VTA1 Human

What is VTA1 and what is its primary function in human cells?

VTA1 (Vesicle Trafficking 1) is a protein-coding gene located on chromosome 6 that plays a crucial role in the endosomal multivesicular body (MVB) pathway. MVBs are responsible for forming intraluminal vesicles that facilitate the breakdown of membrane proteins, including activated growth factor receptors, lysosomal enzymes, and lipids. VTA1 functions as a cofactor of VPS4A/B, enzymes that dismantle membrane-associated ESCRT-III complexes, contributing to the sorting and downregulation of EGFR (epidermal growth factor receptor) . VTA1 is also involved in HIV-1 budding processes . This protein is also known by several alternative names including C6orf55, DRG-1, HSPC228, LIP5, and SBP1 .

What is the structural organization of the VTA1 protein?

VTA1 possesses a modular structure characterized by two well-folded terminal domains connected by a region dominated by random-coil structure . The N-terminal domain (Vta1NTD) consists of two MIT (microtubule interacting and transport) motifs that structurally resemble the MIT domain of Vps4, with root mean square deviations for the Cα atoms of 1.3Å and 1.7Å over 52 and 54 residues, respectively, when compared to human Vps4A . The C-terminal domain (Vta1CTD) mediates VTA1 dimerization and interacts with Vps4 in an adenine nucleotide-dependent manner. This domain arrangement allows VTA1 to function effectively as a regulatory scaffold protein, coordinating multiple components of the ESCRT machinery .

How does VTA1 regulate VPS4 ATPase activity?

VTA1 functions as a positive regulator of Vps4 by stimulating its ATPase activity, which is critical for the proper function of the MVB sorting reaction . Mechanistically, the C-terminal domain of VTA1 promotes the ATP-dependent double ring assembly of Vps4, while the N-terminal domain, projected by the disordered middle linker region, allows contact between the Vps4 disassembly machinery and accessory ESCRT-III proteins to coordinate ESCRT-III assembly and disassembly . This regulatory mechanism appears to be evolutionarily conserved, as VTA1 orthologs have been identified from yeast to humans with highly homologous sequence motifs that mediate interactions with Vps4 .

What is the relationship between VTA1 expression and cancer prognosis?

High expression of VTA1 has been identified as an adverse prognostic factor in lung adenocarcinoma (LUAD) . LUAD is characterized by high heterogeneity, variable prognosis, and high mortality, and VTA1 has been linked to tumor progression in human solid tumors . The association between VTA1 expression and poor prognosis suggests that VTA1 may contribute to cancer progression through its involvement in the multivesicular body pathway, which regulates the degradation of receptor tyrosine kinases and other signaling molecules . This finding indicates that VTA1 could potentially serve as a biomarker for LUAD diagnosis, prognosis classification, therapy response prediction, and possibly even as a target for drug development .

What other diseases are associated with VTA1 dysfunction?

According to research databases, diseases associated with VTA1 include Arthrogryposis, Distal, Type 1B and Arthrogryposis, Distal, Type 1A . These are congenital conditions characterized by joint contractures. The connection between VTA1 and these conditions may relate to its role in cellular signaling pathways and membrane protein trafficking, though more research is needed to fully elucidate these relationships.

How does VTA1 function in the abscission checkpoint during cell division?

VTA1 plays a crucial role in regulating cytokinesis abscission through its interactions with checkpoint proteins. Studies have shown that depletion of VTA1 disrupts VPS4A-ANCHR interactions and accelerates abscission, suggesting VTA1's involvement in the abscission checkpoint . The percentage of intercellular bridges containing lagging chromatin significantly increases in VTA1 knockout (KO) cells, a phenotype that can be restored by exogenous VTA1 expression . VTA1 interacts with checkpoint proteins ANCHR and CHMP4C, and preferentially binds to VPS4A over VPS4B in cells . These findings indicate that VTA1 is an integral component of the VPS4-CHMP4C-ANCHR abscission checkpoint complex that regulates the timing of cell division completion .

What methodologies are effective for investigating VTA1 localization and dynamics?

