VASP Human

What is human VASP and what is its primary function in cellular processes?

Human VASP (Vasodilator-stimulated phosphoprotein) functions as a key regulator of actin filament assembly and organization, influencing cellular processes dependent on cytoskeletal dynamics. To study VASP's function, researchers typically employ immunofluorescence microscopy to visualize its localization at focal adhesions and association with actin filaments . Experimental approaches for investigating VASP function include:

• Genetic rescue experiments: Transgenic expression of VASP in model organisms lacking endogenous Ena/VASP proteins

• Co-localization studies: Visualizing VASP with cytoskeletal components and binding partners

• Protein-protein interaction assays: Identifying VASP binding partners through co-immunoprecipitation or yeast two-hybrid screens

VASP's role extends beyond basic cytoskeletal regulation to specialized functions in various cell types, including platelet adhesion/aggregation and smooth muscle relaxation .

What is the structural organization of human VASP and its functional domains?

Human VASP contains three main functional domains with distinct roles in cytoskeletal regulation:

The EVH1 domain mediates interaction with focal adhesion proteins, notably zyxin. The EVH2 domain contains G-actin and F-actin binding sites and includes a 45-residue tetramerization domain (TD) with a unique right-handed α-helical coiled-coil structure . To study these domains, researchers employ:

• Site-directed mutagenesis to disrupt specific domain functions

• Domain truncation experiments to assess contribution of individual domains

• Crystallography to determine three-dimensional structures (as demonstrated by the 1.3-Å resolution crystal structure of the VASP TD)

Mutations in either the EVH1 or EVH2 domains result in severe functional deficiencies, highlighting their critical importance for VASP's physiological activity .

How does the tetramerization domain of VASP contribute to protein function?

The tetramerization domain (TD) of VASP forms a distinctive right-handed α-helical coiled-coil structure that enables VASP to assemble into tetramers. Methodologically, researchers determined this structure through X-ray crystallography at 1.3-Å resolution, revealing several key features :

• A 15-residue repeat pattern in the amino acid sequence (contrasting with the common 7-residue heptad repeats in left-handed coiled coils)

• Exceptional thermal stability (melting point of 120°C)

• Stabilization through hydrophobic interactions and a network of salt bridges

To experimentally assess tetramerization's functional importance, researchers can:

• Generate VASP mutants with disrupted tetramerization domains

• Perform size exclusion chromatography to verify oligomerization state

• Conduct functional assays comparing wild-type and tetramerization-deficient VASP

A nonsense mutation resulting in C-terminal truncation of Drosophila Ena (lacking the EVH2 domain containing the TD) prevented formation of multimeric complexes and reduced binding to both zyxin and the Abelson Src homology 3 domain , demonstrating that tetramerization is essential for normal VASP function.

What signaling pathways regulate VASP activity in different cellular contexts?

VASP activity is regulated by multiple signaling pathways that converge on its phosphorylation status. Experimental approaches to study these regulatory mechanisms include:

• Phosphorylation-specific antibodies and western blotting

• P-VASP blocking antibody lipofection, as described in the search results :

  • Diluting blocking peptide in DPBS

  • Adding it to dried Pierce Reagent

  • Vortexing and incubating at room temperature

  • Applying the lipo-surrounded blocking peptide to cells

  • Incubating overnight at 37°C with 5% CO2

• Pharmacological manipulations of relevant kinases and phosphatases

Key regulatory pathways include:

Experimental evidence demonstrates that these pathways are functionally significant, as mice lacking both VASP and Mena show more than 50% reduction in cGMP- and cAMP-induced smooth muscle relaxation compared to wild-type controls .

How can researchers assess the evolutionary conservation and functional equivalence between VASP and other Ena/VASP family members?

