Recombinant Human Tubulin alpha-3E chain (TUBA3E)

What is TUBA3E and what is its primary role in cellular function?

TUBA3E (tubulin alpha-3E) is a member of the tubulin protein family that serves as a major constituent of microtubules in the eukaryotic cytoskeleton . Microtubules are essential cylindrical structures formed by the polymerization of alpha and beta tubulin heterodimers arranged in protofilaments .

The primary functions of TUBA3E, as part of the microtubule network, include:

• Providing structural support for the cell

• Facilitating intracellular transport

• Contributing to cell division through spindle formation

• Participating in cell motility mechanisms

TUBA3E belongs to a family of highly conserved proteins with multiple isoforms, each potentially exhibiting tissue-specific expression patterns and specialized functions within the cytoskeletal network .

What are the key structural features and biochemical properties of TUBA3E?

TUBA3E exhibits several important structural and biochemical characteristics:

• Molecular Weight: The predicted molecular weight is approximately 49.8 kDa

• GTP Binding: It binds one mole of GTP at a non-exchangeable site, while the beta-tubulin partner binds one mole of GTP at an exchangeable site

• Domain Organization: Contains conserved protein domains characteristic of the tubulin family

• C-terminal Tail: Possesses an intrinsically disordered alpha-tubulin tail that can undergo post-translational modifications and plays a regulatory role in microtubule dynamics

The protein structure of TUBA3E is highly conserved across species, reflecting its fundamental role in cellular architecture and function. The amino acid sequence contains specific binding regions that facilitate interactions with beta-tubulin subunits and various microtubule-associated proteins (MAPs) .

How do alpha and beta tubulins interact to form functional microtubules?

The formation of microtubules through alpha and beta tubulin interactions follows a specific process:

• Alpha-tubulin (such as TUBA3E) and beta-tubulin first form heterodimers

• These heterodimers polymerize in a head-to-tail arrangement to form protofilaments

• Typically, 13 protofilaments associate laterally to form the hollow cylindrical structure of a microtubule

The polymerization process is dynamic and GTP-dependent:

• Microtubules grow by the addition of GTP-bound tubulin dimers to the plus end

• A stabilizing cap forms at this growing end

• Below the cap, tubulin dimers are in the GDP-bound state due to the GTPase activity of alpha-tubulin

This structural arrangement creates inherent polarity in microtubules, with distinct plus and minus ends that exhibit different polymerization rates and properties, critical for their cellular functions .

What post-translational modifications occur on TUBA3E and what are their functional implications?

TUBA3E, like other alpha-tubulins, can undergo several post-translational modifications that regulate microtubule properties and functions:

Research has shown that these modifications create a "tubulin code" that regulates microtubule dynamics and interactions with binding partners. For example, CLIP-170 preferentially associates with tyrosinated microtubules, while CENP-E motor prefers detyrosinated microtubules .

What experimental approaches are most effective for studying TUBA3E function in vitro?

Several complementary approaches can be employed to study TUBA3E function:

• Recombinant Protein Expression and Purification:

  • Expression in HEK293T cells provides properly folded human TUBA3E with appropriate post-translational modifications

  • Purification via affinity chromatography using tags (e.g., C-Myc/DDK) followed by conventional chromatography methods

  • Buffer composition (25 mM Tris-HCl, 100 mM glycine, pH 7.3, 10% glycerol) is critical for maintaining protein stability

• In Vitro Microtubule Assembly Assays:

  • Turbidity assays to monitor polymerization kinetics

  • Total internal reflection fluorescence (TIRF) microscopy to visualize single microtubule dynamics

  • Co-sedimentation assays to study interactions with binding partners

• Structural Studies:

  • X-ray crystallography or cryo-electron microscopy to determine high-resolution structures

  • Molecular dynamics simulations to study the role of the intrinsically disordered α-tubulin tail in polymerization dynamics

For reliable results, researchers should ensure protein purity >80% as determined by SDS-PAGE and implement appropriate storage conditions (-80°C with minimal freeze-thaw cycles) .

How can researchers design experiments to distinguish the specific functions of TUBA3E from other alpha-tubulin isoforms?

Distinguishing TUBA3E functions from other alpha-tubulin isoforms requires careful experimental design:

• isoform-specific gene knockdown/knockout strategies:

  • CRISPR-Cas9 targeting of TUBA3E-specific regions

  • siRNA approaches targeting unique UTRs

  • Validation of specificity through qRT-PCR and western blotting

• Complementation experiments:

  • Express recombinant TUBA3E in cells depleted of endogenous TUBA3E

  • Compare with expression of other alpha-tubulin isoforms to identify TUBA3E-specific functions

• Isoform-specific antibodies:

  • Generate antibodies against unique epitopes of TUBA3E

  • Validate specificity through immunoblotting and immunoprecipitation

  • Use in immunofluorescence to study subcellular localization

• Chimeric protein approaches:

  • Create chimeras between TUBA3E and other alpha-tubulin isoforms

  • Map functional domains through domain-swapping experiments

What methodologies are available for studying the interactions between TUBA3E and its binding partners?

