Recombinant Mouse Torsin-4A (Tor4a)

What is the molecular classification of Torsin-4A and how does it relate to other Torsins?

Torsin-4A (Tor4A) belongs to the Torsin family of AAA+ (ATPases associated with a variety of cellular activities) ATPases. Like other Torsins, including the well-characterized TorsinA (TorA), Tor4A functions to disassemble protein complexes or unfold proteins . Torsins represent an atypical subclass of AAA+ ATPases with distinctive structural features that differentiate them from conventional members of this superfamily . While TorsinA has been extensively studied due to its association with dystonia, Tor4A research provides comparative insights into the functional diversity within this protein family.

What are the primary subcellular localizations of Tor4A, and how do they influence its functionality?

Based on studies of Torsin family proteins, Tor4A likely localizes primarily to the endoplasmic reticulum (ER) lumen and potentially the nuclear envelope (NE), similar to TorsinA . The protein contains an N-terminal signal sequence that directs it into the ER lumen, where post-translational modifications including glycosylation likely occur . The partitioning between ER and NE compartments is functionally significant as it determines the protein substrates and regulatory partners accessible to Tor4A. For experimental investigations, subcellular fractionation followed by immunoblotting can confirm the precise distribution pattern of Tor4A.

What experimental approaches are recommended for verifying the ATPase activity of recombinant Tor4A?

To assess the ATPase activity of recombinant Tor4A, researchers should consider:

• In vitro ATP hydrolysis assays: Measuring phosphate release from ATP using colorimetric methods such as malachite green assays

• ATP binding studies: Using non-hydrolyzable ATP analogs like ATPγS to assess nucleotide binding capacity

• Comparative analysis: Including Walker B motif mutants (e.g., E171Q in TorsinA) as negative controls

• Co-factor dependence testing: Examining whether regulatory proteins such as LAP1 or LULL1 luminal domains enhance the ATPase activity

These approaches should be performed under different pH and ionic strength conditions to determine optimal enzymatic parameters.

How can researchers effectively design experiments to determine the specific substrate selectivity of Tor4A?

Determining substrate selectivity for Tor4A requires multifaceted approaches:

• Proteomic identification of binding partners:

  • Perform immunoprecipitation of tagged Tor4A followed by mass spectrometry

  • Use ATP-trapped mutants (similar to TorA E171Q) that maintain high-affinity substrate binding

  • Include appropriate controls with ΔE-equivalent mutations that may disrupt interactions

• Direct binding assays:

  • Express and purify the luminal domain of Tor4A

  • Test interaction with candidate substrates using techniques like surface plasmon resonance or microscale thermophoresis

  • Compare binding in ATP, ADP, and nucleotide-free states

• Cellular substrate trapping:

  • Express ATP-hydrolysis deficient Tor4A in cells

  • Perform proximity labeling (BioID or APEX) to identify nearby proteins

  • Validate candidates with in vitro reconstitution experiments

This systematic approach enables identification of physiologically relevant Tor4A substrates and distinguishes them from non-specific interactions.

What are the critical considerations when designing recombinant Tor4A constructs for in vivo studies?

When designing recombinant Tor4A constructs:

• Signal sequence integrity: Maintain the native signal sequence to ensure proper ER targeting, as alterations may redirect the protein to inappropriate compartments

• Tag positioning: Consider that N-terminal tags may interfere with ER targeting, while C-terminal tags could affect ATPase activity or interactions with regulatory partners

• Expression system selection:

  • Mammalian expression systems provide proper post-translational modifications

  • Heterologous systems like yeast may require adaptation for correct localization

• Mutation design:

  • Include Walker B mutations (ATP-trapped) for substrate identification

  • Consider equivalent-to-dystonia mutations (ΔE in TorA) to assess functional consequences

• Solubility considerations: The N-terminal hydrophobic domain affects membrane association; modifications may alter solubility and localization

A thoughtful construct design is critical for ensuring physiologically relevant results.

How can Tor4A research inform our understanding of dystonia and other movement disorders?