To evaluate functional conservation between Ena/VASP family members, researchers employ several complementary methodological approaches:

• Cross-species rescue experiments: The search results describe a powerful methodology where human VASP was tested for its ability to rescue Drosophila Ena null mutations :

  • Generate stable transgenic Drosophila expressing human VASP

  • Express the transgene using the UAS/GAL4 binary expression system

  • Quantify rescue efficiency by calculating percentage of ena mutant progeny surviving to adulthood

  • Compare against control rescues using Drosophila Ena transgenes

This approach demonstrated that human VASP could partially rescue ena mutant lethality (allowing 25-85% survival compared to 79-100% with Drosophila Ena) , providing strong evidence for functional conservation despite limited sequence identity.

• Domain-specific functional analysis through:

  • Site-directed mutagenesis of conserved residues

  • Domain swapping between family members

  • Assessment of binding to common partners like zyxin

• Paralog compensation studies:

  • Generate animals lacking multiple family members (e.g., VASP-/-Mena GT/GT mice)

  • Assess phenotypic severity compared to single mutants

  • Measure specific functional readouts (e.g., smooth muscle relaxation)

These approaches revealed that Mena and VASP functionally compensate for each other in vascular smooth muscle relaxation, as evidenced by the >50% reduction in cGMP- and cAMP-induced relaxation in double-mutant mice .

What methodological approaches can be used to study how mutations in VASP affect its functionality?

Researchers employ multiple complementary approaches to investigate how mutations impact VASP functionality:

• Genetic analysis of naturally occurring mutations:

  • Characterize molecular lesions through sequencing

  • Correlate with phenotypic effects

  • Assess domain-specific impacts

As demonstrated in the search results, researchers characterized two lethal Drosophila ena mutations: a missense mutation in the EVH1 domain that eliminated zyxin binding, and a nonsense mutation creating a C-terminally truncated protein lacking the EVH2 domain that failed to form multimeric complexes and showed reduced binding to interaction partners .

• Structure-function analysis through:

  • Site-directed mutagenesis targeting specific residues

  • Domain deletion constructs

  • Chimeric proteins

• Biochemical characterization:

  • Protein-protein interaction assays with known binding partners

  • Actin polymerization assays

  • Oligomerization state determination

• Cellular phenotype assessment:

  • Localization studies using fluorescently tagged mutant proteins

  • Cell migration and adhesion assays

  • Cytoskeletal organization analysis

• In vivo functional rescue experiments:

  • Express mutant variants in VASP-deficient backgrounds

  • Quantify restoration of function

  • Compare to wild-type rescue efficiency

These approaches provide comprehensive insights into how specific mutations affect VASP's molecular interactions, cellular localization, and physiological functions.

What is the significance of the right-handed coiled-coil structure in VASP's tetramerization domain compared to typical left-handed coiled coils?

The right-handed coiled-coil structure in VASP's tetramerization domain represents an unusual and significant structural motif that can be studied through several methodological approaches:

• Structural analysis techniques:

  • X-ray crystallography (as used to determine the 1.3-Å resolution structure)

  • Nuclear magnetic resonance (NMR) spectroscopy

  • Cryo-electron microscopy

• Sequence analysis methods:

  • Identification of the characteristic 15-residue repeat pattern

  • Comparison with the more common 7-residue heptad repeats

  • Bioinformatic searches for similar patterns in other proteins

• Biophysical characterization:

  • Circular dichroism spectroscopy to confirm α-helical content

  • Thermal stability measurements (revealing the exceptional 120°C melting point)

  • Analytical ultracentrifugation to determine oligomerization state

The significance of this structure includes:

This structural uniqueness may provide VASP with specialized functional properties for cytoskeletal regulation that distinguish it from proteins with conventional left-handed coiled coils .

How does VASP contribute to vascular smooth muscle relaxation and what experimental approaches demonstrate this role?

