear transport machinery (e.g., leptomycin B to inhibit nuclear export) can help determine the dynamics and regulatory mechanisms of this shuttling process.

What are the common pitfalls when expressing TARDBP (1-414) Human, His in cellular models?

Expression of TARDBP (1-414) Human, His in cellular models requires careful consideration of several factors:

• Expression levels: Overexpression can be cytotoxic, as demonstrated in rat models where AAV-mediated TARDBP expression at approximately three times endogenous levels caused neurodegeneration of dopaminergic neurons .

• Cell type specificity: Different neural cell types show varying susceptibility to TDP-43 toxicity, requiring thoughtful selection of experimental models.

• Aggregation propensity: The protein may form aggregates that complicate biochemical analyses, particularly when expressed at high levels.

To address these challenges, researchers should implement titratable expression systems, conduct viability assays in parallel with functional studies, and employ solubility fractionation methods to distinguish between soluble and aggregated protein forms.

How do specific TARDBP mutations affect protein structure and function compared to wild-type TARDBP (1-414) Human, His?

Pathogenic TARDBP mutations, particularly those in the glycine-rich domain such as p.Gly290Ala and p.Gly298Ser, impact protein function through multiple mechanisms . Comparative analysis between wild-type and mutant forms reveals:

Methodologically, researchers should employ circular dichroism spectroscopy and thermal shift assays to assess structural differences between wild-type and mutant proteins. RNA immunoprecipitation followed by sequencing (RIP-seq) can identify differential RNA binding properties, while proximity ligation assays can reveal altered protein interaction networks.

What is the relationship between TARDBP (1-414) Human, His aggregation and neurodegeneration in experimental models?

The aggregation of TARDBP and its relationship to neurodegeneration is complex and likely involves both gain- and loss-of-function mechanisms . When working with TARDBP (1-414) Human, His:

• Solubility analysis shows that pathological TDP-43 becomes increasingly insoluble during disease progression.

• Biochemical fractionation reveals distinct aggregation species with different neurotoxic properties.

• Time-course experiments demonstrate that cytoplasmic mislocalization often precedes visible aggregation and neurodegeneration.

Researchers should implement longitudinal studies in cellular or animal models expressing TARDBP (1-414) Human, His to capture the temporal relationship between protein aggregation and neuronal dysfunction. Sequential extraction protocols using buffers of increasing detergent strength can isolate different protein fractions for analysis. Correlative light and electron microscopy provides valuable insights into the ultrastructural characteristics of aggregates and their relationship to cellular organelles.

How does post-translational modification affect TARDBP (1-414) Human, His behavior in in vitro and in vivo systems?

Post-translational modifications (PTMs) significantly influence TDP-43 function and pathology. For TARDBP (1-414) Human, His research:

• Phosphorylation at specific serine residues (particularly S409/410) serves as a marker for pathological TDP-43 and affects its solubility and cellular localization.

• Ubiquitination patterns differ between normal and disease states, with pathological TDP-43 showing increased ubiquitination.

• SUMOylation may influence protein stability and function.

To study PTMs experimentally, researchers should apply mass spectrometry-based approaches after immunoprecipitation of TARDBP (1-414) Human, His from cellular or tissue lysates. Phospho-specific and ubiquitin-specific antibodies in western blotting and immunohistochemistry provide complementary information. Site-directed mutagenesis of key modification sites (e.g., phosphomimetic mutations) can help determine their functional significance.

What are the optimal conditions for purifying functional TARDBP (1-414) Human, His protein for biochemical studies?

Purification of functional TARDBP (1-414) Human, His requires careful optimization to maintain protein integrity:

• Buffer selection: Use buffers containing 300-500 mM NaCl to prevent non-specific nucleic acid binding, with 10-20 mM imidazole during binding and 250-300 mM for elution when using Ni-NTA chromatography.

• Temperature considerations: Maintain samples at 4°C throughout purification to minimize aggregation and proteolytic degradation.

• Stabilizing additives: Include 5-10% glycerol and 1-2 mM DTT to enhance protein stability.

• Quality control: Assess protein purity by SDS-PAGE and authenticity by western blotting with anti-TDP-43 and anti-His antibodies. Verify functional activity through RNA-binding assays using known TDP-43 target sequences.

• Storage: Flash-freeze aliquots in liquid nitrogen and store at -80°C to maintain activity. Avoid repeated freeze-thaw cycles.

What experimental approaches best capture TARDBP (1-414) Human, His interaction with RNA and other proteins?

To investigate TARDBP (1-414) Human, His interactions with RNA and proteins:

• For RNA interactions:

  • CLIP-seq (UV cross-linking and immunoprecipitation followed by RNA sequencing) provides transcriptome-wide binding sites with nucleotide resolution.

  • Electrophoretic mobility shift assays (EMSAs) determine binding affinities to specific RNA sequences.

  • Surface plasmon resonance offers quantitative binding kinetics to defined RNA motifs.

• For protein-protein interactions:

  • Immunoprecipitation followed by mass spectrometry identifies interaction partners.

