ANKRA2 Human

What is the basic structure of human ANKRA2 protein?

ANKRA2 is an ankyrin-repeat domain (ARD) containing protein characterized by five ankyrin repeat units. These repeats form a concave binding surface that mediates protein-protein interactions. The ARD of ANKRA2 adopts the canonical ankyrin repeat fold consisting of pairs of antiparallel α-helices connected by β-hairpin motifs . The domain exhibits a concave surface formed by the inner helices and hairpin loops, which serves as the primary interface for substrate binding. The structural integrity of ANKRA2 depends on conserved positions that are depleted in missense variants within the human population, indicating their importance for proper protein folding and function .

What are the key binding partners of ANKRA2?

ANKRA2 recognizes proteins containing a specific PxLPxL motif. Notable binding partners include:

• CCDC8: The 3M syndrome protein CCDC8 is a top binder of ANKRA2, with interaction occurring via a PxLPxL motif located at the C-terminal domain of CCDC8 .

• Class IIa Histone Deacetylases: ANKRA2 has been identified as a novel partner of class IIa histone deacetylases, including HDAC4, through protein interaction studies .

These interactions suggest ANKRA2 plays important roles in transcriptional regulation and potentially in developmental processes.

How is ANKRA2 expression regulated in human tissues?

ANKRA2 is widely expressed across human tissues, with notable expression in cells involved in transcriptional regulation. The regulation of ANKRA2 expression involves:

• Transcriptional Control: ANKRA2 is involved in the regulation of transcription by RNA polymerase II .

• Tissue Distribution: While comprehensive tissue-specific expression data for ANKRA2 in humans is still emerging, studies in model organisms like Drosophila show that ankyrin proteins are particularly abundant in neuronal tissues, suggesting possible enrichment of ANKRA2 in the human nervous system .

• Developmental Regulation: Expression patterns may change during development, though more research is needed to fully characterize these temporal patterns in humans.

What are the recommended methods for studying ANKRA2-protein interactions?

Several complementary approaches can be employed to study ANKRA2 interactions:

Structural Methods:

• X-ray crystallography has been successfully used to determine the structure of ANKRA2 in complex with binding partners, revealing the molecular basis of the interaction with the PxLPxL motif .

• Nuclear magnetic resonance (NMR) spectroscopy can provide information about the dynamics of these interactions.

Biochemical Methods:

• Co-immunoprecipitation assays using tagged versions of ANKRA2 and potential binding partners can confirm interactions in cellular contexts.

• Yeast two-hybrid screening can identify novel interaction partners.

• Pull-down assays with recombinant proteins can verify direct interactions.

Cellular Methods:

• Fluorescence resonance energy transfer (FRET) can be used to study interactions in living cells.

• Proximity ligation assays can detect protein-protein interactions with high sensitivity in fixed cells.

Computational Methods:

• Molecular dynamics simulations can predict stability and conformational changes in ANKRA2-partner complexes.

• Sequence-based prediction of PxLPxL motifs can identify potential new binding partners.

How can researchers effectively express and purify ANKRA2 for in vitro studies?

Recombinant expression and purification of ANKRA2 requires careful optimization:

Expression Systems:

• E. coli: BL21(DE3) strain with pET vectors containing His-tagged or GST-tagged ANKRA2 is commonly used.

• Insect cells: Baculovirus expression systems may be preferable for obtaining properly folded protein if bacterial expression proves challenging.

Purification Protocol:

• Affinity chromatography using Ni-NTA for His-tagged proteins or glutathione-agarose for GST-tagged proteins.

• Ion exchange chromatography to separate charged variants.

• Size exclusion chromatography as a final polishing step.

Critical Considerations:

• Include protease inhibitors throughout purification to prevent degradation.

• Consider co-expression with binding partners to stabilize the protein.

• Optimize buffer conditions (pH 7.0-8.0, 150-300 mM NaCl) to maintain protein solubility.

• Storage in small aliquots at -80°C with 10% glycerol to prevent freeze-thaw damage.

What techniques are most effective for characterizing ANKRA2 expression in tissue samples?

Multiple techniques can be employed to assess ANKRA2 expression:

Protein Detection:

• Immunohistochemistry with validated antibodies against ANKRA2.

• Western blotting for semi-quantitative analysis of protein levels.

• Mass spectrometry-based proteomics for unbiased detection.

mRNA Detection:

• RT-qPCR for quantitative analysis of ANKRA2 transcript levels.

• RNA in situ hybridization to visualize spatial distribution within tissues.