To study TUBA3E protein interactions, researchers can employ several complementary approaches:

• Protein-Protein Interaction Assays:

  • Co-immunoprecipitation using tagged recombinant TUBA3E

  • Proximity labeling techniques (BioID, APEX)

  • Yeast two-hybrid screening for novel interaction partners

  • Pull-down assays with immobilized TUBA3E protein

• Functional Interaction Studies:

  • Microtubule co-sedimentation assays with potential binding proteins

  • In vitro reconstitution of microtubule dynamics with purified components

  • Competition assays to identify binding interfaces

• High-throughput Screening:

  • Protein microarrays to identify novel binding partners

  • Mass spectrometry-based proteomics after affinity purification

Based on STRING database analysis, TUBA3E shows strong interactions (confidence score >0.96) with multiple beta-tubulin isoforms including TUBB, TUBB2A, TUBB4A, and TUBB2B . Additionally, it interacts with CDC42 (confidence score 0.923), suggesting roles beyond basic microtubule structure .

How can researchers effectively study the role of α-tubulin tail modifications in TUBA3E function?

Studying α-tubulin tail modifications requires specialized approaches:

Recent research has shown that the intrinsically disordered α-tubulin tail negatively regulates microtubule polymerization by transiently interacting with the α-tubulin longitudinal polymerization interface . Interestingly, detyrosination and Δ2 modification alone do not significantly affect microtubule dynamic parameters, but tyrosination quantitatively tunes the density of CLIP-170 at the microtubule plus end .

What are the critical factors to consider when using recombinant TUBA3E in experimental systems?

When working with recombinant TUBA3E, researchers should consider several factors to ensure experimental success:

• Protein Quality and Handling:

  • Ensure high purity (>80% as determined by SDS-PAGE and Coomassie blue staining)

  • Store at -80°C and minimize freeze-thaw cycles to maintain stability

  • Filter before use in cell culture applications to remove aggregates

• Buffer Composition:

  • Use appropriate buffers (e.g., 25 mM Tris-HCl, 100 mM glycine, pH 7.3, 10% glycerol) to maintain native conformation

  • Consider the effect of buffer components on experimental outcomes (e.g., GTP concentration for polymerization assays)

• Expression System Considerations:

  • HEK293T-expressed TUBA3E may have different post-translational modifications compared to E. coli-expressed protein

  • Tagged versions (e.g., C-Myc/DDK) may affect certain protein interactions

• Experimental Controls:

  • Include appropriate tubulin isoform controls to distinguish TUBA3E-specific effects

  • Consider using tubulin purified from native sources (e.g., brain) as a reference standard

  • Include both positive and negative controls in binding and functional assays

• Incorporation into Microtubules:

  • Recombinant TUBA3E may co-polymerize with endogenous tubulin in cellular systems

  • Consider using labeled TUBA3E to track incorporation into microtubules

For in vitro reconstitution of microtubule dynamics, researchers should ensure that recombinant TUBA3E is properly folded and competent to form heterodimers with beta-tubulin before proceeding with polymerization assays.

How should researchers approach experimental design when studying TUBA3E in the context of disease models?

When investigating TUBA3E in disease contexts, researchers should consider:

• Experimental Design Framework:

  • Define clear hypotheses about TUBA3E's role in the disease mechanism

  • Select appropriate control and experimental groups with sufficient sample sizes

  • Control extraneous variables that might influence results

  • Use both between-subjects and within-subjects designs when appropriate

• Disease-Relevant Experimental Systems:

  • Patient-derived samples (if available)

  • Disease-relevant cell lines

  • Animal models expressing disease-associated TUBA3E variants

  • iPSC-derived models for human relevance

• Relevant Readouts:

  • Microtubule dynamics and stability

  • Cell division and mitotic spindle formation

  • Cellular migration and morphology

  • Protein-protein interactions specific to disease contexts

• Translational Considerations:

  • Connect molecular findings to cellular phenotypes

  • Correlate with clinical observations when possible

  • Consider therapeutic implications of targeting TUBA3E or its interactions

While specific disease associations for TUBA3E are not extensively documented in the provided search results, research on other tubulin family members suggests potential roles in neurological disorders, cancer, and developmental abnormalities that could guide experimental approaches.