Recombinant Mouse Torsin-1A-interacting protein 2 (Tor1aip2)

What is the basic structure and function of Tor1aip2?

Tor1aip2 (also known as LULL1, IFRG15, or Lumenal domain-like LAP1) is a type II integral membrane protein primarily localized in the endoplasmic reticulum (ER). The mouse Tor1aip2 protein consists of 470 amino acid residues with a predicted molecular mass of approximately 28.7 kDa, though the actual mass is typically observed at 29 kDa in experimental settings .

Functionally, Tor1aip2 serves as a crucial cofactor for TorsinA ATPase activity. It plays essential roles in:

• Maintaining endoplasmic reticulum integrity

• Regulating the distribution of TOR1A between the ER and nuclear envelope

• Inducing the ATPase activity of TOR1A, TOR1B, and TOR3A

The protein contributes to cellular architecture by participating in the linkage between the cytoskeleton and nuclear envelope, an interaction that appears particularly important in neuronal cells .

How does recombinant mouse Tor1aip2 differ from the endogenous protein?

Commercially available recombinant mouse Tor1aip2 typically consists of a fragment spanning amino acids Tyr250 to Leu475 (Accession # Q8BYU6) with an N-terminal His-tag for purification purposes . This differs from the endogenous full-length protein in several key aspects:

These differences should be considered when interpreting experimental results, particularly when studying protein-protein interactions or functional assays that might be affected by the absence of the N-terminal domain or native post-translational modifications .

What experimental applications are suitable for recombinant Tor1aip2?

Recombinant mouse Tor1aip2 can be utilized in multiple experimental applications:

• Immunological studies: As a positive control in Western blotting and immunoprecipitation experiments

• Protein interaction studies: For in vitro binding assays with potential partners like TOR1A, TOR1B, and TOR3A

• Immunogen production: For generating specific antibodies against Tor1aip2

• Structure-function analysis: For crystallography or other structural biology techniques

• ATPase activity assays: To evaluate the cofactor function of Tor1aip2 on torsin proteins

When designing experiments, researchers should reconstitute the lyophilized protein in 10mM PBS (pH 7.4) to a concentration of 0.1-1.0 mg/mL without vortexing to maintain protein integrity. For long-term storage, aliquoting and storing at -80°C is recommended to avoid repeated freeze-thaw cycles that can compromise protein activity .

How does Tor1aip2 regulate TorsinA activity at the molecular level?

Tor1aip2 functions as a critical cofactor that stimulates the ATPase activity of TorsinA through direct protein-protein interactions. At the molecular level, this regulation involves several mechanisms:

• Spatial regulation: Tor1aip2 controls the distribution of TorsinA between the endoplasmic reticulum and nuclear envelope compartments, effectively concentrating the enzyme where its activity is required

• Conformational activation: The interaction between Tor1aip2 and TorsinA induces conformational changes in TorsinA that promote its ATPase activity, similar to how AAA+ protein cofactors function

• Oligomerization facilitation: Evidence suggests that Tor1aip2 may facilitate the assembly of TorsinA into functional oligomeric complexes, which represent the active form of many AAA+ ATPases

The strong interaction score (0.999) between TOR1AIP2 and TOR1A in protein interaction databases confirms the biological significance of this relationship . This regulatory mechanism appears to extend to other torsin family members, including TOR1B (interaction score 0.905) and TOR3A (interaction score 0.687), suggesting a conserved mode of action across the torsin family .

What experimental approaches can resolve conflicting data regarding Tor1aip2 cellular localization?

Researchers frequently encounter conflicting data regarding Tor1aip2 localization, with some studies reporting predominantly ER localization while others suggest nuclear envelope association. To resolve these discrepancies, consider implementing the following complementary approaches:

• Multiple detection methods:

  • Immunofluorescence using antibodies targeting different epitopes of Tor1aip2

  • Live-cell imaging with fluorescently tagged Tor1aip2 constructs

  • Subcellular fractionation followed by Western blotting

  • Proximity labeling techniques (BioID or APEX2)

• Cell type-specific analysis:

  • Compare localization patterns across multiple cell types (neurons, fibroblasts, muscle cells)