VASP plays a critical role in vascular smooth muscle relaxation, as demonstrated through the following experimental approaches:

• Genetic knockout models:

  • Generation of VASP-/-Mena GT/GT double-mutant mice

  • Comparison with wild-type controls in functional assays

• Ex vivo vessel ring contractility studies:

  • Isolation of vascular rings

  • Measurement of tension responses to vasoactive agents

  • Quantification of relaxation following contraction

• Pharmacological interventions:

  • Application of cGMP and cAMP analogs to stimulate relaxation

  • Comparison of responses between genotypes

Key findings from these approaches revealed that:

• cGMP- and cAMP-induced relaxation was reduced by more than 50% in VASP-/-Mena GT/GT mice compared to wild-type controls

• PKG- and PKA-dependent smooth muscle relaxation is significantly disturbed in the absence of Mena and VASP

• Mena and VASP functionally compensate for each other in this process

These results establish VASP as an essential component of the signaling pathways linking cyclic nucleotides to smooth muscle relaxation, with important implications for vascular physiology and potential therapeutic interventions for vascular disorders.

What techniques are available for manipulating VASP phosphorylation to study its regulatory effects?

Several sophisticated techniques are available to manipulate and study VASP phosphorylation:

• P-VASP blocking antibody lipofection:

  • Transfection of cells with antibodies specific to phosphorylated VASP

  • Protocol using Pierce Protein Transfection Reagent Kit includes :

    • Diluting blocking peptide in DPBS

    • Adding to dried Pierce Reagent

    • Vortexing and incubating at room temperature

    • Applying the lipo-surrounded blocking peptide to cells

    • Incubating overnight at 37°C with 5% CO2

• Genetic approaches:

  • Generation of phosphomimetic mutants (e.g., serine to aspartate/glutamate)

  • Creation of phosphodeficient mutants (e.g., serine to alanine)

  • Knock-in mice expressing modified VASP

• Pharmacological interventions:

  • Application of cyclic nucleotide analogs to activate PKA/PKG

  • Use of specific kinase inhibitors

  • Phosphatase inhibitors to maintain phosphorylation state

• Analytical methods:

  • Phosphorylation-specific antibodies for western blotting

  • Mass spectrometry to identify and quantify phosphorylation sites

  • Kinase assays to measure phosphorylation dynamics

These approaches allow researchers to precisely manipulate VASP phosphorylation status and determine its effects on cellular processes including cytoskeletal organization, cell migration, and smooth muscle relaxation.

Methodological Approaches for VASP Research

Multiple imaging techniques can be employed to visualize VASP localization and dynamics, each with specific advantages:

• Immunofluorescence microscopy:

  • Fixed cell imaging using VASP-specific antibodies

  • Co-localization studies with cytoskeletal components and binding partners

  • As demonstrated in the search results, this approach revealed colocalization of Ena and VASP at focal adhesions and with actin filaments

• Live-cell imaging with fluorescent protein fusions:

  • GFP-VASP constructs for real-time visualization

  • Photoactivatable or photoconvertible fluorescent proteins for pulse-chase experiments

  • FRAP (Fluorescence Recovery After Photobleaching) to measure dynamics

• Super-resolution microscopy:

  • STED (Stimulated Emission Depletion) microscopy

  • SIM (Structured Illumination Microscopy)

  • PALM/STORM for nanoscale resolution of VASP organization

• FRET (Förster Resonance Energy Transfer):

  • Measuring VASP conformation changes

  • Detecting protein-protein interactions in living cells

  • Biosensors for VASP activity or phosphorylation state

• Correlative light and electron microscopy (CLEM):

  • Combining fluorescence localization with ultrastructural context

  • Precise positioning of VASP relative to cytoskeletal structures

For dynamic studies, fluorescent protein fusions with time-lapse confocal microscopy represent the most widely applicable approach, while super-resolution techniques provide the highest spatial resolution for detailed structural analysis.

What bioinformatic and computational approaches can be applied to analyze VASP structure, interactions, and function?

