Recombinant Human Torsin-4A (TOR4A)

What is Torsin-4A (TOR4A) and how is it classified?

TOR4A, also known as C9orf167, is a member of the Torsin family of proteins. Similar to other Torsins, it belongs to the AAA+ (ATPases Associated with diverse cellular Activities) superfamily of proteins. Unlike the better-studied TorsinA (TorA), which has been extensively characterized in relation to dystonia type 1, TOR4A has distinct tissue expression patterns and cellular functions. TOR4A has emerged as an important protein in various biological processes, including potential roles in cancer progression and infectious disease responses .

What genetic and structural characteristics define TOR4A?

TOR4A is encoded by the TOR4A gene located on chromosome 9 in humans. Like other members of the Torsin family, it likely contains conserved AAA+ domain features including Walker A and B motifs for ATP binding and hydrolysis. While specific structural data for TOR4A is limited, research on related family members suggests it may share the characteristic ring-like hexameric arrangement common to AAA+ ATPases. These structural features are likely critical for its cellular functions, though detailed structural studies specifically on TOR4A are still needed .

How does TOR4A expression vary across tissues?

Expression analysis reveals that TOR4A exhibits a distinctive tissue distribution pattern. It is most abundantly expressed in the lungs and spleen, with relatively lower expression in other tissues. This expression pattern is consistent with its potential role in respiratory infections such as bacterial pneumonia. The tissue-specific expression suggests TOR4A may have specialized functions in pulmonary and immune tissues, potentially related to host defense mechanisms or tissue-specific cellular processes .

What cellular functions has TOR4A been implicated in?

Research indicates that TOR4A functions as a promoter of cell proliferation and an inhibitor of apoptosis in certain contexts. In glioma cells, TOR4A has been shown to enhance the expression of phosphorylated protein kinase B (p-AKT) while inhibiting antiapoptotic proteins. This suggests a role in regulating cell survival pathways and potentially contributing to oncogenic processes. The protein's function may be context-dependent, with different activities in various cellular compartments and tissue types .

How does TOR4A contribute to disease pathogenesis?

TOR4A has been identified as a potential oncogene in gliomas. Studies have demonstrated that high expression levels of TOR4A correlate with worse prognosis in glioma patients. Mechanistically, TOR4A promotes glioma cell proliferation and inhibits apoptosis through the AKT signaling pathway. Additionally, TOR4A has been implicated in bacterial pneumonia susceptibility, suggesting a potential role in infectious disease processes. These diverse associations indicate that TOR4A may function in multiple pathological processes depending on the cellular context .

What signaling pathways interact with TOR4A?

The AKT signaling pathway appears to be a key mediator of TOR4A function, particularly in cancer contexts. Research shows that TOR4A enhances phosphorylated AKT (p-AKT) expression, which is a central regulator of cell survival, proliferation, and metabolism. Additionally, TOR4A has been shown to influence the expression of antiapoptotic proteins, suggesting it may interface with programmed cell death pathways. The relationship between TOR4A and the DNER (Delta and Notch-like epidermal growth factor-related receptor) pathway has also been observed, with DNER potentially acting as a tumor suppressor by inhibiting TOR4A in glioma cells .

What experimental models are suitable for studying TOR4A function?

Several experimental systems have proven valuable for investigating TOR4A function:

Cell culture systems, particularly glioma cell lines, have been successfully used to characterize TOR4A's role in proliferation and apoptosis. While no specific yeast models for TOR4A are documented in the provided literature, related Torsin proteins have been studied in yeast systems, suggesting this could be a viable approach for TOR4A as well .

How can recombinant TOR4A be used in protein-protein interaction studies?

Recombinant TOR4A protein can serve as a valuable tool for identifying protein interaction partners through techniques such as co-immunoprecipitation, pull-down assays, and proximity labeling approaches. When designing such experiments, researchers should consider:

• Using appropriate tags that don't interfere with TOR4A function

• Including proper controls to distinguish specific from non-specific interactions

• Confirming interactions through multiple methodologies

• Validating interaction partners in relevant cellular contexts

Given TOR4A's potential role in AKT signaling, investigating interactions with components of this pathway may be particularly informative. Additionally, exploring interactions with other Torsin family members could provide insights into possible functional redundancy or cooperation .

What approaches can be used to modulate TOR4A expression in experimental settings?

