Recombinant Mouse Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 2 (Asap2), partial

What is the domain structure of mouse ASAP2 and how does it relate to its function?

Mouse ASAP2 is a multidomain protein containing an N-terminal alpha-helical region with a coiled-coil motif (N-BAR domain), followed by a pleckstrin homology (PH) domain, an Arf-GTPase Activating Protein (Arf-GAP) domain, ankyrin repeats, proline-rich regions, and a C-terminal Src homology 3 (SH3) domain . This complex domain architecture enables ASAP2 to interact with multiple cellular components and participate in diverse signaling pathways.

The N-BAR domain mediates dimerization and interacts with membrane lipids, F-actin, and other proteins like Rab11 effector FIP3 and nonmuscle myosin II A . The GAP domain exhibits strong GTPase-activating activity toward ARF1 and ARF5, with weaker activity toward ARF6 . The SH3 domain facilitates interaction with proline-rich regions of other proteins, such as protein tyrosine kinase 2-beta (PYK2) .

The functional importance of each domain has been validated through mutagenesis studies, particularly with the lysine-rich clusters in the N-BAR domain that are critical for actin binding and cellular remodeling .

What are the key structural differences between ASAP1 and ASAP2 that affect their function?

Despite sharing similar domain architecture, ASAP1 and ASAP2 exhibit important functional differences:

• Actin Binding Specificity: High-speed actin sedimentation assays demonstrate that the BAR-PH region of ASAP1 binds to F-actin in a concentration-dependent manner, whereas ACAP1 BAR-PH (a related protein) does not bind to actin filaments . This functional difference stems from specific amino acid sequences within their respective BAR domains.

• Lysine-Rich Clusters: ASAP1 contains critical lysine-rich clusters, particularly at positions 75, 76, and 79 in the N-BAR domain, that are essential for actin binding. Mutations in these residues (both neutralizing and charge-reversal mutations) significantly reduce binding to actin filaments and abrogate actin bundle formation in vitro .

• Cellular Effects: Exogenous expression of ASAP1 BAR-PH induces actin-rich protrusions and microspikes, while ACAP1 cannot rescue the loss of actin structures when ASAP1 expression is reduced . This indicates non-redundant functions despite structural similarities.

These differences highlight the importance of sequence-specific elements within the conserved domain architecture that dictate unique protein functions.

What are the optimal expression systems and purification strategies for recombinant mouse ASAP2?

The optimal expression system for recombinant mouse ASAP2 depends on your experimental requirements:

E. coli Expression System:

• Application: Suitable for partial ASAP2 constructs, particularly the BAR-PH domain which can be successfully expressed and purified from E. coli .

• Expression Vector: Typically uses pET-based vectors with N-terminal 6xHis tags for efficient purification.

• Buffer Conditions: Tris-based buffer containing 50% glycerol maintains stability during storage .

• Purity Assessment: SDS-PAGE analysis should confirm >90% purity .

Mammalian Expression System (HEK-293 Cells):

• Application: Preferred for full-length ASAP2 (AA 1-958) where proper folding and post-translational modifications are critical .

• Advantages: Provides proper protein folding, post-translational modifications, and higher solubility of the full-length protein.

• Vector Design: Contains mammalian-optimized promoters (CMV) and appropriate tag sequences (typically His-tag).

Purification Strategy:

• Affinity chromatography using Ni-NTA columns for His-tagged proteins

• Size exclusion chromatography to remove aggregates

• Ion-exchange chromatography for further purification if needed

Storage Conditions: Store in Tris-based buffer with 50% glycerol at -20°C to maintain stability and activity .

How should experimental designs be structured to assess ASAP2's role in phagocytosis?

