Recombinant Human Ankyrin repeat domain-containing protein 20A4 (ANKRD20A4), partial

What is ANKRD20A4 and where is it located in the human genome?

ANKRD20A4 (ankyrin repeat domain 20 family, member A4) is a protein-coding gene located on Chromosome 9 in humans. It belongs to the ankyrin repeat domain containing (ANKRD) family of proteins and is identified by NCBI Gene ID 728747 . This gene encodes the protein known as A20A4_HUMAN, which contains ankyrin repeat domains—one of the most abundant repeat motifs found in eukaryotic proteins .

How are ankyrin repeat domains structurally organized in ANKRD20A4?

Ankyrin repeat (AR) domains in ANKRD20A4, like other proteins in this family, consist of approximately 33 amino acid residues forming a helix-turn-helix motif followed by a β-hairpin . These domains typically have solvent-exposed regions that facilitate protein-protein interactions. The ankyrin repeat structure allows for significant functional versatility without requiring strict conformity within its primary sequence, enabling interactions with diverse binding partners . The structural organization suggests that ANKRD20A4 can potentially interact with multiple protein partners through its ankyrin repeat domains.

What biological functions are associated with ANKRD20A4?

Based on current research, ANKRD20A4 likely functions in protein-protein interactions mediated by its ankyrin repeat domains . While specific functions of ANKRD20A4 have not been fully characterized, proteins containing ankyrin repeat domains generally play critical roles in various biological processes including the ubiquitylation signaling pathway . The Harmonizome database indicates that ANKRD20A4 has 1,733 functional associations with biological entities spanning 5 categories (molecular profiles, chemicals, structural features, cell lines/tissues, and genes/proteins/microRNAs) extracted from 27 datasets . These associations provide insights into potential functions that warrant further investigation.

What are the recommended methods for expressing recombinant ANKRD20A4?

When expressing recombinant ANKRD20A4, researchers should consider a mammalian expression system, as post-translational modifications may be important for proper folding and function. HEK293T cells have been successfully used for expressing ankyrin repeat domain-containing proteins . For partial ANKRD20A4 expression, it is crucial to preserve key structural elements of the ankyrin repeat domains to maintain functionality. The expression protocol should include:

• Codon optimization for the expression system

• Addition of purification tags (e.g., His-tag or GST) that minimally interfere with protein folding

• Careful selection of buffer conditions to maintain protein stability

• Validation of proper folding using circular dichroism or limited proteolysis

Researchers should monitor protein solubility and stability during the purification process, as ankyrin repeat domains can be prone to aggregation if improperly folded.

How should experimental controls be designed when studying ANKRD20A4 function?

Designing appropriate controls is critical when studying ANKRD20A4 function. A rigorous experimental design should include:

• Positive controls: Known ankyrin repeat domain-containing proteins with well-characterized functions (e.g., ankyrin-G)

• Negative controls: Proteins lacking ankyrin repeat domains or mutated versions of ANKRD20A4 with disrupted ankyrin repeat structures

• Vector-only controls: Cells expressing only the vector backbone without the ANKRD20A4 insert

For functional assays, researchers should include both wild-type ANKRD20A4 and mutant versions with alterations in key residues of the ankyrin repeat domains . When studying protein stability, proteasome inhibitors like MG132 can be used to confirm proteasome-dependent degradation, as has been demonstrated with other ankyrin repeat proteins .

What cell lines are appropriate for studying ANKRD20A4 expression and function?

Based on available research, several cell line options exist for studying ANKRD20A4:

The choice should be guided by your specific research question. If investigating neurological implications, primary cortical neurons or neuronal cell lines would be appropriate, as ankyrin repeat domain proteins have been associated with neurological diseases . For general biochemical characterization, HEK293T cells provide a practical system with good expression levels.

What methods are most effective for identifying ANKRD20A4 binding partners?

Several complementary approaches can be used to identify ANKRD20A4 binding partners:

• Co-immunoprecipitation (Co-IP): This traditional approach can identify stable protein interactions but may miss transient interactions.