Several methodologies can be employed to manipulate TOR4A expression:

• RNA interference (siRNA or shRNA) for transient or stable knockdown

• CRISPR-Cas9 gene editing for knockout or precise mutation introduction

• Overexpression systems using plasmid vectors

• Inducible expression systems for temporal control

When studying TOR4A in glioma contexts, researchers have successfully employed RNA interference approaches to demonstrate its role in cell proliferation and apoptosis. For more detailed mechanistic studies, CRISPR-based approaches might offer advantages in creating complete knockouts or introducing specific mutations .

What is the relationship between TOR4A and infectious disease susceptibility?

Genome-wide and transcriptome-wide association studies have identified TOR4A as significantly associated with bacterial pneumonia. The protein shows highest expression in lung tissue, consistent with its potential role in respiratory infections. TOR4A may represent a host factor that influences susceptibility to specific pathogens or the inflammatory response to infection. Understanding the precise mechanisms by which TOR4A affects infectious disease processes requires further investigation, but may involve interaction with innate immune pathways or cellular stress responses .

How does TOR4A function in cancer biology?

TOR4A has been characterized as an oncogene in glioma research. Key findings include:

• High TOR4A expression correlates with worse prognosis in glioma patients

• TOR4A promotes cell proliferation in glioma models

• TOR4A inhibits apoptosis through AKT pathway modulation

• DNER (Delta and Notch-like epidermal growth factor-related receptor) acts as a tumor suppressor by inhibiting TOR4A

These observations suggest TOR4A could be a potential therapeutic target in gliomas. The p-AKT pathway appears to be a key mediator of TOR4A's oncogenic effects, suggesting potential synergy with AKT inhibitors in treatment approaches. Additional research is needed to determine if TOR4A plays similar roles in other cancer types .

What are the key methodological considerations when using recombinant TOR4A in structural studies?

When planning structural studies of recombinant TOR4A, researchers should consider:

• Expression system selection: Cell-free expression systems have been used successfully for TOR4A production as indicated by commercial sources

• Purification strategy: Aim for ≥85% purity as typically achieved in commercial preparations

• Buffer optimization: Consider conditions that maintain native conformation while preventing aggregation

• Structural technique selection: X-ray crystallography, cryo-EM, and NMR may provide complementary structural insights

Since TOR4A belongs to the AAA+ ATPase family, it likely forms oligomeric structures that should be considered in experimental design. Additionally, the protein's ATP binding and hydrolysis activities may influence structural states, suggesting that studies should examine both nucleotide-bound and unbound forms .

What are the optimal conditions for functional assays using recombinant TOR4A?

When designing functional assays for recombinant TOR4A:

• ATP hydrolysis assays: As an AAA+ family member, TOR4A likely exhibits ATPase activity. Use a malachite green assay or coupled enzyme assay to measure ATP hydrolysis rates.

• Protein refolding assays: Based on studies of related Torsin proteins, TOR4A may function in protein quality control. Consider using denatured luciferase or other aggregation-prone substrates to assess refolding activity.

• Cell-based functional assays: For cellular studies, assess effects on:

  • Cell proliferation (using MTT or BrdU incorporation)

  • Apoptosis (using Annexin V/PI staining)

  • AKT pathway activation (measuring p-AKT levels)

• Critical controls:

  • Include ATP-binding deficient mutants (typically mutations in Walker A motif)

  • Compare with other Torsin family members to assess functional specificity

  • Use physiologically relevant concentrations based on tissue expression data

How can researchers effectively generate and validate TOR4A mutants for functional studies?

Creation and validation of TOR4A mutants should follow these guidelines:

• Strategic mutation selection:

  • Walker A motif mutations to disrupt ATP binding

  • Walker B motif mutations to allow binding but prevent hydrolysis

  • Mutations corresponding to disease-associated variants in related Torsins

  • Mutations in potential interaction interfaces

• Expression system considerations:

  • Test expression in multiple systems (bacterial, insect, mammalian)

  • Assess folding and solubility of mutant proteins

  • Compare protein stability with wild-type controls

• Functional validation:

  • Confirm predicted biochemical defects (e.g., ATP binding/hydrolysis)

  • Assess cellular localization of mutants

  • Evaluate effects on model cellular processes (proliferation, apoptosis)

  • Test interaction with known binding partners

Studies of the related TorsinA protein have demonstrated the value of creating equivalent mutations to those identified in movement disorders (e.g., the ΔE302-303 mutation in TorsinA). Similar approaches could provide insights into TOR4A structure-function relationships .