  • Use primary cells rather than transformed cell lines when possible

  • Examine localization in tissue sections to capture in vivo distribution

• Dynamic localization studies:

  • Investigate localization changes during cell cycle progression

  • Examine effects of cellular stress (ER stress, oxidative stress)

  • Study localization following disruption of the nuclear envelope during mitosis

• High-resolution imaging:

  • Implement super-resolution microscopy (STORM, PALM, or SIM)

  • Use correlative light and electron microscopy (CLEM)

  • Apply expansion microscopy for improved spatial resolution

By combining these approaches and carefully controlling for fixation artifacts, antibody specificity, and expression levels of tagged constructs, researchers can develop a more comprehensive understanding of the true subcellular distribution of Tor1aip2 .

How does RNA modification affect Tor1aip2 gene expression and splicing?

Recent research has implicated N6-methyladenosine (m6A) RNA modification in regulating Tor1aip2 expression and splicing. When investigating this regulatory mechanism, researchers should consider:

• Splicing regulation: Intron 3 of the Tor1aip2 gene was identified as one of the most strongly affected differentially spliced regions following acute depletion of METTL3 (a key m6A methyltransferase), suggesting m6A-dependent regulation of Tor1aip2 alternative splicing

• Methodological approaches to study this phenomenon include:

  • MeRIP-seq or m6A-CLIP to map m6A modifications across Tor1aip2 transcripts

  • RNA-seq following METTL3/METTL14 knockdown or knockout to identify splicing changes

  • Minigene splicing assays to validate the role of specific m6A sites in splicing regulation

  • CRISPR-based mutagenesis of m6A sites to establish causality

• Functional consequences:

  • Different splicing events may generate protein isoforms with distinct subcellular localizations or functional properties

  • Alternative splicing could affect the ratio between the ER-localized isoform and the interferon-responsive isoform mentioned in literature

This area represents an emerging field connecting epitranscriptomics to Tor1aip2 regulation and function, with significant implications for understanding tissue-specific expression patterns and potential dysregulation in disease states .

What is the significance of the TOR1AIP2::ETV6 fusion in hematological malignancies?

The recently identified TOR1AIP2::ETV6 fusion transcript, resulting from the chromosomal translocation t(1;12)(q25;p13), represents a novel genetic aberration in acute myeloid leukemia (AML) that has progressed from myelodysplastic syndrome (MDS) . When investigating this fusion in research settings:

• Structural analysis:

  • The fusion appears to encode a transcript that does not produce a functional fusion protein, suggesting potential regulatory roles through non-coding mechanisms

  • The breakpoints and the exact fusion sequence should be characterized to understand the structural consequences

• Clinical correlations:

  • The fusion was observed in a case with concurrent FLT3-ITD mutation, a known marker of poor prognosis in AML

  • Further studies should examine the frequency of this fusion across larger patient cohorts and determine its prognostic significance

  • Potential cooperation between the fusion and FLT3-ITD in disease progression merits investigation

• Functional studies should address:

  • Effects of the fusion on normal hematopoiesis using in vitro and in vivo models

  • Potential disruption of normal TOR1AIP2 and ETV6 functions

  • Investigation of downstream signaling pathways affected by the fusion

• Monitoring applications:

  • The fusion transcript may serve as a molecular marker for minimal residual disease (MRD) monitoring

  • PCR-based detection methods should be optimized for sensitive detection in patient samples

This discovery highlights the importance of comprehensive genomic characterization in hematological malignancies and opens new research directions regarding the role of TOR1AIP2 in normal and malignant hematopoiesis .

What are the optimal conditions for working with recombinant Tor1aip2 in vitro?