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions within 16 nm distance, making it ideal for identifying spatial relationships between ANKRD20A4 and potential binding partners in cellular contexts .

• Yeast Two-Hybrid Screening: Useful for high-throughput identification of potential interactors, though it requires validation with other methods.

• Affinity Purification-Mass Spectrometry (AP-MS): This approach can identify multiple interaction partners simultaneously and provide quantitative data on binding affinities.

For ANKRD20A4, a focus on ubiquitination pathway components would be particularly relevant, as ankyrin repeat domains have been implicated in ubiquitination processes . When designing these experiments, researchers should consider both phosphorylated and non-phosphorylated states of ANKRD20A4, as phosphorylation can significantly affect protein-protein interactions, as demonstrated with other ankyrin repeat proteins like ankyrin-G and its interaction with Usp9X .

How can researchers characterize the specificity of ANKRD20A4 protein-protein interactions?

Characterizing the specificity of ANKRD20A4 protein-protein interactions requires multiple approaches:

• Domain mapping: Create truncated versions of ANKRD20A4 to identify which ankyrin repeat domains mediate specific interactions. This approach has been successful with other ankyrin repeat proteins .

• Mutational analysis: Introduce point mutations in conserved residues within the ankyrin repeat domains. Key lysine residues are particularly important as they can be targets for ubiquitination .

• Competition assays: Use peptides derived from putative binding regions to compete with protein-protein interactions, allowing quantification of binding specificity.

• Structural biology approaches: X-ray crystallography or cryo-EM of ANKRD20A4-partner complexes can provide detailed insights into binding interfaces.

The ankyrin repeat domains in ANKRD20A4 likely recognize structural features rather than specific primary sequences in their binding partners , so researchers should focus on three-dimensional structural complementarity when analyzing interactions.

How is ANKRD20A4 stability regulated in cells?

While specific data on ANKRD20A4 regulation is limited, insights can be drawn from studies of other ankyrin repeat domain-containing proteins:

Based on research with ankyrin-G, ANKRD20A4 stability may be regulated through the ubiquitin-proteasome system . Ankyrin repeat domains often contain conserved destruction box (D-box) motifs that are recognized by E3 ubiquitin ligases such as Cdc20/APC . Treatment with proteasome inhibitors like MG132 has been shown to increase levels of ankyrin repeat domain-containing proteins, confirming their degradation through the proteasome pathway .

To investigate ANKRD20A4 stability:

• Examine its sequence for potential D-box motifs and lysine residues that may be ubiquitination targets

• Perform cycloheximide chase assays to measure protein half-life

• Use proteasome inhibitors to assess proteasome-dependent degradation

• Investigate interactions with deubiquitinating enzymes like Usp9X that may counteract ubiquitination

What role do phosphorylation events play in modulating ANKRD20A4 function?

Phosphorylation likely plays a critical role in regulating ANKRD20A4 function, similar to other ankyrin repeat proteins:

Studies of ankyrin repeat domain proteins have shown that phosphorylation can significantly enhance protein-protein interactions . For instance, phosphorylation of Usp9X at specific serine residues (S1598, S1600, S1608) significantly increases its interaction with ankyrin-G, with the S1608D phosphomimetic mutation showing the most robust enhancement of interaction .

To investigate phosphorylation of ANKRD20A4:

• Use phospho-specific antibodies or mass spectrometry to identify phosphorylation sites

• Create phosphomimetic (serine/threonine to aspartate) and phospho-null (serine/threonine to alanine) mutants to assess functional consequences

• Investigate kinases that may target ANKRD20A4 based on consensus sequence recognition

• Perform in vitro kinase assays to confirm direct phosphorylation

What neurological conditions are associated with altered ANKRD20A4 expression or function?

Current literature indicates that ankyrin repeat domain-containing proteins are associated with several neurological conditions, though specific associations for ANKRD20A4 remain to be fully characterized . Ankyrin repeat domain-containing proteins play critical roles in neuronal development and function, particularly in dendritic spine development . Mutations or dysregulation of these proteins can contribute to neurological disorders.