While TorsinA has a well-established link to DYT1 dystonia through the ΔE deletion, investigating Tor4A can provide complementary insights:

• Functional redundancy: Tor4A may partially compensate for TorsinA deficiency in certain tissues, explaining tissue-selective phenotypes in dystonia

• Comparative analysis: Structural differences between Tor4A and TorsinA might reveal why mutations in TorsinA, but not Tor4A, lead to movement disorders

• Cellular stress pathways: Like TorsinA, Tor4A may participate in the ER stress response and protein quality control mechanisms relevant to neurodegenerative conditions

• Model systems: Analyzing Tor4A knockout or overexpression in parallel with TorsinA models can reveal unique and overlapping functions

A comprehensive understanding of the entire Torsin family, including Tor4A, is necessary to fully elucidate the pathophysiology of dystonia and develop targeted therapeutic approaches.

What methods should be used to assess whether Tor4A can functionally complement TorsinA deficiency in experimental models?

To determine whether Tor4A can compensate for TorsinA loss:

• Rescue experiments in cellular models:

  • Use CRISPR/Cas9 to generate TorsinA-null cells

  • Express Tor4A at physiological levels

  • Assess rescue of phenotypes such as nuclear envelope morphology and ER stress responses

• Ex vivo tissue analysis:

  • Examine Tor1A−/− or Tor1A Δgag/Δgag tissues with and without Tor4A overexpression

  • Assess nuclear envelope vesicle formation and other ultrastructural features

• In vivo models:

  • Generate conditional double knockout models of TorA and Tor4A

  • Compare phenotypes to single knockouts to assess synergistic effects

  • Perform tissue-specific rescue experiments

• Biochemical complementation:

  • Test whether Tor4A can interact with known TorsinA partners like LAP1 and LULL1

  • Assess whether these interactions are regulated similarly to TorsinA

These approaches can determine whether Tor4A has functional redundancy with TorsinA and in which specific contexts.

How do the regulatory mechanisms of Tor4A differ from those of TorsinA in terms of co-factor interactions?

To investigate differential regulation of Tor4A compared to TorsinA:

• Co-immunoprecipitation studies:

  • Compare binding of Tor4A and TorsinA to LAP1 and LULL1 under identical conditions

  • Determine whether these interactions are similarly ATP-dependent

  • Assess whether disease-associated mutations have comparable effects on these interactions

• In vitro reconstitution:

  • Express and purify recombinant Tor4A and regulatory proteins

  • Compare ATPase stimulation between different Torsin family members

  • Determine binding affinities and kinetic parameters

• Structural biology approaches:

  • Analyze conserved surface patches that might mediate interactions

  • Target mutations to predicted interaction interfaces to verify functional consequences

  • Consider that the acidic patch affected by the dystonia-causing deletion in TorsinA may have different properties in Tor4A

Understanding these differences may explain why mutations in different Torsin family members lead to distinct phenotypes.

What experimental systems best demonstrate the evolutionary conservation or divergence of Tor4A function across species?

To investigate evolutionary aspects of Tor4A function:

• Heterologous expression studies:

  • Express mouse Tor4A in simpler organisms like yeast, similar to studies with TorsinA

  • Assess protection against environmental stressors

  • Test refolding activity for heat-denatured substrates like luciferase

• Comparative genomics and phylogenetic analysis:

  • Analyze conservation of key structural features across species

  • Identify species-specific variations in regulatory domains

  • Map conservation of interaction sites for regulatory partners

• Cross-species complementation:

  • Test whether Tor4A from different species can complement deficiencies in orthologous genes

  • Compare with complementation efficiency of other Torsin family members

• Organoid and primary cell comparisons:

  • Compare Tor4A function in neural organoids or primary cells from different species

  • Assess species-specific differences in subcellular localization and response to stress

These approaches provide insight into both conserved core functions and species-specific adaptations of Tor4A.

What methodological approaches are recommended to differentiate between direct and indirect effects when studying Tor4A's role in protein quality control?