Multiple complementary approaches can be employed to investigate VTA1 localization and dynamics:

Live Cell Imaging Techniques:

• Transfection with fluorescently-tagged VTA1 (e.g., mEmerald-VTA1, GFP-VTA1) allows real-time visualization of protein localization

• Co-expression with markers like mCherry-tubulin enables simultaneous visualization of cellular structures

• Time-lapse microscopy to track VTA1 during dynamic processes like cell division

Biochemical Approaches:

• Immunoprecipitation (IP) using anti-GFP beads for cells expressing GFP-tagged VTA1 to identify binding partners

• Immunoblotting for detection of VTA1 and interacting proteins in cellular fractions

Advanced Techniques:

• Maximum-intensity projections from time-lapse movies to analyze VTA1 arrival at the intercellular bridge during abscission

• Application of Laplacian 2D filters to DNA staining images for better visualization of chromatin bridges in VTA1-depleted cells

These methodologies have successfully demonstrated VTA1's dynamic localization during cytokinesis and identified its interactions with key abscission regulators.

How can researchers effectively design knockout experiments to study VTA1 function?

Effective VTA1 knockout experimental design should include:

• Generation of complete knockout cell lines:

  • CRISPR-Cas9 technology for targeted deletion of VTA1

  • Thorough validation of knockout through immunoblotting and genomic analysis

• Comprehensive phenotypic analysis:

  • Live cell imaging to measure abscission timing (from cleavage furrow formation to microtubule bridge cleavage)

  • Quantification of chromatin bridges using DNA staining and appropriate image filters

  • Analysis of protein-protein interactions through immunoprecipitation experiments

• Critical control conditions:

  • Wild-type cells as baseline controls

  • Rescue experiments with exogenous VTA1 expression to confirm phenotype specificity

  • Comparison of single knockouts versus double knockouts when studying related proteins

• Data collection and analysis:

  • Multiple independent experiments (minimum of 2-3 replicates)

  • Appropriate statistical tests (e.g., Anova (Kruskal–Wallis test) for abscission timing, chi-square for chromatin bridge percentages)

  • Sufficient sample sizes (e.g., n>20 for cell division timing experiments, n>450 for chromatin bridge analysis)

What protein interaction studies are most informative for understanding VTA1 function?

The most informative protein interaction studies for understanding VTA1 function include:

• Co-immunoprecipitation experiments:

  • IP of GFP-tagged VTA1 followed by immunoblotting for potential binding partners

  • Reverse IP using tagged binding partners to confirm interactions

  • Comparison across different cellular conditions (e.g., cell cycle stages)

• Domain-specific interaction analysis:

  • Mapping interaction domains through truncation or point mutation experiments

  • Identifying how the N-terminal domain (MIT motifs) and C-terminal domain contribute to different binding relationships

• Nucleotide dependency studies:

  • Analysis of how ATP/ADP binding affects VTA1 interactions with VPS4A/B

  • Examination of how nucleotide state influences complex formation

• Comparative analysis across VPS4 paralogs:

  • Assessment of VTA1's preferential binding to VPS4A over VPS4B

  • Investigation of how these differential interactions contribute to paralog-specific functions

• Checkpoint complex component analysis:

  • Examination of how VTA1 mediates interactions between VPS4A and ANCHR

  • Investigation of VTA1's role in CHMP4C binding and checkpoint function

These approaches have revealed critical insights, such as the finding that VTA1 depletion disrupts VPS4A-ANCHR interactions while maintaining VPS4A-CHMP4C binding, and that in VPS4A knockout cells, VTA1 loses its ability to interact with CHMP4C but maintains ANCHR binding .

How do the human VPS4 paralogs differentially interact with VTA1?

Human cells uniquely possess two VPS4 paralogs (VPS4A and VPS4B), and research has demonstrated that VTA1 exhibits preferential binding to VPS4A over VPS4B . This selective interaction appears functionally significant, as VPS4A depletion results in a more severe abscission delay than VPS4B depletion, suggesting paralog-specific roles in cytokinesis regulation .