Research into ankyrin-G, another ankyrin repeat domain-containing protein, has shown its importance in dendrite morphogenesis through Cdc20/APC-dependent ubiquitination of its ankyrin repeat domain . Similar mechanisms may apply to ANKRD20A4, suggesting potential roles in neurodevelopmental disorders if dysregulated.

To investigate neurological implications of ANKRD20A4:

• Analyze expression patterns in neuronal tissues and during brain development

• Assess effects of ANKRD20A4 knockdown or overexpression on neuronal morphology

• Examine genetic associations between ANKRD20A4 variants and neurological disorders

• Investigate interactions with known neuronal proteins

How does ANKRD20A4 contribute to cellular stress responses?

While specific data on ANKRD20A4's role in stress responses is limited, ankyrin repeat domain-containing proteins generally participate in cellular adaptation to stress:

The ubiquitination pathway, in which many ankyrin repeat proteins function, is a key component of cellular stress responses . These proteins can regulate the stability of stress-responsive transcription factors and signaling molecules. Additionally, protein quality control mechanisms involving ubiquitination are critical during cellular stress.

To investigate ANKRD20A4's role in stress responses:

• Compare ANKRD20A4 expression levels under various stress conditions (oxidative stress, heat shock, ER stress)

• Identify stress-induced changes in ANKRD20A4 localization or post-translational modifications

• Assess the impact of ANKRD20A4 depletion on cellular survival during stress

• Investigate interactions with known stress-responsive proteins

What bioinformatic approaches are most useful for analyzing ANKRD20A4 structure and function?

Several bioinformatic approaches are valuable for studying ANKRD20A4:

• Structural modeling: Use threading approaches to model ANKRD20A4 structure based on known ankyrin repeat structures . This can identify solvent-exposed residues that may be involved in protein-protein interactions or post-translational modifications.

• Evolutionary analysis: Compare ANKRD20A4 sequences across species to identify conserved regions that may be functionally important. Pay particular attention to conserved lysines and D-box motifs within the ankyrin repeat domains .

• Network analysis: Utilize databases like Harmonizome to explore ANKRD20A4's functional associations with other biological entities across multiple datasets . This can provide insights into potential functions and interactions.

• Protein-protein interaction prediction: Use algorithms that predict potential binding partners based on structural complementarity and known interaction motifs.

When interpreting bioinformatic analyses, researchers should consider the three-dimensional structure of the ankyrin repeat domains, as their function is often determined by structural features rather than specific primary sequences .

How can researchers overcome challenges in analyzing ANKRD20A4 expression data?

Analyzing ANKRD20A4 expression data presents several challenges that can be addressed through careful experimental design and data analysis:

• Tissue specificity: ANKRD20A4 may have tissue-specific expression patterns. Researchers should consult tissue expression databases and validate findings with qPCR or Western blotting in relevant tissues.

• Isoform complexity: Consider potential alternative splicing of ANKRD20A4. Design primers or antibodies that can distinguish between isoforms.

• Cross-reactivity: The ankyrin repeat family has multiple members with similar domains. Use highly specific antibodies and validate specificity with knockdown or knockout controls.

• Data normalization: When analyzing expression across different conditions or tissues, use multiple reference genes for normalization and consider using both RNA and protein level measurements.

• Copy number variations: The CCLE Cell Line Gene CNV Profiles and COSMIC Cell Line Gene CNV Profiles datasets indicate variable copy numbers of ANKRD20A4 across cell lines . These variations should be considered when interpreting expression data.

What are the key considerations for designing knockout or knockdown experiments for ANKRD20A4?