Several sophisticated computational approaches can enhance VASP research:

• Structural analysis and prediction:

  • Homology modeling of VASP domains

  • Molecular dynamics simulations of conformational changes

  • Protein-protein docking to predict interaction interfaces

• Network and pathway analysis:

  • STRING database for protein interaction networks

  • Gene Ontology (GO) enrichment analysis

  • KEGG pathway mapping

The search results describe using STRING v10 for GO and KEGG pathway analysis , which can be applied to datasets involving VASP to understand its functional networks.

• Gene set enrichment analysis (GSEA):

  • Identification of biological pathways involving VASP

  • Integration of proteomics and transcriptomics data

  • As mentioned in the search results, GSEA 4.0 software can be employed for this purpose

• Sequence analysis approaches:

  • Identification of conserved motifs across species

  • Prediction of post-translational modification sites

  • Evolutionary analysis of Ena/VASP family proteins

• Quantum mechanical modeling:

  • While primarily designed for materials science, computational chemistry tools like the Vienna Ab initio Simulation Package (VASP) can be adapted to study protein structures:

    • Electronic structure calculations

    • Density functional theory approaches

    • Simulation of spectroscopic properties

These computational approaches complement experimental data and can guide hypothesis generation for targeted experimental validation.

What biochemical assays are most informative for characterizing VASP interactions with actin and focal adhesion proteins?

Several biochemical assays provide critical insights into VASP's interactions with actin and focal adhesion proteins:

• Actin polymerization assays:

  • Pyrene-actin fluorescence to monitor polymerization kinetics

  • Total Internal Reflection Fluorescence (TIRF) microscopy of individual actin filaments

  • Sedimentation assays to quantify F-actin binding

• Protein-protein interaction assays:

  • Co-immunoprecipitation for endogenous protein complexes

  • GST pulldown assays for domain-specific interactions

  • Yeast two-hybrid screening for novel binding partners

  • Surface plasmon resonance for binding kinetics

The search results detail how researchers demonstrated the interaction between Ena and zyxin through in vitro binding assays, revealing that a missense mutation in the EVH1 domain eliminated this interaction .

• Crosslinking approaches:

  • Chemical crosslinking followed by mass spectrometry

  • Proximity-dependent biotin identification (BioID)

  • APEX2-based proximity labeling

• Structural studies:

  • X-ray crystallography of protein complexes

  • NMR spectroscopy for dynamic interactions

  • Hydrogen-deuterium exchange mass spectrometry

• Functional reconstitution:

  • Reconstitution of actin structures with purified components

  • Single-molecule studies of VASP-actin interactions

  • Microfluidics-based force measurements

These complementary approaches provide a comprehensive view of VASP's molecular interactions and mechanisms of action in cytoskeletal regulation.

How does VASP research contribute to understanding vascular pathophysiology and potential therapeutic targets?

VASP research provides significant insights into vascular pathophysiology and therapeutic development through several methodological approaches:

• Genetic models of vascular dysfunction:

  • Studies in VASP-/-Mena GT/GT mice revealed that cGMP- and cAMP-induced relaxation was reduced by more than 50% compared to wild-type controls

  • This demonstrates that VASP is a critical component of vasodilatory signaling pathways

• Mechanistic investigation of smooth muscle relaxation:

  • PKG- and PKA-dependent pathways require VASP for optimal function

  • Understanding these mechanisms identifies potential intervention points in vascular disorders

• Biomarker development:

  • VASP phosphorylation status may serve as a biomarker for vascular responsiveness

  • P-VASP-specific antibodies can be used in clinical samples

• Target validation approaches:

  • Pharmacological manipulation of VASP phosphorylation

  • Assessment of effects on vascular tone

  • Identification of pathway-specific interventions

These findings suggest potential therapeutic applications including:

• Development of drugs targeting VASP phosphorylation or function

• Personalized medicine approaches based on VASP pathway integrity

• Novel interventions for vascular disorders characterized by impaired relaxation

Understanding the molecular mechanisms by which VASP mediates vascular smooth muscle relaxation provides a foundation for developing targeted therapeutics for hypertension, vasospasm, and other vascular disorders.

