Recombinant Mouse Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit C (Ankrd52), partial

What is the functional relationship between ANKRD52 and the PPP6C phosphatase complex?

ANKRD52 serves as a regulatory ankyrin repeat subunit that forms a complex with the catalytic subunit PPP6C. This ANKRD52-PPP6C phosphatase complex plays a critical role in microRNA (miRNA) biogenesis by dephosphorylating Argonaute 2 (AGO2), which restores AGO2's miRNA loading activity. The complex counterbalances casein kinase 1α (CK1α)-induced phosphorylation that would otherwise cause AGO2 to dissociate from the active complex . This regulation maintains proper miRNA-mediated gene silencing, which impacts numerous cellular processes including immune response pathways.

How do ANKRD52 mutations affect miRNA machinery function in experimental models?

Mutations in ANKRD52 significantly disrupt the miRNA machinery's function by impairing the dephosphorylation of AGO2. Re-introduction of frequent ANKRD52 patient mutations dampens the JAK-STAT-interferon-γ signaling and antigen presentation in cancer cells, largely by abolishing miR-155-targeted silencing of suppressor of cytokine signaling 1 (SOCS1) . This dysregulation has profound effects on gene expression profiles, particularly those involved in immune response pathways. In experimental models, ANKRD52 mutations can lead to altered T cell recognition of cancer cells, potentially contributing to immune evasion mechanisms.

What role does ANKRD52 play in tumor suppression?

ANKRD52 has been identified as a suppressor of tumor metastases, with reduced ANKRD52 levels associated with late-stage lung cancer . As part of the miRNA machinery, ANKRD52 helps maintain proper gene expression by ensuring functional miRNA-mediated silencing. When ANKRD52 function is compromised, the resulting dysregulation can promote oncogenic pathways and suppress tumor-inhibitory mechanisms. Its role in maintaining JAK-STAT-interferon-γ signaling and antigen presentation further supports its tumor suppressive function by facilitating immune surveillance mechanisms.

What are the optimal methods for expressing and purifying recombinant mouse ANKRD52?

For successful expression and purification of recombinant mouse ANKRD52:

The recommended workflow includes:

• Clone the partial mouse Ankrd52 sequence into an expression vector with an N-terminal tag

• Express in the chosen system (mammalian cells preferred for functional studies)

• Lyse cells in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease inhibitors

• Purify using affinity chromatography followed by size exclusion chromatography

• Validate purified protein by SDS-PAGE, Western blot, and functional phosphatase assays with AGO2 as substrate

How can researchers effectively develop and validate ANKRD52 knockout mouse models?

To develop and validate ANKRD52 knockout mouse models, researchers should consider a strategy similar to the Ppp6c knockout models described in the literature :

• Design strategy:

  • Use Cre-loxP recombination systems (e.g., Pdx1-CreER^T2 for pancreas-specific deletion)

  • Include tamoxifen-inducible systems for temporal control of gene deletion

  • Consider crossing with cancer-prone models (e.g., KRAS^G12D/Trp53-deficient) to study cancer contexts

• Validation methods:

  • PCR genotyping to confirm recombination of floxed alleles

  • Western blot analysis to verify protein depletion

  • qRT-PCR to assess mRNA levels

  • Immunohistochemistry to examine tissue-specific knockout efficiency

• Phenotypic assessment:

  • Monitor for spontaneous tumor development

  • Examine tissue-specific changes in miRNA profiles by small RNA sequencing

  • Analyze changes in ANKRD52-dependent signaling pathways (JAK-STAT, NFκB)

  • Evaluate immune cell infiltration in relevant tissues

What methods are most effective for studying phosphorylation dynamics in the ANKRD52-PPP6C-AGO2 axis?