• RNA-seq for comprehensive transcriptomic profiling.

Considerations for Accurate Results:

• Use multiple antibodies targeting different epitopes to confirm specificity.

• Include appropriate positive and negative control tissues.

• Normalize expression data to validated housekeeping genes or proteins.

• Consider cell type-specific expression patterns when analyzing heterogeneous tissues.

How does ANKRA2 contribute to the pathogenesis of 3M syndrome through CCDC8 interaction?

The interaction between ANKRA2 and CCDC8 has significant implications for 3M syndrome, a primordial growth disorder:

Mechanistic Insights:

• ANKRA2 recognizes a specific PxLPxL motif in the C-terminal domain of CCDC8 .

• This interaction is part of a larger complex involving CUL7 and OBSL1, which together form a ubiquitin ligase complex implicated in growth regulation .

• Mutations in CCDC8 that disrupt the PxLPxL motif or affect its presentation may prevent proper binding to ANKRA2, potentially contributing to 3M syndrome pathology.

Experimental Approaches to Study This Relationship:

• Generate point mutations in the PxLPxL motif of CCDC8 and assess binding affinity to ANKRA2 using isothermal titration calorimetry or surface plasmon resonance.

• Perform co-immunoprecipitation studies in patient-derived cells with CCDC8 mutations.

• Develop cellular models using CRISPR/Cas9 to introduce 3M syndrome-associated mutations and assess downstream signaling effects.

• Use proximity-dependent biotinylation (BioID) to identify additional components of the ANKRA2-CCDC8 complex in disease-relevant cell types.

What are the structural determinants of ANKRA2 specificity for the PxLPxL motif?

The specific recognition of the PxLPxL motif by ANKRA2 involves several key structural features:

Key Structural Elements:

• Binding Pocket Composition: The ankyrin repeats of ANKRA2 form a specialized concave surface with hydrophobic pockets that accommodate the leucine residues in the PxLPxL motif .

• Critical Residues: Based on structural studies, specific residues within the ankyrin repeats make direct contacts with the PxLPxL motif, including both hydrophobic interactions and potential hydrogen bonds.

• Conserved Positions: Five positions within the ankyrin repeats are highly conserved across homologs and depleted in missense variants within the human population, suggesting their crucial role in maintaining the structural integrity necessary for binding specificity .

Table 1: Key Residues in ANKRA2 Important for PxLPxL Recognition

Experimental Approaches to Study Specificity:

• Alanine scanning mutagenesis of both ANKRA2 and PxLPxL-containing peptides.

• Hydrogen-deuterium exchange mass spectrometry to map the binding interface.

• Computational modeling and molecular dynamics simulations to predict the energetic contributions of specific residues.

How does ANKRA2 function in the regulation of class IIa HDACs compare to RFXANK?

Both ANKRA2 and RFXANK are ankyrin repeat proteins that interact with class IIa HDACs, but they exhibit distinct functional properties:

Comparative Features:

• Structural Similarities: Both ANKRA2 and RFXANK contain five ankyrin repeat units and recognize the PxLPxL motif present in class IIa HDACs .

• Functional Divergence: While both interact with HDACs, RFXANK has an established role in immune regulation as part of the RFX complex, whereas ANKRA2 appears to have broader cellular functions.

• Binding Preferences: Subtle differences in their ankyrin repeat structures may confer different affinities for specific HDAC family members or other binding partners.

Methodological Approaches to Compare Functions:

• Comparative binding assays with different HDACs to determine relative affinities.

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genomic regions where each protein is recruited in complex with HDACs.

• RNA interference or CRISPR knockout of each protein followed by transcriptomic analysis to determine overlapping and distinct gene expression changes.

• Protein localization studies to determine if ANKRA2 and RFXANK recruit HDACs to different subcellular compartments.

What is the significance of ANKRA2 in neuronal development and function?

While direct evidence for ANKRA2's role in human neuronal development is limited, research on related ankyrin proteins suggests potential functions:

Current Understanding:

• Ankyrin2 in Drosophila (which shares structural features with human ANKRA2) is required for neuronal morphogenesis and long-term memory formation .

• In Drosophila, Ankyrin2 is widely expressed throughout the brain and localizes predominantly to axon tracts .

• Knockdown of Ankyrin2 in the mushroom body of Drosophila results in defects in axon morphogenesis and impairs long-term memory .

Translational Relevance to Human ANKRA2:

• Human ANKRA2 may have conserved functions in neuronal development and plasticity.

• Its interaction with HDACs suggests a potential role in epigenetic regulation of genes involved in neuronal function.