Successful experimentation with recombinant mouse Tor1aip2 requires careful attention to handling and experimental conditions:

• Reconstitution protocol:

  • Use 10mM PBS (pH 7.4) for reconstitution to a final concentration of 0.1-1.0 mg/mL

  • Avoid vortexing during reconstitution as this can lead to protein denaturation

  • Allow complete dissolution by gentle rotation at 4°C

• Storage considerations:

  • Store reconstituted protein at 2-8°C for up to one month

  • For long-term storage, prepare small aliquots and store at -80°C

  • Avoid repeated freeze-thaw cycles which significantly reduce protein activity

• Buffer optimization for functional assays:

  • For ATPase activity assays: 20mM HEPES (pH 7.4), 150mM NaCl, 10mM MgCl₂, 1mM DTT

  • For binding studies: PBS with 0.05% Tween-20 or 0.1% BSA to minimize non-specific interactions

  • For structural studies: Consider buffer screening to identify conditions that enhance protein stability

• Thermal stability:

  • The thermal stability of recombinant Tor1aip2 should be assessed through thermal shift assays

  • Accelerated thermal degradation tests can determine the protein loss rate under various conditions

  • This information is crucial for planning experiments and interpreting results from functional assays

Following these guidelines will help ensure experimental reproducibility and maximize the functional activity of recombinant Tor1aip2 in various research applications.

How can researchers effectively study Tor1aip2 interactions with torsin family proteins?

To investigate the interactions between Tor1aip2 and torsin family proteins (TOR1A, TOR1B, TOR3A), researchers should consider a multi-faceted approach:

• In vitro binding assays:

  • Pull-down assays using His-tagged recombinant Tor1aip2 and GST-tagged torsin proteins

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinity constants

  • Isothermal titration calorimetry (ITC) for thermodynamic characterization of interactions

  • AlphaScreen or ELISA-based methods for high-throughput interaction screening

• Cellular interaction studies:

  • Co-immunoprecipitation from cells expressing tagged variants of both proteins

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • FRET or BiFC to monitor interactions in living cells

  • BioID or APEX2 proximity labeling to identify interaction interfaces

• Functional interaction analysis:

  • ATPase activity assays to quantify Tor1aip2-mediated activation of torsins

  • Structural studies (X-ray crystallography, cryo-EM) of the complex

  • Mutagenesis of predicted interaction interfaces followed by binding and activity assays

  • Domain mapping to identify minimal regions required for interaction

The strong predicted interaction scores from STRING database (0.999 for TOR1A, 0.905 for TOR1B, and 0.687 for TOR3A) provide a foundation for these studies, and researchers should use these values to prioritize investigations while remaining open to discovering novel interaction partners .

What controls are essential when studying Tor1aip2 in cellular models?

Robust experimental design for Tor1aip2 studies in cellular models requires comprehensive controls to ensure valid interpretation of results:

• Expression controls:

  • Include vector-only controls when overexpressing Tor1aip2

  • Use multiple siRNA/shRNA sequences targeting different regions when performing knockdown studies

  • Verify knockdown/overexpression efficiency by both qRT-PCR and Western blot

  • Consider inducible expression systems to control expression timing and level

• Localization controls:

  • Use established organelle markers (calnexin for ER, lamin for nuclear envelope)

  • Include both N- and C-terminally tagged constructs to assess tag interference

  • Compare results from fixed and live cell imaging

  • Validate antibody specificity using knockout cells or blocking peptides

• Functional controls:

  • Include known TOR1A mutants (e.g., ΔE302/303) when studying Tor1aip2-TOR1A interactions

  • Use catalytically inactive mutants in ATPase assays

  • Complement knockout studies with rescue experiments using wild-type and mutant constructs

  • Include relevant cell types (neurons for dystonia-related studies)

• Disease model controls:

  • When studying dystonia-related mechanisms, include both wild-type and disease-associated mutant forms

  • For hematological malignancy studies, compare findings in normal and malignant cell lines

  • Consider patient-derived cells versus established cell lines

Implementation of these controls will strengthen data interpretation and improve reproducibility across different experimental systems and research groups.

How does Tor1aip2 contribute to neuromuscular disease pathogenesis?