Designing effective knockout or knockdown experiments for ANKRD20A4 requires careful consideration of several factors:

• Method selection:

  • CRISPR-Cas9 for complete knockout

  • siRNA or shRNA for temporary knockdown

  • Inducible systems for temporal control of expression

• Target specificity:

  • Design guide RNAs or siRNAs that specifically target ANKRD20A4 without affecting other ankyrin repeat family members

  • Include validation of specificity by measuring expression of related family members

• Controls:

  • Non-targeting control guides or siRNAs

  • Rescue experiments with ANKRD20A4 cDNA resistant to the targeting strategy

  • Include wild-type and mutant (non-functional) rescue constructs

• Phenotypic analysis:

  • Assess multiple cellular functions, including those related to known ankyrin repeat protein functions (protein-protein interactions, ubiquitination pathways)

  • Consider both acute and long-term consequences of ANKRD20A4 depletion

  • If studying neurons, include dendrite morphology analysis, as ankyrin repeat proteins influence dendrite development

How should researchers design experiments to investigate ANKRD20A4 function in the ubiquitylation pathway?

Based on the knowledge that ankyrin repeat domain-containing proteins often function in the ubiquitylation signaling pathway , researchers can design experiments to investigate ANKRD20A4's specific role:

• Ubiquitination assays:

  • In vitro ubiquitination assays with purified components

  • Cellular ubiquitination assays using tagged ubiquitin

  • Analysis of ubiquitination patterns under conditions of ANKRD20A4 overexpression or depletion

• E3 ligase interaction studies:

  • Investigate interactions with E3 ligases, particularly Cdc20/APC, which recognizes D-box motifs in ankyrin repeat domains

  • Use co-immunoprecipitation and proximity ligation assays to detect interactions in cells

• Deubiquitinase interactions:

  • Examine potential interactions with deubiquitinases like Usp9X

  • Investigate how phosphorylation states affect these interactions

• Substrate identification:

  • Use proteomics approaches to identify proteins with altered ubiquitination status upon ANKRD20A4 manipulation

  • Validate key substrates with targeted experiments

• Functional readouts:

  • Measure protein stability using cycloheximide chase assays

  • Assess proteasome inhibitor sensitivity

  • Monitor cellular responses to ubiquitination pathway perturbations

What emerging technologies could advance our understanding of ANKRD20A4?

Several cutting-edge technologies hold promise for deepening our understanding of ANKRD20A4:

• Cryo-electron microscopy: This technology could provide high-resolution structural insights into ANKRD20A4 and its complexes with interaction partners, revealing binding interfaces and conformational changes.

• Proximity labeling proteomics: Technologies such as BioID or APEX2 can identify proteins in close proximity to ANKRD20A4 in living cells, providing a more comprehensive view of its protein interaction network.

• Single-cell approaches: Single-cell RNA-seq and proteomics can reveal cell type-specific expression patterns and functions of ANKRD20A4, particularly important if studying heterogeneous tissues like brain.

• CRISPR screens: Genome-wide or targeted CRISPR screens can identify genetic interactions with ANKRD20A4, revealing functional connections and potential redundancies with other proteins.

• Structural prediction using AI: New AI-based structural prediction tools like AlphaFold2 could provide insights into ANKRD20A4 structure and potential interaction surfaces, guiding experimental design.

How can systems biology approaches enhance our understanding of ANKRD20A4 function?

Systems biology approaches offer powerful frameworks for understanding ANKRD20A4 within broader cellular contexts:

• Network analysis: Integrate ANKRD20A4 into protein-protein interaction networks, particularly those involved in ubiquitination pathways. The Harmonizome database indicates ANKRD20A4 has 1,733 functional associations across multiple biological entities , which can form the basis for network construction.

• Multi-omics integration: Combine transcriptomics, proteomics, and ubiquitinomics data to understand how ANKRD20A4 perturbation affects multiple layers of cellular regulation.

• Pathway enrichment analysis: Identify biological pathways enriched among ANKRD20A4-interacting proteins or genes affected by ANKRD20A4 manipulation.

• Mathematical modeling: Develop computational models of ANKRD20A4-containing protein complexes to predict how perturbations affect system behavior.

• Comparative analysis across ankyrin repeat proteins: Systematically compare functions and interactions of different ankyrin repeat domain-containing proteins to identify common and unique features of ANKRD20A4.