Based on established methodologies, the following experimental design approach is recommended to investigate ASAP2's role in phagocytosis:

Step 1: Define Variables

• Independent Variables: ASAP2 expression levels (wildtype, knockout, overexpression), domain mutations

• Dependent Variables: Phagocytosis efficiency, actin recruitment to phagocytic cups

• Control Variables: Cell type, particle type, opsonization conditions

Step 2: Cell Model Selection

• Primary Models: Human neutrophils, mouse macrophages

• Cell Lines: CHO cells stably expressing FcγRIIA receptor (CHO-IIA), PLB-985 cells differentiated toward neutrophil-like phenotype

Step 3: Experimental Approaches

Step 4: Data Analysis Plan

• Quantify phagocytosis index (number of particles internalized per cell)

• Measure fluorescence intensity at phagocytic cups

• Analyze recruitment kinetics using time-lapse imaging

• Perform statistical analysis using appropriate tests (Mann-Whitney U test, Student's t-test)

Recent research demonstrated that ASAP2 transiently accumulates at actin-rich phagocytic cups and increases Fcγ receptor-mediated phagocytosis, with the GAP domain being essential for this function . GAP-deficient ASAP2 failed to localize at phagocytic cups, while the GAP-deficient [R618K]ASAP2 mutant (which alters GAP activity but not the domain structure) retained localization and enhanced phagocytosis .

How does ASAP2 regulate actin cytoskeleton dynamics and what methods can assess this function?

ASAP2 plays a critical role in regulating actin cytoskeleton dynamics through several mechanisms:

Mechanisms of Actin Regulation by ASAP2:

• Direct Binding to F-actin: The N-BAR domain of ASAP2 directly binds to and bundles actin filaments through lysine-rich clusters (particularly residues K75, K76, and K79) .

• Membrane-Cytoskeleton Interface: ASAP2 functions at the interface between membranes and the cytoskeleton, localizing to focal adhesions and phagocytic cups .

• ARF GTPase Regulation: Through its GAP activity, ASAP2 controls the activity of ARF family small GTPases, which are key regulators of membrane trafficking and actin remodeling .

Methods to Assess ASAP2's Role in Actin Dynamics:

Research has shown that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 (which is structurally similar to ASAP2) reduced binding to actin filaments and abrogated actin bundle formation in vitro . Exogenous expression of actin-binding defective mutants prevented cellular actin remodeling in U2OS cells, and full-length [K75E, K76E, K79E] mutants failed to rescue the reduction of cellular actin fibers following knockdown of endogenous protein .

What are the key methodological considerations for studying ASAP2 in cancer cell models?

When investigating ASAP2 in cancer cell models, several methodological considerations are essential:

Cell Line Selection:

• Choose cell lines relevant to ASAP2-associated cancers (e.g., pancreatic ductal adenocarcinoma cell lines like Panc1 and MiaPaCa2)

• Include both cell lines with high and low endogenous ASAP2 expression for comparison

• Consider patient-derived primary cells when possible for increased clinical relevance

Gene Manipulation Approaches:

• CRISPR-Cas9 Technology: For complete knockout of ASAP2, as demonstrated in MiaPaCa2 and Panc1 cells

• siRNA Knockdown: For transient reduction of ASAP2 expression

• Stable Overexpression: Using pcDNA3.3 or similar vectors with appropriate selection markers (e.g., geneticin at 1000 μg/mL)

Functional Assays:

Molecular Pathway Analysis:

• RNA-seq: To identify gene expression changes in ASAP2 knockout cells

• Western Blotting: For assessing effects on key pathways (e.g., EGFR, ERK1/2)

• Validation Methods: qPCR to confirm gene expression changes using appropriate reference genes (e.g., GAPDH)

Data Analysis Considerations:

• Use appropriate statistical tests (Mann-Whitney U test, Student's t-test) depending on data distribution

• Consider survival analysis using Kaplan-Meier curves with log-rank tests when analyzing patient data

• Apply Gene Set Enrichment Analysis (GSEA) for pathway identification

Recent research demonstrated that ASAP2 promotes cell cycle progression in pancreatic cancer cells, with knockout cells showing G1 phase arrest and reduced proliferation . RNA-seq analysis revealed significant changes in pathways related to cell cycle, cytoskeleton, and migration in ASAP2 knockout cells .