What methodological challenges exist in translating basic VASP research findings to clinical applications?

Translating basic VASP research to clinical applications presents several methodological challenges:

• Model system limitations:

  • Cell culture systems lack physiological complexity

  • Animal models may not fully recapitulate human vascular physiology

  • Human challenge models (like those used for vaccine testing) raise ethical and safety considerations

• Molecular complexity challenges:

  • Functional redundancy between Ena/VASP family members

  • Context-dependent functions across different tissues

  • Multiple phosphorylation sites with distinct regulatory effects

• Therapeutic targeting difficulties:

  • Achieving specificity for VASP versus other Ena/VASP proteins

  • Targeting specific functions while preserving others

  • Cell type-specific delivery to vascular tissues

• Clinical assessment obstacles:

  • Limited accessibility of vascular tissue for direct analysis

  • Need for reliable biomarkers of VASP activity or function

  • Heterogeneity of vascular diseases

• Technological requirements:

  • Development of phosphorylation-specific monitoring tools

  • Real-time assessment of VASP function in vascular tissues

  • Integration with existing clinical measurements

Addressing these challenges requires multi-disciplinary approaches combining advanced molecular techniques, sophisticated animal models, and innovative human study designs to bridge the gap between basic VASP biology and clinical applications.

What are the most promising future directions for VASP research in both basic and translational contexts?

The study of VASP presents several promising research directions that bridge basic science and translational applications:

• Advanced structural studies:

  • Cryo-electron microscopy of full-length VASP in different phosphorylation states

  • Structural analysis of VASP interactions with actin and focal adhesion proteins

  • Further investigation of the unique right-handed coiled-coil tetramerization domain

• Comprehensive phospho-regulation mapping:

  • Identification of all phosphorylation sites and responsible kinases

  • Temporal dynamics of phosphorylation in response to different stimuli

  • Functional consequences of site-specific modifications

• Cell type-specific functions:

  • Conditional knockout approaches in specific vascular beds

  • Single-cell analysis of VASP function in heterogeneous tissues

  • Tissue-specific compensation mechanisms among Ena/VASP family members

• Integration with mechanobiology:

  • VASP's role in mechanotransduction

  • Force-dependent regulation of VASP function

  • Contribution to cellular responses to mechanical stress

• Therapeutic development:

  • Small molecule modulators of VASP function or phosphorylation

  • Gene therapy approaches for vascular disorders

  • Biomarkers based on VASP phosphorylation status

The discovery that VASP and Mena are required for vascular smooth muscle relaxation opens particularly promising avenues for cardiovascular disease research, potentially leading to novel therapeutic strategies for hypertension, atherosclerosis, and other vascular pathologies.

How can researchers best integrate multi-disciplinary approaches to advance understanding of VASP biology?

Advancing VASP research requires integrating diverse methodological approaches:

• Multi-scale experimental integration:

  • Molecular: Crystal structures , protein interactions , phosphorylation studies

  • Cellular: Localization , dynamic imaging, cytoskeletal organization

  • Tissue/organ: Ex vivo vessel studies , tissue-specific functions

  • Organismal: Genetic models , physiological responses

• Technology synthesis:

  • Combining structural biology with live cell imaging

  • Integrating omics approaches (proteomics, transcriptomics)

  • Merging computational modeling with experimental validation

• Cross-disciplinary collaboration frameworks:

  • Cell biologists with vascular physiologists

  • Structural biologists with clinical researchers

  • Computational scientists with experimental biologists

• Standardized research resources:

  • Validated antibodies and genetic tools

  • Reproducible experimental protocols

  • Shared data repositories and analysis pipelines

• Translational research pipelines:

  • Basic discovery → mechanistic understanding → therapeutic development

  • Biomarker identification → clinical validation → diagnostic implementation

  • Target validation → drug discovery → clinical testing

By adopting these integrated approaches, researchers can accelerate progress in understanding VASP biology and develop applications that address important clinical needs in vascular medicine and beyond.