Immunoprecipitation experiments with GFP-tagged VPS4 constructs co-expressed with mApple-VTA1 have confirmed this preferential binding pattern . The molecular basis for this selective interaction likely involves structural differences between the VPS4 paralogs that affect their binding interfaces with VTA1.

The differential binding has significant functional implications:

• VTA1's interaction with VPS4A specifically mediates VPS4A-ANCHR binding

• The VTA1-VPS4A complex appears particularly critical for abscission checkpoint regulation

• VPS4A's more severe abscission delay phenotype compared to VPS4B may be directly related to its stronger VTA1 interaction

These findings suggest a specialized regulatory relationship between VTA1 and VPS4A that may have evolved to provide finer control over specific cellular processes, particularly the timing and fidelity of cell division.

What is the molecular mechanism of VTA1's action in cytokinesis regulation?

The molecular mechanism of VTA1's action in cytokinesis regulation involves its role as a critical component of the abscission checkpoint:

• Temporal regulation of abscission:

  • VTA1 knockout results in accelerated abscission (73.3 ± 30 min vs. wild-type ~90 min)

  • This acceleration leads to increased frequency of chromatin bridges, indicating premature cytokinesis completion

• Complex formation with checkpoint proteins:

  • VTA1 forms a complex with ANCHR and CHMP4C, key abscission checkpoint proteins

  • VTA1 mediates the interaction between VPS4A and ANCHR, as this interaction is lost in VTA1 KO cells

  • In VPS4A KO cells, VTA1 loses its ability to interact with CHMP4C but maintains ANCHR binding

• Spatial dynamics during abscission:

  • Live cell imaging reveals VTA1 arrives at the intercellular bridge during abscission

  • This localization is temporally regulated during the abscission process

• Functional consequences of disruption:

  • VTA1 depletion disrupts the abscission checkpoint, leading to chromatin bridge formation

  • The checkpoint failure phenotype can be rescued by exogenous VTA1 expression

This mechanism highlights VTA1's role as an essential scaffold protein that coordinates the assembly and function of the abscission checkpoint complex, ensuring proper timing of cytokinesis completion to prevent chromosome missegregation.

How does the MIT domain structure of VTA1 contribute to its functional versatility?

The MIT (microtubule interacting and transport) domain structure of VTA1 contributes significantly to its functional versatility:

• Structural homology with Vps4 MIT domains:

  • VTA1's N-terminal domain contains two MIT motifs (MIT1 and MIT2) that structurally resemble the MIT domain of Vps4

  • When aligned with the MIT domain helices of Vps4, root mean square deviations for the Cα atoms are remarkably similar: 1.3Å and 1.7Å over 52 and 54 residues, respectively, for human Vps4A

• Interaction mechanism with ESCRT-III-like proteins:

  • Similar to how Vps4's MIT domain interacts with ESCRT-III subunits, VTA1's MIT domains likely mediate interactions with Vps60 and Vps46/Did2

  • The interaction likely involves a surface groove formed by helix 2 and helix 3 of the MIT domain

  • ESCRT-III subunits interact with Vps4 by providing a fourth helix to complete a non-canonical tetratricopeptide repeat (TPR) fold, and VTA1 likely employs a similar mechanism

• Functional implications:

  • The dual MIT domain structure allows VTA1 to simultaneously interact with multiple ESCRT-III-like proteins

  • The structural similarity to Vps4's MIT domain facilitates coordinated function within the ESCRT machinery

  • The presence of two MIT domains may provide specificity for different binding partners or enhance binding affinity

This domain architecture allows VTA1 to serve as a molecular bridge between the VPS4 ATPase machinery and its substrates, contributing to the protein's ability to function in diverse cellular processes including MVB formation, receptor degradation, viral budding, and cytokinesis regulation.

What are the key challenges in studying VTA1 in primary human tissues?

