Recombinant Mouse Tankyrase-1 (Tnks), partial

Basic Research Questions

• What is the molecular structure and domain organization of mouse Tankyrase-1?

Mouse Tankyrase-1 consists of 1320 amino acids with 96% sequence identity and 97% homology to human Tankyrase-1. The protein contains four distinct domains:

• An N-terminal HPS (histidine, proline, and serine-rich) domain with currently unknown function

• An ankyrin repeat domain composed of 24 ankyrin repeats organized into five ankyrin repeat clusters (ARCs)

• A SAM (sterile alpha motif) domain that mediates oligomerization

• A C-terminal PARP catalytic domain responsible for poly(ADP-ribosyl)ation activity

The ankyrin domain mediates protein-protein interactions through the recognition of specific motifs in target proteins, while the SAM domain forms novel antiparallel double helical structures essential for tankyrase polymerization and function .

• What are the species-specific differences between mouse and human Tankyrase-1?

Despite high sequence homology, significant functional differences exist:

These differences suggest evolutionary divergence in telomere maintenance mechanisms, as mice and rats have much longer telomeres and higher telomerase activity in somatic tissues than humans .

• How does the absence of TRF1 binding affect the functional applications of recombinant mouse Tankyrase-1?

The inability of mouse Tankyrase-1 to bind TRF1 has several implications for experimental design:

• Mouse models may not accurately reflect human telomere biology when studying tankyrase inhibition

• Research applications should focus on non-telomeric functions such as Wnt signaling and YAP pathway regulation

• Experiments examining telomere maintenance should account for species differences

• Mouse Tankyrase-1 remains valuable for studying conserved functions in Wnt/β-catenin signaling, glucose metabolism, and mitotic regulation

Researchers should utilize this difference to discriminate between telomeric and non-telomeric functions of Tankyrase-1 when designing experiments .

• What methods are recommended for assessing the enzymatic activity of recombinant mouse Tankyrase-1?

Standard enzymatic activity assays include:

• In vitro PARsylation assay: Using NAD+ (optionally 32P-labeled) to measure auto-PARsylation or substrate PARsylation

• Western blot detection: Anti-PAR antibodies to detect modified proteins

• PARP inhibitor controls: Include 3-aminobenzamide (3AB) as negative control

• Substrate comparison: Use known substrates like AXIN1/2 or TAB182

• Colorimetric/fluorometric NAD+ consumption assays: Measuring NAD+ depletion

Validation of activity should include controls with PARP inhibitors (XAV939, IWR1) and analysis of known downstream effects such as AXIN stabilization and β-catenin reduction .

Advanced Research Questions

• What are the structural determinants of substrate recognition by mouse Tankyrase-1 ARCs?

The substrate recognition mechanism involves:

• Recognition of consensus binding motifs (primarily RXXPDG, RXXAXG, or RxxxxG)

• A critical "glycine-selection gate" formed by two parallel tyrosine side chains that accommodate the glycine residue of the binding motif

• Mutation of either gate-forming tyrosines or the glycine in substrates abolishes binding

• ARCs 1, 2, 4, and 5 can bind substrates with similar recognition modes

• Bivalent binding (as seen with Axin) where a substrate contains two binding segments that interact simultaneously with two ARC domains

This structural arrangement is essential for substrate specificity and underlies the selectivity of Tankyrase-1 for its various binding partners .

• How does SAM domain-mediated polymerization affect the catalytic activity of recombinant mouse Tankyrase-1?

The SAM domain forms a novel antiparallel double helix structure that:

• Positions catalytic domains for recurring head-to-head and tail-to-tail interactions

• The head interactions are highly conserved and induce an allosteric switch in the active site that promotes catalysis

• Tail interactions, while having limited effect on catalysis, are essential for function in Wnt/β-catenin signaling

• Disruption of polymerization significantly reduces catalytic activity

• SAM-mediated assembly controls both catalytic and non-catalytic functions

This polymerization mechanism represents a unique regulatory feature that distinguishes tankyrases from other PARP family members and presents opportunities for developing inhibitors that target this aspect of Tankyrase function .

• What methodological approaches are recommended for studying functional redundancy between Tankyrase-1 and Tankyrase-2 in mouse models?

The functional redundancy between these proteins requires specific experimental designs:

• Genetic models: Single knockouts show mild phenotypes, while double knockout is embryonically lethal by day 10

• Conditional knockouts: Tissue-specific deletion to bypass embryonic lethality

• siRNA approaches: Simultaneous knockdown of both proteins is required to observe significant phenotypes

• Inhibitor studies: Use of tankyrase inhibitors affecting both isoforms (XAV939, IWR1, G007-LK)

• Rescue experiments: Expression of one isoform in cells depleted of both to identify unique functions

Quantitative analysis should include measurement of target protein stability (AXIN, AMOTL2) and downstream pathway activity (Wnt/β-catenin and YAP signaling) .

• How can researchers distinguish between catalytic and non-catalytic functions of recombinant mouse Tankyrase-1?

To differentiate these functions:

• Catalytic-dead mutants: Express PARP domain mutants that maintain structure but lack enzymatic activity

• Domain-specific inhibitors: Compare effects of inhibitors targeting different functional domains

• Substrate mutants: Use substrates with mutations in the tankyrase binding motif versus PARsylation sites

• Time-course experiments: Separate rapid non-catalytic effects from slower PARsylation-dependent outcomes

• Proteasome inhibition: Block degradation of PARsylated proteins to distinguish between binding and degradation effects

Researchers should monitor both immediate protein-protein interactions and subsequent PARsylation-dependent protein turnover to fully characterize tankyrase functions .