Defects in Tor1aip2 have been implicated in early-onset primary dystonia, a neuromuscular disorder characterized by sustained or intermittent muscle contractions leading to abnormal movements or postures . Current research suggests several mechanistic pathways:

• Nuclear envelope dynamics:

  • Tor1aip2 regulates TorsinA distribution between the ER and nuclear envelope

  • Dystonia-causing mutations may disrupt this regulatory function, leading to abnormal nuclear envelope architecture

  • This affects mechanotransduction in neurons particularly sensitive to nuclear mechanics

• Protein quality control:

  • As a cofactor for TorsinA, Tor1aip2 participates in protein folding and quality control

  • Disruption may lead to accumulation of misfolded proteins, triggering cellular stress responses

  • Neurons with high protein synthesis rates may be particularly vulnerable to such disruptions

• Synaptic vesicle recycling:

  • TorsinA regulates synaptic vesicle recycling, and Tor1aip2 may indirectly affect this process

  • Alterations in neurotransmitter release could explain the circuit-level dysfunction in dystonia

  • Research should focus on electrophysiological studies in neurons with Tor1aip2 perturbations

• Cytoskeletal interactions:

  • The TorsinA-Tor1aip2 system links the cytoskeleton with the nuclear envelope

  • This connection is crucial for nuclear positioning and movement in migrating neurons

  • Disruption could affect neuronal development and circuitry formation

Future research should employ neuron-specific conditional knockout models, patient-derived iPSCs differentiated into relevant neuronal subtypes, and high-resolution imaging of nuclear envelope dynamics to further elucidate these mechanisms.

What is the role of Tor1aip2 in interferon responses and immune function?

One isoform of Tor1aip2 has been identified as an interferon alpha responsive protein (denoted by the alternative name IFRG15), suggesting important functions in immune response . This represents an understudied aspect of Tor1aip2 biology with potential implications for both infectious disease and cancer research:

• Expression regulation:

  • Characterize the transcriptional and post-transcriptional regulation of Tor1aip2 in response to type I interferons

  • Map the interferon-stimulated response elements (ISREs) in the Tor1aip2 promoter

  • Investigate tissue-specific expression patterns following interferon stimulation or viral infection

• Functional roles in immune cells:

  • Examine Tor1aip2 functions in different immune cell populations (macrophages, dendritic cells, T cells)

  • Investigate potential roles in antigen presentation, cytokine production, or cellular activation

  • Determine whether Tor1aip2 participates in pattern recognition receptor signaling pathways

• Viral infection contexts:

  • Study Tor1aip2 expression and function during viral infections that trigger strong interferon responses

  • Investigate whether viruses have evolved mechanisms to counteract Tor1aip2-mediated antiviral effects

  • Examine potential interactions with viral proteins that target the nuclear envelope or ER

• Relationship to ER stress:

  • Explore the intersection between interferon signaling, ER stress responses, and Tor1aip2 function

  • Investigate whether Tor1aip2 participates in the integrated stress response during viral infection

  • Examine potential connections to the unfolded protein response pathway

This research direction may reveal novel functions for Tor1aip2 beyond its established roles in nuclear envelope biology and potentially identify new therapeutic targets for inflammatory or infectious diseases.

How can Tor1aip2 research inform therapeutic approaches for related disorders?

Research on Tor1aip2 has significant translational potential for developing therapeutic strategies for dystonia and potentially other disorders:

• Small molecule modulators:

  • Screen for compounds that enhance Tor1aip2-TorsinA interactions or bypass defective interactions

  • Develop structure-based drug design approaches targeting the Tor1aip2-TorsinA interface

  • Evaluate compounds that modulate ATPase activity in physiologically relevant contexts

• Gene therapy approaches:

  • Evaluate AAV-mediated delivery of Tor1aip2 to relevant neuronal populations

  • Develop CRISPR-based strategies to correct disease-causing mutations

  • Investigate the therapeutic window during development for intervention in early-onset dystonia

• Hematological malignancy applications:

  • Explore whether the TOR1AIP2::ETV6 fusion represents a druggable target

  • Develop diagnostic assays for fusion detection in patient samples

  • Investigate whether targeting this fusion could sensitize cells to conventional therapies, particularly in FLT3-ITD positive leukemias

• Biomarker development:

  • Assess whether Tor1aip2 protein levels or post-translational modifications correlate with disease status

  • Explore Tor1aip2 as a potential biomarker for treatment response in neurological disorders

  • Investigate circulating autoantibodies against Tor1aip2 in autoimmune conditions

These translational research directions build upon the basic and mechanistic understanding of Tor1aip2 function and could potentially lead to novel therapeutic strategies for currently challenging disorders.