How can mouse ASAP2 be used in phagocytosis studies related to immune cell function?

Mouse ASAP2 serves as a valuable model protein for investigating phagocytosis mechanisms in immune cells, with several advanced applications:

Experimental Models:

• Mouse Primary Macrophages: Isolation from bone marrow or peritoneal cavity for physiologically relevant studies

• Neutrophil-like Cell Lines: PLB-985 cells differentiated toward neutrophil phenotype provide a reliable model for phagocytosis studies

• CHO-IIA Cells: Chinese Hamster Ovary cells stably expressing FcγRIIA receptors offer a controlled system for studying specific receptor-mediated phagocytosis

Advanced Methodological Approaches:

Integration with Immune Function Studies:

• Cytokine Production: Measure inflammatory cytokine release in relation to ASAP2-mediated phagocytosis

• Pathogen Clearance: Use live bacteria to assess the role of ASAP2 in antimicrobial responses

• Resolution of Inflammation: Investigate ASAP2's role in efferocytosis (clearance of apoptotic cells)

Research has demonstrated that ASAP2 positively regulates FcγR-dependent uptake of opsonized particles by phagocytic cells . Notably, particulate agonists such as opsonized zymosan or monosodium urate crystals induce ASAP2 phosphorylation in human neutrophils, suggesting integration with inflammatory signaling pathways .

What are the latest techniques for investigating ASAP2 interactions with membrane trafficking components?

Cutting-edge techniques for studying ASAP2's interactions with membrane trafficking machinery include:

Advanced Imaging Techniques:

• Super-Resolution Microscopy: Techniques like STORM or PALM provide nanoscale resolution of ASAP2 localization at membrane microdomains

• Lattice Light-Sheet Microscopy: Enables long-term 3D imaging of membrane trafficking events with minimal phototoxicity

• Correlative Light and Electron Microscopy (CLEM): Combines fluorescence and electron microscopy to correlate ASAP2 localization with ultrastructural features

Protein-Protein Interaction Analysis:

Membrane Trafficking Assays:

• Vesicle Isolation: Differential centrifugation or immunoisolation to purify ASAP2-associated vesicles

• Cargo Tracking: Pulse-chase experiments with fluorescently labeled trafficking cargoes

• In vitro Reconstitution: Purified components on supported lipid bilayers to study minimal requirements for ASAP2 function

Computational Approaches:

• Molecular Dynamics Simulations: Model ASAP2-membrane interactions at atomic resolution

• Systems Biology Analysis: Integrate proteomic and functional data to build comprehensive trafficking networks

Studies have shown that ASAP2 regulates the formation of post-Golgi vesicles and modulates constitutive secretion . It also functions at the interface of endosomal trafficking and actin remodeling, highlighting its dual role in coordinating membrane dynamics with cytoskeletal reorganization .

How should researchers address discrepancies in ASAP2 functional studies across different experimental systems?

When confronting conflicting results in ASAP2 studies across different experimental systems, researchers should implement a systematic approach:

Sources of Experimental Variability:

• Expression System Differences: Protein folding, post-translational modifications, and activity may vary between E. coli, mammalian, and insect cell expression systems

• Protein Fragment vs. Full-Length: Partial constructs may exhibit different functionalities compared to the full-length protein

• Cell Type Specificity: ASAP2 function may depend on cell-specific interaction partners or signaling pathways

• Experimental Conditions: Buffer compositions, temperature, pH, and ionic strength can affect protein behavior

Systematic Validation Strategy:

Resolution Framework for Contradictory Data:

• Evaluate methodological differences (protein source, purity, assay conditions)

• Determine if discrepancies reflect genuine biological differences or technical artifacts

• Design bridging experiments that systematically vary one parameter at a time

• Consider combining approaches (e.g., in vitro reconstitution with cellular validation)

For example, studies investigating the actin-binding properties of ASAP2 should consider that the BAR-PH domain alone may behave differently than the full-length protein in cellular contexts . Similarly, when studying phagocytosis, different opsonizing conditions can lead to engagement of different receptors and downstream pathways , potentially leading to variable results regarding ASAP2's contribution.