Research Approaches to Explore Neuronal Functions:

• Study ANKRA2 expression in human neuronal cultures or brain organoids.

• Use CRISPR/Cas9 to knock out ANKRA2 in human neural progenitor cells and assess effects on differentiation and morphology.

• Investigate potential associations between ANKRA2 variants and neurological disorders through genomic analyses.

• Employ proximity labeling techniques to identify neuron-specific interaction partners of ANKRA2.

What are the most effective CRISPR-based approaches for studying ANKRA2 function?

CRISPR/Cas9 technology offers powerful tools for investigating ANKRA2 function:

Recommended Approaches:

• Complete Knockout Studies:

  • Design gRNAs targeting early exons to create frameshift mutations.

  • Verify knockout using Western blotting and genomic sequencing.

  • Assess phenotypic consequences on cell morphology, proliferation, and protein-protein interactions.

• Domain-Specific Editing:

  • Use base editing or prime editing to introduce specific mutations in the ankyrin repeat domain without disrupting the entire protein.

  • Target conserved residues identified in structural studies to disrupt specific interactions.

• Endogenous Tagging:

  • Knock-in fluorescent or affinity tags to track endogenous ANKRA2 localization and interactions.

  • Consider using split-GFP or HaloTag systems for studying dynamic interactions.

• Inducible Systems:

  • Implement degron-based approaches for acute protein depletion.

  • Use CRISPRi/CRISPRa systems for reversible manipulation of ANKRA2 expression levels.

Technical Considerations and Troubleshooting:

• Validate multiple gRNA designs to identify optimal targeting sequences.

• Screen multiple clones to account for heterogeneity in editing outcomes.

• Consider potential off-target effects by performing whole-genome sequencing.

• For essential functions, use inducible or tissue-specific systems to circumvent lethality.

How can researchers resolve contradictory findings about ANKRA2 interactions with its binding partners?

Contradictory results regarding ANKRA2 interactions may arise from differences in experimental conditions:

Common Sources of Discrepancies:

• Cell Type Differences: Interaction strength may vary across cell types due to expression levels of competing binding partners or post-translational modifications.

• Technical Variations: Different assay sensitivities (e.g., co-IP vs. yeast two-hybrid) can yield apparently conflicting results.

• Experimental Conditions: Buffer composition, salt concentration, and pH can significantly affect interaction strength.

• Protein Tags and Constructs: N- or C-terminal tags might interfere with specific interactions, and protein fragments may behave differently than full-length proteins.

Methodological Approaches to Resolve Contradictions:

• Comprehensive Interaction Analysis:

  • Use multiple complementary techniques (co-IP, FRET, proximity ligation assay).

  • Test interactions under various conditions (different salt concentrations, pH values).

  • Compare tagged and untagged versions of the proteins.

• Structural Validation:

  • Perform mutation studies based on structural data to confirm the molecular basis of interactions.

  • Use hydrogen-deuterium exchange mass spectrometry to map interaction interfaces.

• In-Cell Validation:

  • Use fluorescence correlation spectroscopy to measure interaction dynamics in living cells.

  • Implement BRET/FRET-based assays to detect real-time interactions.

  • Apply proximity-dependent biotinylation to capture transient interactions.

• Systematic Reporting:

  • Clearly document all experimental conditions.

  • Report negative results alongside positive findings.

  • Consider publishing detailed protocols to facilitate reproducibility.

What bioinformatic approaches can predict novel functions or interactions of ANKRA2?

Advanced computational methods can provide new insights into ANKRA2 biology:

Recommended Bioinformatic Approaches:

• Structural Prediction and Analysis:

  • AlphaFold2 or RoseTTAFold for predicting full-length ANKRA2 structure.

  • Molecular docking to identify potential new binding partners.

  • Molecular dynamics simulations to explore conformational dynamics.

• Network-Based Analyses:

  • Construct protein-protein interaction networks incorporating ANKRA2.

  • Apply graph theory algorithms to identify potential functional modules.

  • Integrate tissue-specific expression data to contextualize interactions.

• Evolutionary Analysis:

  • Compare ankyrin repeat sequences across species to identify conserved functional surfaces.

  • Analyze positions showing evidence of positive selection, which may indicate functionally important sites.

  • Study co-evolution patterns between ANKRA2 and potential binding partners.

• Integration of Multi-omics Data:

  • Correlate ANKRA2 expression with transcriptomic, proteomic, and phosphoproteomic datasets.

  • Look for condition-specific changes in expression or phosphorylation that might indicate regulatory mechanisms.