For studying phosphorylation dynamics in the ANKRD52-PPP6C-AGO2 axis, researchers should employ a multi-faceted approach:

• Phospho-specific antibodies and Western blotting:

  • Use antibodies targeting specific phosphorylation sites on AGO2

  • Compare phosphorylation levels in wild-type vs. ANKRD52-deficient conditions

  • Utilize phosphatase inhibitors to preserve phosphorylation status during sample preparation

• Mass spectrometry-based approaches:

  • Employ phospho-proteomic analysis to identify and quantify specific phosphorylation sites

  • Use SILAC (Stable Isotope Labeling by Amino acids in Cell culture) for quantitative comparison

  • Apply targeted MS methods for improved sensitivity of specific phosphorylation events

• In vitro dephosphorylation assays:

  • Purify recombinant ANKRD52-PPP6C complex and phosphorylated AGO2

  • Perform time-course experiments to measure dephosphorylation kinetics

  • Use phosphomimetic and phospho-dead AGO2 mutants as controls

• Live-cell imaging techniques:

  • Develop FRET-based biosensors for real-time monitoring of AGO2 phosphorylation

  • Use fluorescently tagged proteins to track complex formation and localization

How does ANKRD52 dysfunction contribute to pancreatic cancer progression?

While ANKRD52 was not directly studied in the pancreatic cancer models from the search results, its function as part of the PPP6C complex suggests a significant role. PPP6C deficiency in mice with KRAS^G12D mutation and Trp53 loss promoted aggressive pancreatic tumorigenesis . Since ANKRD52 is essential for PPP6C function, we can infer that ANKRD52 dysfunction would similarly contribute to pancreatic cancer progression through:

• Enhanced signaling pathway activation:

  • Increased phosphorylation of Erk and RelA (NFκB pathway component)

  • Upregulation of DUSP4, 5, 6, and 10 due to negative feedback mechanisms

  • Dysregulation of inflammatory signaling pathways

• Accelerated tumor development and progression:

  • Increased number and size of tumors

  • More numerous precancerous lesions (acinar-to-ductal metaplasia)

  • Development of pancreatic ductal adenocarcinoma (PDAC)

  • Evidence of epithelial-mesenchymal transition (EMT)

• Inflammatory and metabolic changes:

  • Enhanced cancer-specific glycolytic metabolism

  • Increased expression of inflammatory cytokines

  • Elevated circulating TNF-α and IL-6 levels

  • Systemic inflammation and cachexia

What is the relationship between ANKRD52 and immune evasion mechanisms in cancer?

ANKRD52, as part of the miRNA machinery, plays a crucial role in cancer cell sensitivity to T cell-mediated cytotoxicity . The relationship between ANKRD52 and immune evasion includes:

• Regulation of JAK-STAT-interferon-γ signaling:

  • Genetic inactivation of the miRNA machinery (including ANKRD52) dampens this signaling pathway

  • This occurs largely by abolishing miR-155-targeted silencing of SOCS1, a negative regulator of JAK-STAT signaling

  • Impaired interferon signaling reduces cancer cell responsiveness to T cell activity

• Impact on antigen presentation:

  • Disruption of ANKRD52 function impairs antigen presentation mechanisms in cancer cells

  • This reduces T cell recognition of cancer cells, facilitating immune evasion

  • Expression of miRNA machinery components strongly correlates with intratumoral T cell infiltration in nearly all human cancer types

• Resistance to immunotherapy:

  • Cancer cells with ANKRD52 mutations may be less responsive to immune checkpoint blockade

  • These mutations can drive resistance to PD-1-independent T cell-mediated cytotoxicity

  • Inactivation of the miRNA machinery represents an evolutionary advantage for cancer cells under immune pressure

How do ANKRD52 mutations affect response to immunotherapy in experimental models?