What statistical approaches and controls are essential for rigorous interpretation of ASAP2 phenotypic data?

Rigorous statistical analysis of ASAP2 phenotypic data requires careful experimental design and appropriate analytical approaches:

Essential Experimental Controls:

• Negative Controls: Non-targeting siRNA, empty vector transfections, or isotype-matched antibodies

• Positive Controls: Known modulators of the pathway being studied

• Rescue Experiments: Re-expression of wild-type ASAP2 in knockout cells

• Domain Mutants: Functional domain mutations as specificity controls (e.g., GAP-deficient vs. actin-binding deficient)

Statistical Approaches for Different Experimental Designs:

Quantification Methods for Complex Phenotypes:

• Cell Morphology: Automated image analysis with multiple parameters (cell area, circularity, protrusion number)

• Actin Structures: Fluorescence intensity measurements, stress fiber counts, focal adhesion size/number

• Phagocytosis: Phagocytic index (particles/cell), percentage of phagocytic cells, kinetics of uptake

• In Vivo Tumor Growth: Tumor volume measurement frequency, accounting for initial tumor establishment

Data Reporting Requirements:

• Report both effect sizes and p-values

• Include confidence intervals where appropriate

• Present individual data points alongside means/medians

• Report all experimental conditions in detail, including negative results

• Show representative images alongside quantification

Recent studies examining ASAP2's role in pancreatic cancer employed multiple statistical approaches, including Student's t-test for comparing cell proliferation between knockout and control cells, Pearson correlation coefficient for assessing relationships between DNA copy number and mRNA expression, and Kaplan-Meier analysis with log-rank tests for survival outcomes .

How can recombinant ASAP2 studies inform therapeutic targeting strategies?

Recombinant ASAP2 research provides critical insights for developing targeted therapeutic approaches:

Druggable Features of ASAP2:

• Enzymatic GAP Activity: The GAP domain represents a potential target for small molecule inhibitors that could modulate ARF GTPase regulation

• Domain-Specific Functions: Targeting specific protein-protein interactions rather than the entire protein may provide more selective therapeutic approaches

• Expression Level Modulation: In cancers where ASAP2 is overexpressed, strategies to reduce expression levels could have therapeutic value

Therapeutic Development Strategies:

Case Example: Niclosamide as a Repositioned Drug
Research has identified niclosamide, an antiparasitic drug, as a potential ASAP2-targeting agent in pancreatic ductal adenocarcinoma . This was accomplished using the gene perturbation correlation (GPC) method, demonstrating that:

• Niclosamide suppressed PDAC growth by inhibiting ASAP2 expression

• In vivo studies using xenograft models showed tumor growth inhibition with niclosamide treatment (25 mg/kg daily)

• Mechanistically, ASAP2 promoted tumor growth by facilitating cell cycle progression through phosphorylation of EGFR

This example illustrates how recombinant ASAP2 studies can facilitate drug repurposing strategies, providing faster paths to clinical applications compared to de novo drug development.

What are the methodological considerations for translating mouse ASAP2 findings to human disease contexts?

Translating mouse ASAP2 research to human applications requires careful consideration of species differences and experimental design:

Cross-Species Comparison Approaches:

• Sequence Alignment Analysis: Compare mouse and human ASAP2 sequences to identify conserved and divergent regions

• Domain Function Validation: Test whether equivalent domains perform identical functions across species

• Expression Pattern Mapping: Compare tissue-specific expression patterns between mouse and human

Translational Research Methodology:

Critical Assessment of Mouse Models:

• Evaluate whether the mouse phenotype fully recapitulates human disease features

• Consider differences in cellular pathways and interaction partners between species

• Assess whether the experimental timeframe in mice reflects human disease progression

Data Integration Strategies:

• Multi-Omics Approaches: Combine genomic, transcriptomic, and proteomic data from both species

• Pathway-Centric Analysis: Focus on conserved pathway functions rather than individual protein differences

• Clinical Correlation: Link experimental findings with human patient data when available

For example, research has demonstrated that high ASAP2 expression correlates with poor prognosis in human pancreatic ductal adenocarcinoma . These clinical findings were validated in mouse models, where ASAP2 knockout reduced tumor growth in vivo, providing a robust translational link between mouse experimental models and human disease .