Studying VTA1 in primary human tissues presents several methodological challenges:

• Protein abundance and detection:

  • VTA1 may be expressed at variable levels across different tissues

  • Developing highly specific antibodies for immunohistochemistry can be challenging

  • Distinguishing VTA1 from its binding partners in complex tissue samples requires careful validation

• Functional redundancy:

  • Potential compensatory mechanisms may mask VTA1 dysfunction in tissue samples

  • The interplay between VTA1 and related proteins in the ESCRT machinery complicates functional analysis

• Context-dependent regulation:

  • VTA1's function may vary significantly across different tissue types

  • Its role in pathological processes likely depends on tissue-specific expression of binding partners

  • Post-translational modifications may alter VTA1 function in a tissue-specific manner

• Technical considerations for clinical samples:

  • Limited availability of fresh tissue samples with preserved protein integrity

  • Variability in fixation methods affecting epitope recognition

  • Need for appropriate controls to establish baseline expression levels

Addressing these challenges requires combining multiple methodological approaches, including immunohistochemistry, RNA-seq analysis, laser capture microdissection of specific cell populations, and validation in relevant model systems.

How can researchers differentiate between VTA1's roles in MVB formation versus cytokinesis?

Differentiating between VTA1's roles in MVB formation versus cytokinesis requires sophisticated experimental design:

• Cell cycle-specific analysis:

  • Synchronize cells at specific cell cycle stages to isolate MVB formation (predominantly interphase) from cytokinesis events

  • Use cell cycle markers (e.g., pH3, cyclin B) to distinguish cells in different phases

• Domain-specific mutation approaches:

  • Generate VTA1 mutants with selective disruption of interactions with:

    • VPS4 (affecting both MVB and cytokinesis functions)

    • ESCRT-III components (primarily affecting MVB formation)

    • ANCHR/CHMP4C (primarily affecting cytokinesis checkpoint)

  • Express these mutants in VTA1 KO backgrounds to assess rescue of specific functions

• Quantitative phenotypic analysis:

  • For MVB formation: Assess endosomal morphology, receptor degradation rates, and ILV formation

  • For cytokinesis: Measure abscission timing, frequency of multinucleation, and chromatin bridge formation

  • Compare phenotypes between wild-type, complete VTA1 KO, and domain-specific mutants

• Interaction network mapping:

  • Compare VTA1's interactome during interphase versus cytokinesis

  • Identify binding partners unique to each process

  • Determine how these interactions are spatiotemporally regulated

This approach allows researchers to dissect the molecular mechanisms by which a single protein contributes to distinct cellular processes through differential protein interactions and regulatory mechanisms.

What are the most promising future directions for VTA1 research in human disease?

Several promising research directions could significantly advance our understanding of VTA1 in human disease:

• Cancer biology and prognostic applications:

  • Further investigate VTA1 as a prognostic biomarker in multiple cancer types beyond lung adenocarcinoma

  • Explore whether VTA1 inhibition could sensitize cancer cells to existing therapies

  • Determine if VTA1 expression correlates with specific cancer subtypes or treatment responses

• Neurodevelopmental disorders:

  • Given that mutations in VPS4A cause severe neurodevelopmental defects , investigate VTA1's role in neural development

  • Examine whether VTA1 variants contribute to neurological disorders through disrupted receptor trafficking

  • Study VTA1 function in neuronal pruning and synapse formation

• Therapeutic targeting strategies:

  • Develop small molecule inhibitors that disrupt specific VTA1 interactions (e.g., VTA1-VPS4A binding)

  • Explore the therapeutic potential of targeting VTA1 in cancers with high VTA1 expression

  • Design peptide-based inhibitors that compete with specific VTA1 binding interfaces

• Systems biology approaches:

  • Map the complete VTA1 interactome across different cell types and disease states

  • Integrate transcriptomic, proteomic, and functional data to create predictive models of VTA1 dysfunction

  • Identify synthetic lethal interactions that could be exploited therapeutically

• Role in cellular stress responses:

  • Investigate how VTA1 function changes under various cellular stresses (oxidative stress, nutrient deprivation, etc.)

  • Determine if VTA1 contributes to stress adaptation mechanisms through altered receptor trafficking

  • Explore potential roles in autophagy regulation, particularly selective autophagy pathways

These research directions hold significant potential for translating our understanding of VTA1 biology into clinical applications for cancer diagnosis, prognosis, and potentially novel therapeutic strategies.