ANKRD52 mutations significantly impact response to immunotherapy in experimental models by modulating cancer cell visibility to the immune system :

• Impact on T cell-mediated cytotoxicity:

  • Genetic inactivation of the miRNA machinery (including ANKRD52) confers resistance to PD-1-independent T cell-mediated cytotoxicity

  • This suggests ANKRD52-mutant tumors may be inherently resistant to certain immunotherapies

  • The resistance mechanism operates independently of the PD-1/PD-L1 immune checkpoint

• Effects on tumor evolution under immune pressure:

  • Cancer cells acquire genetic heterogeneity to escape immune surveillance during tumor evolution

  • ANKRD52 mutations may be selected for during this process, particularly under immunotherapy pressure

  • Mutations may facilitate tumor escape from T cell-mediated elimination and immunotherapy

• Experimental evidence from mouse models:

  • Tumors from immunocompetent mice with and without immunotherapy show differential mutation patterns

  • Some mutations are eliminated by T cells after PD-1/PD-L1 blockade, while others persist

  • CRISPR library screens in immunocompetent murine tumors can identify genes like ANKRD52 that mediate immune evasion

How can researchers distinguish between direct and indirect effects of ANKRD52 dysfunction on miRNA networks?

Distinguishing between direct and indirect effects of ANKRD52 dysfunction on miRNA networks requires sophisticated experimental approaches:

Additionally, researchers should:

• Use inducible knockout systems to capture immediate (direct) versus long-term (indirect) effects

• Combine with AGO2 phospho-mutants (phosphomimetic or phospho-dead) to bypass ANKRD52 regulation

• Perform time-course experiments to track the propagation of effects through the regulatory network

• Focus on specific miRNAs known to be affected by AGO2 phosphorylation, such as miR-155

What are the key considerations for designing clinical studies involving ANKRD52 biomarkers?

When designing clinical studies involving ANKRD52 as a biomarker, researchers should consider:

How can ANKRD52 function be effectively restored in experimental disease models?

Restoring ANKRD52 function in experimental disease models requires sophisticated approaches tailored to the specific defect:

• Gene therapy approaches:

  • Viral vectors (AAV, lentivirus) expressing wild-type ANKRD52

  • Tissue-specific promoters to target expression to relevant cell types

  • CRISPR-based approaches for correcting specific mutations

  • mRNA delivery systems for transient expression

• Small molecule modulators:

  • Inhibitors targeting CK1α to reduce AGO2 phosphorylation

  • Compounds that stabilize the ANKRD52-PPP6C complex

  • Molecules that mimic ANKRD52 function by facilitating AGO2 dephosphorylation

  • Drugs that modulate downstream pathways (JAK-STAT, NFκB)

• Peptide-based therapeutics:

  • Peptides derived from ANKRD52 functional domains

  • Cell-penetrating peptide conjugates to improve delivery

  • Stapled peptides for enhanced stability and target binding

  • Peptide-nucleic acid conjugates to target specific miRNA-mRNA interactions

• RNA-based approaches:

  • miRNA mimics to compensate for defective miRNA loading

  • Anti-miRs targeting upregulated miRNAs resulting from ANKRD52 dysfunction

  • siRNAs targeting overexpressed genes normally suppressed by ANKRD52-dependent miRNAs

  • Modified mRNAs encoding functional ANKRD52

• Validation methods:

  • Rescue of miRNA loading onto AGO2 (assessed by CLIP-seq)

  • Restoration of normal phosphorylation dynamics of AGO2

  • Correction of downstream gene expression profiles

  • Functional readouts specific to the disease model (e.g., tumor growth reduction, immune cell infiltration)

What are common technical challenges in ANKRD52 immunoprecipitation experiments and how can they be overcome?

Researchers often encounter several challenges when performing ANKRD52 immunoprecipitation experiments:

• Low antibody specificity and cross-reactivity:

  • Challenge: Commercial antibodies may have limited specificity for mouse ANKRD52

  • Solution: Validate antibodies using ANKRD52 knockout controls

  • Alternative: Use epitope-tagged recombinant ANKRD52 (FLAG, HA, or V5) and corresponding high-affinity antibodies

• Preservation of protein complex integrity:

  • Challenge: The ANKRD52-PPP6C-AGO2 complex may dissociate during extraction

  • Solution: Use gentle lysis conditions (0.5% NP-40 or 0.1% Triton X-100)

  • Alternative: Apply crosslinking reagents (DSP, formaldehyde) prior to lysis

• Maintaining phosphorylation status:

  • Challenge: Phosphorylation events may be lost during sample processing

  • Solution: Include phosphatase inhibitors (sodium orthovanadate, β-glycerophosphate) in all buffers

  • Alternative: Use rapid extraction methods to minimize dephosphorylation

• Low yield of co-immunoprecipitated proteins:

  • Challenge: Detecting interacting partners may be difficult due to low abundance

  • Solution: Scale up starting material and optimize IP conditions

  • Alternative: Use more sensitive detection methods (fluorescent Western blotting, mass spectrometry)

• Non-specific binding to beads:

  • Challenge: High background due to proteins binding non-specifically to beads

  • Solution: Pre-clear lysates with beads, use more stringent washing conditions

  • Alternative: Employ tandem affinity purification approaches

How can researchers effectively assess miRNA function in the context of ANKRD52 studies?

To effectively assess miRNA function in ANKRD52 studies, researchers should implement a multi-layered approach:

• Global miRNA profiling methods:

  • Small RNA sequencing to identify differentially expressed miRNAs

  • miRNA microarrays for targeted profiling

  • qRT-PCR arrays for validation of specific miRNA changes

  • AGO2-CLIP-seq to identify actively loaded miRNAs

• Target validation approaches:

  • Luciferase reporter assays with wild-type and mutant 3'UTR sequences

  • Western blotting to confirm protein-level changes of miRNA targets

  • qRT-PCR to measure target mRNA expression changes

  • Correlation analysis between miRNA and target expression in various conditions

• Functional assays to link miRNA changes to biological outcomes:

  • Cell proliferation, migration, and invasion assays

  • Apoptosis assays

  • Immune cell co-culture experiments to assess T cell recognition

  • Animal models to evaluate in vivo relevance

• Experimental designs specific to ANKRD52 function:

  • Compare wild-type, ANKRD52-knockout, and ANKRD52-reconstituted conditions

  • Introduce phosphomimetic or phospho-dead AGO2 mutants to bypass ANKRD52-PPP6C regulation

  • Implement miRNA mimics or inhibitors to rescue or phenocopy ANKRD52 deficiency effects

  • Use inducible systems to capture temporal dynamics of miRNA function

What are the best experimental designs for studying ANKRD52's role in immune recognition of tumor cells?

For studying ANKRD52's role in immune recognition of tumor cells, researchers should consider these experimental designs:

• In vitro co-culture systems:

  • Set up co-cultures of ANKRD52-wildtype or ANKRD52-mutant cancer cells with T cells

  • Measure T cell activation markers (CD69, CD25, IFN-γ production)

  • Quantify cancer cell killing using real-time impedance-based systems

  • Perform blocking experiments with neutralizing antibodies against key mediators

• In vivo tumor models:

  • Establish syngeneic mouse models with ANKRD52-wildtype or ANKRD52-mutant cancer cells

  • Compare tumor growth in immunocompetent versus immunodeficient mice

  • Test response to immune checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

  • Analyze tumor-infiltrating lymphocytes by flow cytometry and spatial transcriptomics

• Antigen presentation assessment:

  • Measure MHC class I surface expression in ANKRD52-manipulated cells

  • Assess antigen processing machinery components (TAP, tapasin, ERAP)

  • Use model antigens (OVA, SIY) and cognate T cell receptors to quantify presentation

  • Perform peptide elution and mass spectrometry to identify presented antigens

• Immune signaling pathway analysis:

  • Monitor JAK-STAT-interferon-γ signaling pathway activation

  • Measure expression of interferon-stimulated genes

  • Assess SOCS1 levels and its relationship to miR-155 expression

  • Evaluate inflammatory cytokine production and response

• Clinical sample correlation:

  • Analyze ANKRD52 expression/mutation status in patient samples

  • Correlate with T cell infiltration metrics

  • Examine relationship to response to immunotherapy

  • Perform spatial analyses of immune cell localization relative to ANKRD52-expressing cells