  • Apply machine learning algorithms to predict functional relationships from integrated datasets.

Validation Strategies for Computational Predictions:

• Prioritize predictions based on convergent evidence from multiple approaches.

• Design targeted experiments to test specific predictions.

• Implement medium-throughput screening to validate multiple predictions simultaneously.

How might single-cell techniques advance our understanding of ANKRA2 function?

Single-cell technologies offer unprecedented resolution for studying ANKRA2 biology:

Applicable Single-Cell Methods:

• Single-Cell RNA Sequencing (scRNA-seq):

  • Map ANKRA2 expression across cell types and states.

  • Identify co-expressed genes that may function in the same pathways.

  • Discover cell type-specific expression patterns of ANKRA2 binding partners.

• Single-Cell ATAC-Seq:

  • Correlate chromatin accessibility with ANKRA2 expression.

  • Identify potential regulatory regions controlling ANKRA2 expression.

• Single-Cell Proteomics:

  • Quantify ANKRA2 protein levels at the single-cell level.

  • Detect correlations between ANKRA2 and other proteins.

• Spatial Transcriptomics/Proteomics:

  • Map ANKRA2 expression within tissue architecture.

  • Identify spatial relationships between ANKRA2-expressing cells and their microenvironment.

• Live Single-Cell Imaging:

  • Track ANKRA2 dynamics in individual cells over time.

  • Correlate protein localization with cellular behaviors or responses.

Experimental Design Considerations:

• Include relevant cell states or developmental timepoints.

• Apply computational trajectory analysis to map dynamic changes in ANKRA2 function.

• Integrate multiple single-cell modalities for comprehensive understanding.

• Validate key findings using traditional approaches in selected cell populations.

What is the evidence linking ANKRA2 variants to human disease?

Current evidence connecting ANKRA2 to human disease is emerging but still limited:

Current Understanding:

• Through its interaction with CCDC8, ANKRA2 may indirectly contribute to 3M syndrome pathophysiology .

• Population genetic analysis shows that certain positions within ANKRA2's ankyrin repeats are depleted of missense variants in humans, suggesting functional constraint and potential disease relevance when mutated .

• Given its interaction with HDACs, which are involved in epigenetic regulation, ANKRA2 variants might influence conditions associated with dysregulated gene expression.

Research Approaches to Strengthen Disease Associations:

• Perform targeted sequencing of ANKRA2 in patient cohorts with relevant phenotypes (growth disorders, neurodevelopmental conditions).

• Analyze existing whole-exome and whole-genome sequencing datasets for enrichment of rare ANKRA2 variants in specific disease cohorts.

• Create cellular or animal models expressing disease-associated variants to assess functional consequences.

• Apply phenome-wide association studies (PheWAS) to identify conditions associated with common ANKRA2 variants.

How should researchers design experiments to investigate ANKRA2's role in specific cellular pathways?

Systematic experimental design is crucial for elucidating ANKRA2's role in cellular pathways:

Recommended Experimental Framework:

• Pathway Perturbation Analysis:

  • Perform ANKRA2 knockout or knockdown followed by phosphoproteomic analysis to identify affected signaling pathways.

  • Use small molecule inhibitors or activators of suspected pathways in combination with ANKRA2 manipulation.

  • Employ reporter assays for key transcription factors to detect pathway activation changes.

• Interactome Analysis:

  • Implement BioID or APEX proximity labeling to identify the complete ANKRA2 interactome.

  • Perform quantitative interaction proteomics under different cellular conditions (e.g., stress, differentiation).

  • Use crosslinking mass spectrometry to capture transient or weak interactions.

• Functional Genomics:

  • Conduct RNA-seq after ANKRA2 manipulation to identify transcriptional programs affected.

  • Perform ChIP-seq for HDAC4 and other binding partners with and without ANKRA2 to determine its influence on chromatin association.

  • Use CRISPR screens to identify synthetic lethal or synthetic viable interactions with ANKRA2.

• Dynamic Analysis:

  • Employ live-cell imaging with fluorescently tagged ANKRA2 to track subcellular localization changes in response to stimuli.

  • Use FRET sensors to detect conformational changes or interactions in real time.

  • Implement optogenetic approaches to acutely modulate ANKRA2 function in specific subcellular locations.

Controls and Validation Strategies:

• Include rescue experiments with wild-type ANKRA2 to confirm specificity of observed phenotypes.

• Use structurally-informed mutants that disrupt specific interactions to dissect pathway contributions.

• Verify key findings across multiple cell types to distinguish general from context-specific functions.