What are the key considerations for designing CRISPR/Cas9 knockout models for ASAP2 functional studies?

When designing CRISPR/Cas9 knockout models for ASAP2 research, several critical factors must be addressed:

Target Site Selection and gRNA Design:

• Exon Targeting Strategy: Target early exons (e.g., exon 2) to ensure complete functional disruption

• Domain-Specific Targeting: Consider targeting specific functional domains to create partial loss-of-function models

• Off-Target Analysis: Use computational tools to predict and minimize potential off-target effects

• Efficiency Considerations: Select target sites with optimal GC content and minimal secondary structure

Cell Type and Delivery Considerations:

Validation Strategies:

• Genomic Verification: PCR amplification and sequencing of the target region

• Transcript Analysis: RT-PCR and RNA-seq to confirm loss of functional transcripts

• Protein Validation: Western blotting to verify complete protein loss

• Functional Assays: Cellular phenotyping to confirm functional consequences

• Rescue Experiments: Re-expression of wild-type ASAP2 to confirm specificity

Phenotypic Analysis Considerations:

• Developmental Effects: Consider potential embryonic lethality, as observed in some ArfGAP family member knockouts

• Compensatory Mechanisms: Assess potential upregulation of related proteins (e.g., ASAP1)

• Tissue-Specific Phenotypes: Examine effects across multiple tissues and cell types

• Time-Dependent Effects: Monitor phenotypes over time to capture both acute and chronic consequences

Recent CRISPR/Cas9 studies targeting ASAP2 in pancreatic cancer cell lines demonstrated successful knockout with verifiable genomic alterations and complete protein loss . These models revealed that ASAP2 knockout significantly suppressed cell proliferation and colony formation, highlighting the protein's role in promoting tumor growth by facilitating cell cycle progression .

How can researchers effectively integrate in vitro and in vivo approaches for comprehensive ASAP2 functional characterization?

Integrating in vitro and in vivo approaches provides a more complete understanding of ASAP2 function:

Hierarchical Experimental Framework:

• Biochemical Characterization: Purified recombinant protein to define intrinsic properties

• Cellular Models: Cultured cells for mechanistic studies and pathway analysis

• Organoid Systems: 3D cultures to bridge the gap between 2D cultures and animal models

• Animal Models: In vivo validation of cellular findings in physiological context

• Human Samples: Correlation with patient specimens for clinical relevance

Integration Strategies:

Specific Combined Approaches for ASAP2:

• Domain Function Analysis: Test mutant constructs in vitro (binding assays, structural studies) and then express in cells and animals to assess functional consequences

• Pathway Validation: Identify interaction partners and pathways in cells, then verify their relevance in vivo using genetic or pharmacological approaches

• Translational Bridge: Use mouse models to validate findings from cell culture before moving to human patients or samples

Case Study: ASAP2 in Cancer Research
A comprehensive approach has been demonstrated in pancreatic cancer research, where:

• In vitro studies identified ASAP2's role in cell cycle progression and EGFR signaling

• CRISPR/Cas9 knockout cell lines revealed growth inhibition in culture

• These cells were then used for in vivo xenograft studies, demonstrating reduced tumor growth

• Patient data analysis showed correlation between ASAP2 expression and survival outcomes

• Drug repositioning studies identified niclosamide as an ASAP2-targeting compound, which was then validated in both in vitro and in vivo models

This integrated approach provided multiple lines of evidence supporting ASAP2 as a druggable target in pancreatic cancer, exemplifying how coordinated in vitro and in vivo strategies can accelerate translational research.