To distinguish direct from indirect effects of Tor4A in protein quality control:

• In vitro reconstitution:

  • Purify recombinant Tor4A and potential substrate proteins

  • Assess direct refolding or disaggregation activity using spectroscopic methods

  • Compare activity to known chaperones and disaggregases

• Substrate-specific assays:

  • Monitor degradation of model ERAD substrates (e.g., mutant CFTR) with and without Tor4A modulation

  • Use pulse-chase experiments to track protein stability

  • Apply proteasome inhibitors to distinguish between effects on retrotranslocation versus degradation

• Proximity-based approaches:

  • Use split fluorescent protein complementation to assess direct interaction with substrates

  • Employ FRET/BRET to monitor real-time interactions during stress conditions

  • Apply crosslinking strategies to capture transient interactions

• Domain mapping:

  • Generate chimeric proteins between Tor4A and other Torsins

  • Identify domains responsible for specific quality control functions

  • Create point mutations in putative substrate-binding regions

These methodologies help establish causality rather than correlation in Tor4A's contribution to protein quality control pathways.

How should researchers design experiments to investigate Tor4A's potential role in nuclear envelope dynamics and nuclear pore complex assembly?

To investigate Tor4A's role in nuclear envelope functions:

• High-resolution imaging approaches:

  • Employ super-resolution microscopy to localize Tor4A relative to nuclear pore complexes (NPCs)

  • Use electron microscopy to visualize nuclear envelope ultrastructure in Tor4A-deficient cells

  • Apply live-cell imaging to monitor NE dynamics during cell division

• Nuclear envelope protein trafficking:

  • Assess nuclear import kinetics using photoactivatable reporters

  • Monitor NPC component localization in Tor4A-deficient backgrounds

  • Investigate interaction with ESCRT machinery implicated in NE functions

• Biochemical fractionation:

  • Isolate nuclear envelopes to quantify Tor4A enrichment

  • Perform proteomic analysis of NE composition with and without Tor4A

  • Analyze post-translational modifications of NE proteins dependent on Tor4A

• Cell cycle-specific investigations:

  • Synchronize cells to examine Tor4A function during specific cell cycle phases

  • Compare interphase versus mitotic functions

  • Assess NE reassembly following mitosis

These approaches can determine whether Tor4A, like other Torsins, participates in maintaining nuclear envelope integrity and function.

What are the optimal conditions for reconstituting and storing recombinant mouse Tor4A to ensure maximum protein stability and activity?

Based on handling recommendations for similar recombinant proteins :

Reconstitution protocol:

• Reconstitute lyophilized Tor4A at 100 μg/mL in sterile PBS

• For enhanced stability, consider adding 0.1% human or bovine serum albumin as a carrier protein

• Allow complete solubilization by gentle rotation at 4°C for 30 minutes

• Filter through a 0.22 μm filter if absolute sterility is required

Storage recommendations:

• Store reconstituted Tor4A at -80°C in small single-use aliquots

• Avoid repeated freeze-thaw cycles which can compromise activity

• For working solutions, maintain at 4°C for no more than 1 week

• Consider addition of stabilizing agents such as glycerol (10%) for increased stability

Activity preservation:

• Validate activity after storage using functional assays relevant to AAA+ ATPases

• Include ATP or non-hydrolyzable ATP analogs when appropriate to stabilize conformation

• Monitor for aggregation using dynamic light scattering before experimental use

Proper handling ensures reliable experimental outcomes when working with recombinant Tor4A.

What analytical methods are most appropriate for confirming the structural integrity and correct folding of recombinant Tor4A?

To verify proper folding and structural integrity:

• Spectroscopic methods:

  • Circular dichroism (CD) to assess secondary structure composition

  • Fluorescence spectroscopy to monitor tertiary structure through intrinsic tryptophan fluorescence

  • FTIR spectroscopy to complement CD analysis for secondary structure

• Hydrodynamic analysis:

  • Size exclusion chromatography to verify oligomeric state

  • Analytical ultracentrifugation to assess homogeneity and oligomerization properties

  • Dynamic light scattering to detect aggregation

• Functional verification:

  • ATP binding assays using fluorescent ATP analogs

  • ATPase activity measurements compared to established references

  • Interaction studies with known binding partners like LAP1 or LULL1 luminal domains

• Thermal stability assessment:

  • Differential scanning fluorimetry (thermofluor) to determine melting temperature

  • Monitoring stability in different buffer conditions to optimize experimental conditions

  • Testing ligand-induced stabilization with ATP or interaction partners

These analytical approaches provide complementary information to ensure that recombinant Tor4A maintains its native structure and functional properties.