• What are the optimal conditions for producing and purifying active recombinant mouse Tankyrase-1?

For optimal production and purification:

For structural studies, the construct comprising residues 1001-1327 (catalytic domain) provides good solubility and activity, while full-length protein may have stability issues .

• How can researchers effectively use tankyrase inhibitors in mouse models to study non-telomeric functions?

To effectively use tankyrase inhibitors in mouse models:

• Consider inhibitor specificity: Different inhibitors target distinct binding sites (nicotinamide subsite, adenosine subsite, or dual-site)

• Use multiple inhibitor classes: Compare effects of structurally distinct inhibitors (XAV939, IWR1, G007-LK)

• Monitor appropriate endpoints: Focus on Wnt signaling (AXIN stabilization, β-catenin reduction) or YAP signaling

• Account for compensatory mechanisms: Combine with genetic approaches or other pathway inhibitors

• Evaluate pharmacokinetics: Mouse-specific dosing may differ from human applications due to metabolism differences

In vivo studies should include controls to distinguish tankyrase-specific effects from off-target activities, particularly when studying combinatorial treatments .

• What role does tankyrase-1 play in DNA repair pathways and how can this be studied using recombinant mouse proteins?

Tankyrase-1's role in DNA repair can be investigated through:

• Sensitization assays: Measuring increased sensitivity to ionizing radiation after tankyrase depletion

• DNA-PKcs stability: Monitoring DNA-PKcs protein levels, as tankyrase-1 PARP activity prevents proteasome-mediated DNA-PKcs degradation

• Chromosome aberration analysis: Assessing telomere fusion and genetic instability

• DNA damage response: Examining γ-H2AX foci formation after DNA damage

• Interaction studies: Analyzing binding between tankyrase-1 and DNA repair proteins

Research indicates that tankyrase-1 knockdown results in defective damage response including increased sensitivity to ionizing radiation, mutagenesis, and chromosome aberrations, potentially through DNA-PKcs regulation .

• How does tankyrase inhibition affect immune responses in mouse cancer models?

Recent research demonstrates that:

• Tankyrase inhibitor G007-LK decreases WNT/β-catenin and YAP signaling in murine melanoma models

• Combined tankyrase inhibition and anti-PD-1 therapy shows synergistic effects in reducing tumor volume

• The mechanism involves loss of β-catenin in tumor cells, enhanced T cell infiltration, and induction of IFNγ- and CD8+ T cell-dependent anti-tumor immunity

• Tankyrase inhibition alters intratumoral cytokine composition

• This approach can overcome β-catenin-mediated resistance to immune checkpoint blockade

These findings suggest tankyrase inhibitors have potential in combination immunotherapy strategies, particularly in cancers with elevated WNT/β-catenin signaling .

• What are the best approaches for studying the role of tankyrase-1 in YAP signaling using recombinant mouse protein?

To investigate tankyrase-1 in YAP signaling:

• Proteomics approach: Identify angiomotin family proteins as tankyrase substrates

• Genetic tools: Use CRISPR/Cas9 to screen for YAP pathway regulators

• Inhibitor studies: Apply tankyrase inhibitors (TNKS656, IWR1) to suppress YAP activity

• Target gene analysis: Monitor expression of YAP targets (CTGF, CYR61, ANKRD1)

• Binding assays: Characterize interactions between tankyrase and angiomotins

• Combination studies: Test tankyrase inhibitors with targeted therapies such as EGFR inhibitors

Research shows that tankyrase inhibition stabilizes angiomotins (negative regulators of YAP signaling), reducing YAP nuclear translocation and decreasing downstream signaling, which may sensitize cancer cells to targeted therapies .

• What technical considerations are important when designing experiments to compare mouse and human tankyrase function?

Key considerations include:

• Substrate specificity: Account for species-specific differences in binding partners (particularly TRF1)

• Telomere biology: Mouse somatic cells have much longer telomeres (~40-50kb) than human cells (~5-20kb)

• Telomerase activity: Mice maintain high telomerase activity in somatic tissues unlike humans

• Compensatory mechanisms: Consider potential redundancy with tankyrase-2

• Readout selection: Choose appropriate endpoints based on conserved versus divergent functions

• Inhibitor selection: Some inhibitors may have species-specific potency differences

Experimental designs should leverage these differences to identify both conserved functions (Wnt signaling, YAP pathway) and divergent functions (telomere maintenance) .

• How can structural insights into tankyrase-substrate interactions guide the development of specific inhibitors?

Structural knowledge can inform inhibitor development through:

• Targeting specific subsites: Small molecules can bind the nicotinamide subsite, adenosine subsite, or both

• Exploiting unique features: The adenosine subsite of tankyrases diverges from other PARP family members

• Structure-guided hybridization: Combining privileged fragments from different inhibitor classes

• Linker optimization: Novel linkers (e.g., cyclobutyl) between binding moieties can improve affinity

• Specificity engineering: Designing inhibitors that exploit structural differences between tankyrases and other PARPs

Recent advances include the development of dual-site inhibitors with improved potency, selectivity, and pharmacokinetic properties, highlighting the value of structure-based approaches in tankyrase inhibitor design .