  • Proximity-dependent biotin identification (BioID) captures both stable and transient interactions in living cells.

  • Bimolecular fluorescence complementation visualizes interactions in their native cellular context.

The data from search results suggests particular attention to heterogeneous ribonucleoproteins (hnRNPs) as important interaction partners mediated through the glycine-rich domain .

What are the key considerations when designing a TARDBP (1-414) Human, His overexpression model for studying neurodegeneration?

When developing overexpression models using TARDBP (1-414) Human, His, researchers should consider:

• Expression level control: Tatom et al. demonstrated that approximately three-fold overexpression of human TDP-43 in rat substantia nigra was sufficient to induce cytoplasmic mislocalization and neurodegeneration . Use inducible promoters (e.g., tetracycline-responsive) to titrate expression levels.

• Cellular specificity: Employ cell type-specific promoters to target expression to relevant populations (e.g., motor neurons for ALS models).

• Temporal considerations: Implement longitudinal studies to capture disease progression, as cytoplasmic mislocalization (observed in approximately 1% of transduced cells) precedes neurodegeneration .

• Readout selection: Include multiple assays spanning molecular (protein solubility, PTMs), cellular (viability, morphology), and functional (electrophysiology, behavior) domains.

• Controls: Include both non-transgenic controls and those expressing an inert protein at similar levels to distinguish specific TDP-43 effects from those of protein overexpression generally.

How can researchers effectively distinguish between gain- and loss-of-function effects when studying TARDBP (1-414) Human, His?

Distinguishing between gain- and loss-of-function effects is crucial for understanding TARDBP pathophysiology and developing targeted therapeutics. The evidence suggests that TARDBP mutations may cause neurodegeneration through both mechanisms . Researchers should:

• Implement parallel overexpression and knockdown/knockout approaches to separate phenotypes.

• Utilize domain-specific mutants that selectively disrupt particular functions (e.g., RNA binding, protein-protein interactions).

• Perform rescue experiments where wild-type TARDBP (1-414) Human, His is introduced following endogenous TDP-43 depletion.

• Analyze RNA processing patterns of known TDP-43 targets to assess functional impact.

• Compare cellular phenotypes between models expressing disease-associated mutations and those with artificial mutations that specifically abolish known functions.

This comprehensive approach allows researchers to determine whether pathological effects stem from novel toxic properties (gain-of-function) or from impairment of normal TDP-43 activities (loss-of-function), facilitating more targeted therapeutic development.

What are the most effective methods for detecting oligomeric species of TARDBP (1-414) Human, His in experimental samples?

Detecting oligomeric species of TARDBP (1-414) Human, His requires specialized approaches:

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) provides absolute molecular weight determination of different oligomeric species.

• Native PAGE preserves non-covalent protein-protein interactions that would be disrupted in SDS-PAGE.

• Analytical ultracentrifugation offers high-resolution separation of distinct oligomeric states in solution.

• For in situ detection, proximity ligation assays can visualize proteins in close proximity, suggesting oligomerization.

• Single-molecule fluorescence techniques, including Förster resonance energy transfer (FRET), can detect transient oligomeric species that may be missed by bulk measurements.

Importantly, sample preparation conditions significantly influence oligomerization state. Researchers should standardize buffer composition, protein concentration, temperature, and time intervals between preparation and analysis to ensure reproducible results.

How do clinical TARDBP mutations from familial ALS cases affect aggregation propensity compared to wild-type TARDBP (1-414) Human, His?

Clinical mutations in TARDBP, particularly those identified in familial ALS cases, show altered aggregation properties. Based on the provided search results and broader research:

To experimentally assess aggregation differences:

• Perform in vitro aggregation kinetics using purified proteins and thioflavin T fluorescence assays.

• Implement cellular models expressing either wild-type or mutant proteins tagged with fluorescent reporters to visualize aggregation dynamics.

• Use biochemical fractionation to quantify the distribution between soluble and insoluble protein fractions over time.

• Apply super-resolution microscopy to characterize the ultrastructural features of aggregates formed by different variants.

Understanding these differences provides insights into mutation-specific pathogenic mechanisms and may guide personalized therapeutic approaches.

What are the emerging research directions for TARDBP (1-414) Human, His in neurodegenerative disease research?

Future research with TARDBP (1-414) Human, His is likely to focus on several promising directions:

• Phase separation biology: Investigating the role of TDP-43 in liquid-liquid phase separation and its transition to pathological aggregation.

• Interactome dynamics: Mapping how disease mutations alter the protein and RNA interaction networks of TDP-43.

• Post-translational modification landscapes: Comprehensive characterization of how PTMs regulate TDP-43 function and pathology.

• Therapeutic targeting: Developing strategies to specifically modulate TDP-43 function or clearance without disrupting essential activities.

• Biomarker development: Utilizing TDP-43 species as diagnostic or prognostic indicators for ALS and FTLD.

Methodologically, integrating multi-omics approaches (proteomics, transcriptomics, interactomics) with high-resolution structural studies and in vivo disease modeling will be crucial for advancing understanding of TDP-43 biology and pathology.